INTRODUCTION

Introduction to the Biology of Apolipoprotein M (ApoM)

Cholesterol is an integral part of cell membranes and in the synthesis of steroids, bile, and vitamin D. Cholesterol found in the human body comes from two sources: biosynthesis and diet [1]. Cholesterol is a hydrophobic molecule and cannot travel through the bloodstream without assistance from lipoproteins. Lipoprotein particles consist of protein components called apolipoproteins that help solubilize the hydrophobic lipids [2]. Apolipoproteins transport cholesterol through the bloodstream to target organs and tissues. A schematic of cholesterol transport and cycling of lipoproteins is shown in

Figure 1 [3]. Lipoprotein particles are generally classified as high, intermediate, low, and very low density lipoproteins (HDL, IDL, LDL, and VLDL, respectively), and chylomicrons [1]. Lipid-free apolipoprotein AI and lipid-poor pre β-HDL particles are precursors to the generation of mature, lipid-containing HDL particles. Apolipoprotein

M is a component of pre β-HDL and is present in a sub-population of mature HDL particles. Pre β-HDL particles are thought to function primarily to induce cholesterol efflux from cells and act as the initial acceptors of cholesterol from peripheral tissues in reverse cholesterol transport (RCT) [2]. In RCT, HDL accepts cholesterol from peripheral cells, transports, and delivers it to the liver for degradation and excretion [2].

Pre β-HDL accepts unesterfied cholesterol and phospholipids, then the enzyme LCAT

(lecithin cholesterol acyltransferase) esterfies the free cholesterol to modify the pre β-

HDL into the spherical α-HDL (cholesterol ester core) [3]. The pre-beta form of HDL

1 exists in a lipid-poor state and may be indicative of the cholesterol-carrying capacity of the HDL in circulation.

Human apoM was discovered by Xu, N. et al in 1999 [4] and is mainly associated with a small sub-population of HDL particles [5]. HDL particles lacking apoM showed a

50% decrease in cholesterol efflux from macrophages compared to apoM-containing

HDL particles in vitro [2]. Human apoM is primarily associated with lipid-poor pre β-

HDL, mature HDL, and to a lesser extent LDL and VLDL [4]. Wolfrum et al demonstrated that apoM is necessary for the formation of these pre β-HDL particles [2].

It has been shown that elevated levels of LDL, VLDL, and chylomicrons in circulation can lead to atherosclerotic lesion formation, whereas HDL has protective anti- atherogenic effects [6]. As a component of HDL, apoM is thought to have a significant influence on the development of coronary heart disease (CHD), caused by atherosclerosis

[7]. Atherosclerotic lesions are a product of the accumulation of macrophage foam cells in blood vessels. Foam cells can be formed when excess LDL in circulation is oxidized and engulfed by macrophages [8]. Accumulation of chylomicron remnants in macrophages can also promote foam cell formation without oxidation [9]. The apoM present in LDL particles is thought to play a protective role against CHD by reducing oxidation of LDL and it has been shown that apoM-containing LDL particles are more resistant to oxidation [10]. LDL receptor knockout (ldlr-/-) mice over-expressing apoM had 70% less atherosclerotic lesion formation compared to ldlr-/- mice with endogenous normal apoM levels [2], supporting the anti-atherogenic benefits of apoM and making it a significant apolipoprotein in the study of CHD.

2 ApoM is also studied in association with other lipid metabolic diseases including diabetes, specifically mature-onset diabetes of the young (MODY3) [11, 12] and obesity

[13]. Since apoM is a recently characterized protein [4], a quantitative assay for apoM has not yet been well established. Western blots have been used to demonstrate changes in apoM levels [2], but this method is not highly quantitative and suffers from low throughput. To date there is not a commercially-available quantitative assay for apoM in any species. The ability to quantitatively analyze apoM will enable a better understanding of its behavior in relationship with certain diseases. Even though development and use of an ELISA has been reported [14], the assay is not widely available. In-house efforts by a collaborator to replicate an ELISA measuring apoM in human serum has proven difficult and was ultimately unsuccessful (unpublished data).

To address the need for a quantitative assay for apoM in serum, an antibody-free, high throughput, mass spectrometry-based assay was developed.

Introduction to Mass Spectrometry

The use of high performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) has been gaining popularity for development of quantitative assays over the past five years [15]. However, this approach often requires an antibody for selective enrichment of the target protein, especially when the protein of interest is in low abundance [16]. We developed a MS-based targeted assay to quantify apoM in human and rodent (mouse or rat) serum that does not require the use of an antibody. This label- free method to quantify apoM in serum provides a powerful tool to further understand the biology of apoM with many research applications.

3 In the initial stages of the development of a quantitative assay for a specific protein or proteins using a targeted LC-MS method, an unbiased global MS approach can be used to identify the target protein in serum. Global profiling studies are typically done to identify the entire protein content of a sample. Global profiling of a biological sample

(i.e. serum) begins with enzymatic digestion of the proteins, typically with trypsin to create tryptic peptides. These peptides can be separated based on hydrophobicity using a simple, two and a half hour HPLC gradient prior to MS analysis. The peptides in the effluent of the HPLC column are ionized and sprayed into an on-line ion trap MS, such as an LTQ (Thermo). Proteins are identified from this analysis based on the identification of unique tryptic peptides from a specific protein.

The ionized tryptic peptides are measured by MS as a mass-to-charge ratio ( m/z ) using the molecular mass and charge status of each peptide. In a global profiling study, a triple-play method can be used to collect ion spectra using three MS scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan.

The full MS scan captures the mass-to-charge ( m/z ) ratios of all ionized peptides eluted at a specific time point. The most abundant peptide in the scan is selected as a precursor ion and a zoom scan of this peptide is used to estimate the charge status and monoisotopic and average masses of the peptide. The zoom scan also evaluates the quality of the selected peptide to avoid false positive protein identifications and eliminates low quality data from further analysis. The selected peptide is then fragmented, and the spectra of these product ions are collected by a MS/ MS scan [17]. The product ions from fragmentation of the precursor ion at the amide bond of the peptide backbone are classified as b-ions and y-ions. A b-ion is observed when the proton is retained by the N-

4 terminal fragment of the peptide, and a y-ion is observed with the retention of the proton by the C-terminal fragment of the peptide [18]. The fragmentation spectra is searched against species-specific computerized protein databases and used for peptide sequencing and protein identification.

The data output from the database search yields protein identifications based on the detection of a tryptic peptide that is unique to the protein from which it was derived.

A targeted MS method can be created for a specific protein or proteins of interest using the results from these global profiling studies. Identification of the protein of interest and the MS/ MS spectra generated from the fragmentation of the precursor ion are collected by the global studies and are used to set up a targeted Multiple Reaction Monitoring

(MRM) assay using the same instrument [19].

The m/z values of the precursor ion and respective fragmentation y- and b-ions obtained from the global profiling studies are used to set up a targeted assay to measure only specific m/z values. Only these m/z values will be collected by the MS while the m/z values of precursor ions that do not fall within the set m/z window are filtered out. The

MS peptide signal from the precursor ion can be integrated to obtain the area-under-curve value (AUC) which can be used to quantify the target protein.

These MS methods were used to develop a targeted assay for the quantification of apoM in human and rodent serum. This assay spans multiple species to streamline the use of this assay between pre-clinical and clinical measurements. The resulting assay is versatile and quantitative for apoM and will provide a powerful tool to expand research in

CHD and other diseases and provide a deeper understanding of the biology of apoM in many different applications.

5 Figure 1

Figure 1: Lipoprotein involvement in lipid transport

The transport of lipid through the bloodstream to target cells is achieved by lipoproteins.

This schematic of lipid transport was from Brewer, HB Jr., N Engl J Med 2004;

350:1491-1494, Apr 8, 2004.

6 EXPERIMENTAL SECTION

Reagents

Human serum was purchased from Biowhittaker (cat #14-491E and 14-402E) and

Bioreclamation (cat #HMSRM). Rabbit serum was purchased from Biomeda (cat

#MS008). Horse serum was from GIBCO (cat #16050-122). Rat serum was from Pel- freeze (cat #36125-3), Harlan (cat #4511) and Biomeda (cat #MS009) and mouse serum was from Lampire (cat #SI-1409). Dulbecco’s Phosphate Buffered Saline (PBS) was from Invitrogen (cat #10010-023). PHM-Liposorb was from CalBiochem (cat #524371).

Ammonium bicarbonate (cat #A6141), DL-Dithiothreitol (cat #D9779), iodoacetamide

(cat #A3221) and urea (cat #208884) were from Sigma. Modified trypsin was purchased from Promega (cat #V5280) and NP40 detergent was from Pierce (cat #28324). HPLC- grade 0.1% formic acid in water (cat #HB523-4) was from Fisher Scientific. HPLC- grade water (cat #365-4) and acetonitrile (cat #015-4) were from Burdick & Jackson.

Formic acid was from J.T. Baker (cat #0129-01). Synthetic peptides were from Midwest

Biotech (Fishers, IN) and 15 N-labeled human apolipoprotein A-IV was prepared in-house by purification of the recombinant protein from E. coli grown in a medium containing

15 N-urea. Mouse anti-apoM primary antibody was from BD Transduction Labs (cat

#612333) and an ECC anti-mouse IgG-Horseradish peroxidase secondary antibody was from Amersham (cat #NA931V). ECL kit was from Amersham (cat #RPN303D).

Human recombinant apoM from E. coli was grown in-house and given by Dr. Thomas

Lee (Eli Lilly and Co.).

7 Sample Preparation for Global LC-MS/ MS Analysis of Human and Rat Serum

Serum was enriched with lipoprotein-binding beads to selectively purify apolipoproteins from serum prior to digestion with trypsin. The digested proteins were separated by reverse-phase HPLC and analyzed by MS using a global profiling method to identify the entire protein content of each sample.

Aliquots of human and rat serum were prepared with PHM-Liposorb in PBS to selectively remove lipoproteins from serum. A method of lipoprotein removal using

Liposorb has been described previously [20] and was adapted for selective removal of apolipoproteins from serum prior to MS analysis by Dr. Bomie Han (Eli Lilly and Co.).

Liposorb powder (one gram) was suspended in 50 mL PBS and filtered through a 250 µm filter. The flow-through was used as the Liposorb working stock solution (1 g/ 50 mL).

Ten microliters (10 µL) of serum was diluted in 90 µL of PBS and incubated with 100 µL of Liposorb stock for 10 minutes at room temperature with shaking to keep Liposorb suspended. The samples were spun down to pellet the Liposorb and supernatant was removed. The pellet was washed three times with 500 µL of 100 mM ammonium bicarbonate (ABC) and digested overnight at 37°C with 1 µg of modified trypsin prior to

MS analysis. Samples were filtered and 50 µL of 1 mL final volume (0.5 µL of serum) was analyzed by triple-play LC-MS/ MS (LTQ from Thermo) in global profiling studies.

8 Global LC-MS/ MS Analysis of Human and Rat Serum or Plasma

Global profiling studies were used to evaluate the entire protein content of each sample using a triple-play method to collect spectral data from the fragmentation of unique peptides to identify each respective protein. The HPLC gradient was controlled using a Surveyor MS pump (Thermo) equipped with a sample loop and a Zorbax SB300-

C18 (3.5 µm particle size) 1 mm x 50 mm reverse phase column (Agilent Technologies, cat #865630-902). The column was kept at 27°C and sample tray was 4°C during analysis. 50 µL of 1 mL total sample volume (0.5 µL of human or rat serum) was injected into a 100 µL sample loop using the partial loop method. The HPLC gradient ran for 142 minutes per injection and utilized a three buffer system [0.1% formic acid in

H2O (Buffer A), 0.1% formic acid in 50% acetonitrile (Buffer B), and 0.1% formic acid in 80% acetonitrile (Buffer C)]. The gradient ran at a 50 µL per minute flow rate of: 90%

A, 10% B from 0.0-5.0 min, then ran a sloped gradient from 90% A, 10% B to 5% A,

95% B from 5.0 to 125.0 min, then 100% C from 125.1-130.0 min, and 90% A, 10% B from 130.1 min-142.0 min. This gradient separated the peptides based on hydrophobicity. The peptides were eluted, ionized, and sprayed directly into an online

MS for mass and charge state determination.

Mass spectrometry data were collected in triple-play mode with three scans per peptide: centroid peptide full MS scan, profile zoom scan, and centroid fragment MS/ MS scan. Protein identification was performed using a Sequest and X! Tandem algorithm that combined the protein identifications output from each search [17] and these results were searched against a reverse database to confirm protein identifications. The p-value

9 of each tryptic peptide was used to determine the quality of the peptide identification to help avoid using false-positive identifications for method development.

Sample Preparation for Targeted MS Analysis of ApoM

A heavy isotope-labeled apolipoprotein standard was spiked into each sample at the beginning of sample preparation to normalize variations in protein recovery from serum that may occur during sample preparation or instrument analysis. Serum or plasma was enriched with lipoprotein-binding beads and the bound proteins were denatured with urea prior to digestion with trypsin in the presence of detergent.

15 N-labeled human apolipoprotein A-IV (15 N-Apo A-IV) was used as an internal standard (iSTD). The labeled protein was diluted into PBS and 300 µL (250ng) was spiked into each experimental and external calibration sample. Rabbit or horse serum was mixed with PBS (1:6) and used as a dilution matrix for human or mouse experimental samples, respectively. 10 µL of human or mouse serum was mixed with

140 µL of the dilution matrix to dilute the experimental sample in the background serum at a 1:2 ratio (3x dilution of the experimental sample). The total serum-to-PBS ratio was

1:4 to mimic the ratio of total serum-to-PBS in the calibration samples. 50 µL of the diluted sample was mixed into the internal standard solution. 200 µL of Liposorb was used per 10 µL of serum. Samples were incubated in Liposorb at 4°C for 20 minutes with shaking to keep Liposorb suspended.

The Liposorb was spun down and supernatant aspirated. The Liposorb pellet was washed one time with 100 mM ABC. The washing step can also be done in a filter plate for higher throughput and will be described later. The Liposorb pellet was resuspended

10 in either 100 µL of 8 M urea, or 10 mM ABC prior to reduction/ alkylation (R/A), with repeated pipetting of the sample in solution.

In the urea-containing method, samples were incubated at room temperature for

15 minutes with shaking. 200 µL of 100 mM ABC was added. In an alternative protocol, proteins were reduced with 100 µL of 10 mM dithiothreitol (DTT) at 37°C for

45 minutes and alkylated with 100 µL of 60 mM iodoacetamide at room temperature for

30 minutes. In both protocols, modified trypsin was added at 2 µg in 200 µL of 0.1%

NP40 in 100 mM ABC. Samples were incubated at 37°C overnight with shaking.

Peptides were eluted from the Liposorb directly with digestion. Samples were filtered after digestion to remove Liposorb from the final sample. 50 µL of 500 µL final sample volume (1 µL serum) was injected to a 100 µL sample loop for the MS measurement of apoM.

Preparation of External Calibration Standards

A series of external calibration standards were prepared using large pools of purchased human, rat, and mouse sera. The first sample in the calibration set was equivalent to 100% human, mouse, or rat serum (no dilution matrix) and then a series of

22 dilutions were made at a fixed ratio into rabbit or horse serum, respectively. Each calibration sample was then mixed 1:4 with PBS to maintain the same fixed dilution in

PBS as the experimental samples. In the human and mouse calibration sets, two samples were prepared using a higher volume of total serum in PBS (without matrix) than the

100% serum-equivalent samples, to create 125% and 156% of human and mouse serum- equivalents. These two samples did not have the same fixed dilution factor into PBS as

11 the rest of the calibration samples. The total volume of human or mouse serum was larger than the total volume of serum in the rest of the calibration samples. Every other calibration sample (12 total) in the dilution series was used to make up one calibration set, called mouse calibration A (Cal-A) and human Cal-G, and the remaining 12 samples created another calibration set, called mouse validation B (Val-B) and human Val-H.

The rat serum calibration samples were prepared in the same manner, but without preparation of samples that contained greater-than-100% rat serum. The rat serum calibration sets were rat Cal-A and Val-B.

Serial dilutions of synthetic apoM tryptic peptides were used as a calibration standard to measure the molar concentration of apoM in these human, mouse, and rat serum calibration samples. The molar concentration of apoM in the serum calibration sets was then used as a standard for absolute quantification of apoM in experimental samples. One or both sets of species-specific calibration samples were included in each

96-well plate of experimental samples. A calibration set was added to the experimental plate at the beginning of sample preparation to undergo the same preparation as the experimental samples prior to MS analysis.

12 LC-MS/ MS of ApoM-Derived Tryptic Peptides

The apoM-derived tryptic peptides used for quantification of apoM include: one peptide common between rat, mouse, and human species (FLLYNR), one human-unique peptide (AFLLTPR), and one rat and mouse-unique peptide (AFLVTPR). The amino acid sequence of apoM was searched against a protein database using Basic Local

Alignment Search Tool (BLAST) from the National Center for Biotechnology

Information (NCBI) [21] to confirm that each tryptic peptide (FLLYNR, AFLLTPR, and

AFLVTPR) was unique to apoM. A tryptic peptide from the 15 N-Apo A-IV

(LEPYADQLR) was used for normalization. All of these peptides were measured in a single HPLC gradient and MS method, making this a versatile assay that spans multiple species.

The HPLC gradient was optimized using a Surveyor MS pump equipped with an

XBridge C18 (2.5 µm particle size) 2.1 mm x 50 mm reverse phase column (Waters, cat

#186003085). The column was kept at 50°C and sample tray was 4°C during the instrument run. 50 µL of 500 µL total sample volume (1 µL of serum) was injected into a 100 µL sample loop using a partial loop method. The HPLC gradient runs 7.5 minutes per sample and includes the mass-to-charge ( m/z) measurement of four tryptic peptides as

2H +-charged precursor ions: FLLYNR (413.50), AFLVTPR (402.49), AFLLTPR

(409.51), and LEPYADQLR (559.58) in a single MS method. The HPLC gradient utilized two buffers [0.1% formic acid in H 2O (Buffer A) and 0.1% formic acid in acetonitrile (Buffer B)]. The final gradient was: 100% A at 250 µL/min for 0.5 min,

100% A at 300 µL/min for 0.1 min, 0-16% B at 300 µL/min for 0.4 min, 16% B at 300

µL/min for another 4.7 min, 16-80% B at 300 µL/min for 0.3 min, 80% B at 300 µL/min

13 for 0.01 min, 80% B at 600 µL/min for 0.49 min, 80-0% B at 600 µL/min for 0.1 min,

100% A at 600 µL/min for 0.9 min, then 100% A at 250 µL/min for 0.1 min. The 15 N-

Apo A-IV-derived tryptic peptide LEPYADQLR eluted around 3.15 minutes and apoM- derived peptides AFLVTPR eluted around 3.89 minutes, FLLYNR close to 4.35 minutes, and AFLLTPR eluted around 5.15 minutes. The total run time per injection was 8.5 min including a 1-minute injection, so 170 samples can be analyzed per day. Samples were analyzed in tandem to keep HPLC column conditions consistent for all samples. The sample was diverted away from the MS source during the first 2.5 minutes of the analysis. The column effluent was also diverted away from the instrument after 5.8 min to avoid spraying NP40 into the MS source, which was eluted from the column around

6.0 min under this gradient condition.

Ion spectra were collected in positive ion mode using an electrospray ionization source (ESI) and a LTQ mass spectrometer. The ion capillary temperature was kept at

250°C. The maximum ion trap time was set to 25.00 ms. The isolation width used to collect the isotopic distributions of the fragmented tryptic peptides was m/z of 3.00. The normalized collision energy was 35.00, activation Q was 0.250, and activation time was

25 ms for the MS method. Three MS transitions were measured for each apoM-derived tryptic peptide and two for the internal standard-derived tryptic peptide, listed below as: tryptic peptide, m/z of precursor ion, m/z of transition (fragmentation site, ion charge).

AFLLTPR was from m/z of 409.51 to m/z of 599.39 (y5, M+H + ion), m/z of 486.30 (y4,

M+H + ion), and m/z of 373.22 (y3, M+H + ion). FLLYNR was from m/z of 413.50 to m/z of 565.31 (y4, M+H + ion), m/z of 452.23 (y3, M+H + ion), and m/z of 678.39 (y5, M+H + ion). AFLVTPR was from m/z 402.49 to m/z of 585.37 (y5, M+H + ion), m/z of 472.29

14 (y4, M+H + ion), and m/z of 373.22 (y3, M+H + ion). All tryptic peptides derived from apoM and transitions had an isolation width of 3.00. The internal standard tryptic peptide

LEPYADQLR was from m/z of 559.58 to m/z of 437.19 (y7, M+2H + ion) and m/z of

873.37 (y7, M+H + ion) with isolation widths of 3.00 and 4.00, respectively.

Tryptic Peptide Peak Identification, Integration, and Quantification

The ion spectra were collected from the fragmentation of each apoM derived tryptic peptide in the targeted assay and were used for peptide identification and apoM quantification. Integration of the chromatographic peak of each tryptic peptide was performed using Xcalibur Processing method. The chromatographic peak of each fragment ion was integrated independently from the other two fragments from each precursor ion and the area of each fragment ion chromatographic peak was added together with the areas of the other two fragment ions from the same peptide prior to curve fitting. The peak detection algorithm used in the processing method was

Interactive Chemical Information System (ICIS) [22]. ICIS peak integration was set to 3 smoothing points, area noise factor of 1, and a peak noise factor of 10. Peak width was constrained to 10% of the peak height with a tailing factor of 3. The baseline window was adjusted to ensure each peak was fully integrated for consistent quantification. The fragment ion with the strongest MS signal (transition A) was detected as the highest peak, and the subsequent fragment ions (transitions B, C) were detected using the nearest retention time based on transition A. The minimum peak height of signal to noise ratio was set at 3.0. Noise was filtered out using the repetitive noise function. Mass range for integration was selected for each transition using a narrower window than the m/z

15 collection width used in the MS method. Transition A from FLLYNR peptide was integrated using an integration window of m/z 564.81 →566.81 amu (M+H +) transition B between m/z 451.73 →453.73 amu (M+H +) and transition C at 677.89 →680.89 amu

(M+H +). AFLLTPR transition A was integrated at m/z 598.89 →600.89 amu (M+H +), transition B at m/z 485.80 →487.80 amu (M+H +), and transition C at m/z 372.72 →374.72 amu (M+H +). The mouse peptide, AFLVTPR, had integration windows between m/z

584.87 →586.87 amu (M+H +), m/z 471.79 →473.79 amu (M+H +), and m/z

372.72 →374.72 amu (M+H +). The internal standard tryptic peptide LEPYADQLR transition A had an integration window of m/z 436.69 →438.69 amu (M+2H +), and transition B at m/z 872.87 →874.87 amu (M+H +). The chromatographic peak of each transition was integrated to obtain the area-under-the-curve (AUC). The apoM-derived tryptic peptide AUCs were normalized within each sample as a ratio with the 15 N-Apo A-

IV-derived tryptic peptide AUC. GraphPad Prism software (GraphPad Software, Inc.) was used to fit the calibration sample data to a standard curve and to quantify apoM in the experimental samples using the standard curve. A nonlinear regression was used to interpolate the unknown concentrations from the standard curve on a log scale. The standard curve was fit as a sigmoidal dose response with variable slope. The fitting method was set using the least squares regression and a weighting factor of 1/Y was also used to fit the data.

ApoM concentration was reported in relative or absolute concentration. In spike- recovery experiments used for method validation, human apoM concentration in Val-H was measured using the known human apoM concentrations in Cal-G (see Results). A three-day spike recovery assay was performed to evaluate the consistency between

16 triplicates of Cal-G and Val-H (inter-assay) and preparations across three separate experiments (intra-assay). The coefficient of variation (% CV) was calculated between duplicate plates within an experiment and across three separate experiments to assess the behavior of the assay and its accuracy, precision, and consistency. Multiple sets of calibration and validation samples were used for spike recovery. The standard deviation and % CV were derived from the comparison of measured apoM concentration against the known apoM concentrations in the validation standard samples.

Adaptation of the Assay to a High-Throughput Format

This assay was optimized for a 96-well plate format to increase throughput. The use of a Beckman Coulter Biomek FX P Laboratory Automation Workstation equipped with a Dual Arm system (Span-8 and Multi-Channel Pipettor) (Beckman Coulter, cat

#A31844) robot was incorporated into parts of the sample preparation procedure to further increase throughput and eliminate manual pipetting of samples and reagents to reduce the incidence of sample to sample variations that may occur with multiple manual pipetting steps. Preparation of the serum samples was performed entirely in 96-well

Captiva filter plates (Varian, cat #A5960045). The bottom of the filter plate was capped with a Captiva Duo Seal plate mat (Varian, cat #A8961008) and fit into a wide mouth 96- well plate (Corning, cat #3433) to keep solution from leaking through the filter plate.

The Biomek FX P robot delivered 300 µL (250 ng) of iSTD to each well. Rabbit serum pre-mixed with PBS (1:6) was used as a background matrix for the serum samples and calibration standards. The robot transferred 10 µL of serum samples into 140 µL of dilution matrix to dilute the experimental serum samples 1:2 (3x dilution) with the serum

17 matrix. Samples were diluted to bring the apoM concentration within the range of the standard curve produced by the calibration samples. A 3x dilution brings control serum samples (100% human serum) to 33%. At this point on the calibration curve there is approximately a 3-fold range above and below the concentration of apoM in the diluted serum. Typically, the experimental samples fell within the calibration range at this dilution factor. The robot delivered 50 µL of either the diluted samples or calibration standards to the filter plate containing the internal standard solution.

Liposorb stock solution was added to the filter plates at 200 µL per 10 µL of total serum (sample plus matrix). The tops of the filter plates were sealed with adhesive aluminum foil (Beckman Coulter, cat #538619). Each plate (including bottom cap mat and holder plate) was taped with duct tape in an A to H direction on each end of the filter plate to prevent the filter plates from coming loose at the bottom and leaking during shaking. The plates were incubated at 4°C for 30 minutes with shaking to keep Liposorb suspended. During this incubation period, the Liposorb beads selectively bound the lipoproteins, purifying them from the complex serum sample in one step. This eliminated the need to incorporate an additional high-abundant protein removal step in this protocol.

The Liposorb fraction contained the apolipoproteins and the unbound fraction was washed through the filter plate.

After incubation with Liposorb, the duct tape and bottom cap mat with holder plate were all removed and the filter plates were placed upright on top of deep 96-well plates. The unbound proteins in the supernatant were filtered through the plate, using a centrifuge at 1500 rpm for five minutes to spin down the liquid and collect it in the deep- well plate. Operation of the centrifuge at a higher speed resulted in a tight Liposorb

18 pellet that made resuspension more difficult and possibly incomplete. Lower speeds left a loose pellet to fully resuspend for a more even exposure to trypsin for a consistent digestion. The Liposorb pellet with bound apolipoproteins remained in the filter plate and the flow-through was discarded. The pellet was washed once with 500 µL of 100 mM ABC and liquid was removed and discarded as before. The apolipoprotein- containing Liposorb pellet was resuspended in either 8 M urea or 100 mM ABC using a

Biomek FX P robot.

The sample prep protocol diverges at this point into either (1) urea or (2) reduction and alkylation (R/A). In the urea-containing protocol, the bottom cap mats and holder plate were replaced and samples were resuspended in 100 µL of 8 M urea. The filter plate was sealed with adhesive foil and wrapped in duct tape. Plates were shaken for 15 minutes at room temperature to keep Liposorb suspended. Shaking speeds varied by plate shaker, so the shaking speed was adjusted per apparatus so that the apolipoprotein-containing Liposorb beads remained in suspension, but the sample solution did not come into contact with the adhesive foil to avoid cross-contamination between wells. Modified trypsin was added at 2 µg per 10 µL total serum in a final concentration of 0.1% NP40 in 100 mM ABC (pH 8). The final sample was in 500 µL to dilute the final urea concentration to 1.6 M prior to digestion with trypsin. Adhesive aluminum foil seals and duct tape were used to keep each filter plate from leaking during digestion. At this step it was important to make sure the samples did not reach the adhesive foil. The detergent in the sample can cause the adhesive foil top to unseal and leak. Plates were incubated overnight at 37°C, shaking to keep Liposorb suspended to ensure even digestion. After digestion, top and bottom seals were removed and samples

19 were filtered. A filter plate ‘sandwich’ was assembled by placing the bottom of the

Varian filter plate into the wells of an additional Solvinert 96-well filter plate (Millipore, cat #MSRPN0410). This assembly was placed on the top of a 96-well plate (Analytical

Services Inc, cat #96965) to collect the final digested samples. The 96-well plates containing the final samples were heat-sealed using a non-adhesive foil (AbGene, cat

#AB-0757) compatible with the Surveyor autosampler (Thermo).

In the R/A preparation protocol, the Liposorb pellet was resuspended in 100 µL

10 mM ABC using the Biomek FX. Due to the presence of the Liposorb beads, a non- volatile reduction and alkylation protocol was used to avoid a drying step. 10 mM DTT was freshly prepared in 10 mM ABC and added to the suspended Liposorb using a

MultiDrop (Thermo) liquid dispenser. Plates were sealed with adhesive foil and incubated at 37°C for 45 minutes with shaking. 60 mM Iodoacetamide in 10 mM ABC was then added to the filter plate using the MultiDrop dispenser. Plates were incubated at room temperature for 30 minutes in the dark since iodoacetamide is light-sensitive.

Trypsin was added at the same concentration and solution as the urea protocol (2 µg trypsin per 10 µL total serum) in a final concentration of 0.1% NP40 in 100 mM ABC.

The final volume in this protocol was also 500 µL. Proteins were digested for 2 hours at

37°C in this protocol. Post-digestion samples were filtered in the high-throughput format described above. 50 µL of 500 µL final volume (1 µL of serum) was injected to the LTQ for measurement of apoM using the MRM assay.

20 Human Clinical Study Samples for Human ApoM Measurement

The conditions of the human serum samples used in this experiment have been described earlier [23]. Briefly, serum samples were collected from participants in a 16- week clinical study. Each participant was treated with placebo, statin (atorvastatin),

PPAR-α agonist (LY518674), or with combination treatments at different doses. Human serum samples were prepared using the urea-containing sample preparation protocol and analyzed for apoM-derived tryptic peptide FLLYNR using the MRM assay. ApoM was quantified using GraphPad Prism statistical software. One-way ANOVA was used to determine statistical difference in group means with p<0.05. Serum was drawn at baseline (pre-treatment) and at four and 16 weeks post-treatment. Measurement of triglycerides, LDL cholesterol, HDL cholesterol, and total cholesterol (mg/ dL) were made and have been described previously [23].

MTTP-Inhibitor Pre-clinical Study for Mouse ApoM Measurement

Mouse apoM was measured in serum samples drawn from mice receiving different doses of a microsomal triglyceride transfer protein (MTTP) inhibitor or control.

Mouse serum was prepared using the urea-containing sample preparation protocol and analyzed by MS to measure apoM-derived tryptic peptide AFLVTPR. Mouse apolipoproteins A1, B and E (apoAI, apoB, and apoE) were also measured in these samples using a modified LC-MS method to include measurement of tryptic peptides from additional apolipoproteins in a panel assay. HDL, LDL, VLDL, and total cholesterol were measured independently from this assay.

21 Sample Preparation for SDS-PAGE and Western Blot of Human ApoM

Human serum and human recombinant apoM were prepared for SDS-PAGE and

Western blot to evaluate the use of human recombinant apoM as a standard to quantify apoM in human serum. Two sets of dilutions of the recombinant protein were made at

100 µg/mL, 20 µg/mL, and 4 µg/mL, one using PBS as the dilution matrix and the other using 12.5 mg/mL bovine serum albumin, BSA (Pierce, cat #77171). 10 µL of the diluted recombinant apoM, neat human serum, and BSA was incubated with 200 µL of

Liposorb to bind to the apolipoproteins from serum and human recombinant apoM.

Liposorb supernatant from human serum and all Liposorb fractions were saved and diluted to the same final volume prior to loading on the gel. All samples were prepared in NU PAGE SDS sample buffer (Invitrogen, cat #NP0007) containing NU PAGE reducing agent (Invitrogen, cat #NP0009) at the same final volume. Samples were boiled at 70°C for 10 minutes. Samples were mixed thoroughly and spun down to collect all droplets. 0.3 µL of total human serum-equivalent and 1 µg, 0.2 µg, and 0.04 µg of recombinant apoM were loaded onto a NU PAGE 4-12% pre-cast 1.5 mm Bis-Tris gel

(Invitrogen, cat #NP0336BOX). A pre-stained molecular weight standard See Blue Plus2

(Invitrogen, cat #LC5925) was also loaded. NU PAGE MES-SDS running buffer was used (Invitrogen, cat #NP0002) with antioxidant (cat #NP0005) included due to reduced sample conditions. The samples were run through the gel under reducing conditions according to the Invitrogen NU PAGE Novex Bis-Tris Mini Gels protocol. The gel ran at a constant 200 V for 35 minutes. The proteins were transferred to nitrocellulose using the iBlot system from Invitrogen. The nitrocellulose was blocked with casein-TBS blocking buffer (Pierce, cat #37532) overnight at 4°C. The primary mouse anti-apoM antibody

22 was added to the nitrocellulose at a 250x dilution in casein TBS buffer and 0.05% Tween

20 detergent (Pierce, cat #PI-28320). The nitrocellulose was washed in 0.05% Tween 20 in TBS. The secondary antibody, ECC goat anti-mouse IgG-HRP, was prepared at a

5000x dilution in the same solution as the primary antibody. The secondary antibody was removed and washed prior to exposure and film development. An ECL Western blot kit (Amersham, cat #RPN2106V1/2) was used for chemiluminescent detection. The ECL reagents were mixed 1:1 and added to the nitrocellulose for one minute. Kodak BioMax

Light film was exposed to the nitrocellulose at different time intervals and developed.

In an experiment to evaluate the selective nature of Liposorb for apolipoproteins from serum, aliquots of 10 µL of human serum and rat plasma (in-house Long-Evans) were incubated with 200 µL of Liposorb and analyzed along with neat human and rat serum by SDS-PAGE. The gel was stained with Invitrogen Simply Blue SafeStain (cat

#LC6060) and washed with H 2O overnight. The visible protein bands were excised, destained with 50% ACN in 10 mM ABC and digested with modified trypsin. The gel slices were removed from the digested sample by filtration. The peptides from each gel slice were analyzed by nanospray LC-MS/ MS (LTQ) in a global profiling experiment using a triple-play data collection method and searched against human and rat protein databases for protein identification.

In another experiment, apoM concentration was measured in several human serum samples using the MS-based assay and compared to the apoM concentration measurements from a more conventional proteomic method, Western blot, in an orthogonal validation of the MS assay. Aliquots of the same human serum samples containing different concentrations of apoM were analyzed using both methods. The MS

23 measurement was performed using the urea-containing method as described above.

Serum samples were also prepared for SDS-PAGE. Five microliters (5 µL) of human serum was diluted into 27.5 µL H 2O, 5 µL of 10x NU PAGE reducing agent, and 12.5 µL of 4x NU PAGE SDS sample buffer to a total volume of 50 µL. Ten microliters (10 µL) of each sample (1 µL serum equivalent) was loaded onto a NU PAGE 4-12% pre-cast 1.5 mm Bis-Tris gel. 10 µL of SeeBlue Plus2 pre-stained MW standard was loaded into the first well. Serum samples (numbered 1-12) were loaded in order into wells 2-13. Well

14 contained 10 µL of the MW standard and well 15 contained the same sample as well

10 (sample 9). A negative control for primary antibody was included in this blotting procedure. Mouse anti-apoM primary antibody was incubated with a 10x excess of recombinant apoM before incubation with a selected portion of the blot (well 15). The remaining portion of the blot was probed with the primary antibody without the presence of recombinant apoM, as usual. The two sections were then placed together during the secondary antibody probe and ECL exposure. The SDS-PAGE was run according to the

NU PAGE Novex Bis-Tris Mini Gels protocol from Invitrogen (described above). The proteins in the gel were transferred to nitrocellulose using the iBlot system from

Invitrogen. The nitrocellulose was kept in Pierce Superblock in TBS (cat #37535) overnight at 4°C to block non-specific antibody binding sites. The nitrocellulose membrane was incubated for one hour with primary antibody mouse anti-apoM diluted

250x in casein-TBS with 0.05% Tween 20. The blots were washed and then incubated for 45 minutes with anti-mouse IgG secondary antibody. The blots were washed a second round. Amersham ECL Western blot kit was used to expose and develop the

Western blot as described above.

24 RESULTS

Enrichment of Apolipoproteins from Serum using Liposorb

Liposorb is known to capture lipids and lipid-binding proteins from serum or plasma and has been used previously to quantitatively capture apolipoproteins [20]. We tested the selective nature of Liposorb for apolipoproteins and also tested if this procedure can be used to quantitatively capture apoM and applied to other apolipoproteins as well. The selective nature of Liposorb was tested by SDS-PAGE comparison of Liposorb-bound human serum and neat human serum to confirm that apolipoproteins were the primary proteins identified from the Liposorb-bound fraction. apoB100, apoAI, apoE, apoAIV, apoCIII, and apoAII were the major proteins identified by global MS analysis of the resulting protein bands from the Liposorb-bound fraction of human serum (Figure 2). A few other lipid-binding proteins were also identified in these bands, but the Liposorb primarily bound apolipoproteins from serum. The Liposorb- bound fraction contained visibly less protein bands than the neat serum when 1 µL of human serum-equivalent was loaded for each condition and directly compared. The protein bands in the Liposorb-bound fraction of 1 µL human serum were not intense enough to be excised and digested for MS preparation and analysis, so 10 µL of

Liposorb-bound human serum-equivalent was also loaded and this lane was used for protein identification by global proteomics using MS. ApoM was not identified in any of the protein bands from this experiment, likely due to its low abundance which can result in a very light or absent protein band. The results of this experiment confirmed the selective nature of Liposorb for removal of apolipoproteins from serum and can be

25 applied to other apolipoprotein assays, but did not address the selective or quantitative capture of apoM by Liposorb for use in this assay.

Quantitative capture of apoM by Liposorb was evaluated using Western blot.

Human serum and recombinant human apoM purified from E.coli were incubated with

Liposorb and both Liposorb-bound and unbound supernatant fractions were analyzed for apoM by Western blot. An apoM band was not visible in the supernatant of either sample, but both Liposorb-bound recombinant and human serum had an intense apoM band around 25 kDa (Figure 3). The intense presence of apoM in the Liposorb-bound fraction, in conjunction with its absence in the supernatant, demonstrated the selective and quantitative capture of apoM from serum by Liposorb. Apolipoproteins were selectively removed from serum and apoM was quantitatively captured by Liposorb.

ApoM protein band intensity from Liposorb-bound apoM from human serum was not compared to the band intensity of neat human serum in this analysis, but quantitative capture of apoM was later confirmed using MS analysis.

Thus, Liposorb was included in sample preparation prior to MS analysis. Serum was incubated with Liposorb to selectively capture apolipoproteins. The sample was spun down to pellet the Liposorb, the supernatant containing unbound proteins was removed, and the Liposorb pellet was washed prior to trypsin digestion and injection into the MS for analysis. Liposorb treatment was also be used prior to Western blot analysis or other proteomic method in which selective analysis of apolipoproteins was desired.

26 Figure 2

Human Serum Human + Liposorb Serum 10 µL 1 µL 1 µL MW kDa

ApoB-100 --188 --98

--62

Serotransferrin --49

ApoL-1, ApoA-1 --38 ApoA-1 PON-1, ApoA-4 --28 ApoE, ApoL-1, PON-1 --17 ApoE, ApoA-1, ApoL-1 --14 ApoA-1 ApoA-1, ApoE --6 Amyloid A-4 Amyloid A/A-4 ApoC-3 --3 ApoA-2, ApoA-1

Figure 2: Selective capture of apolipoproteins from human serum using Liposorb

SDS-PAGE demonstrates the selective capture of apolipoproteins and other lipid- associated proteins from human serum using PHM-Liposorb (rat serum SDS-PAGE not shown). The indicated gel bands were excised from the gel, digested, and proteins were identified using nanospray LC-MS/ MS. The major proteins in each band were mostly identified as apolipoproteins and are listed next to the corresponding arrows. Protein bands were analyzed from 10 µL of Liposorb-bound human serum and the major protein identifications are shown above. 1 µL of Liposorb-bound human serum was compared to

1 µL of neat human serum and was shown that the Liposorb-bound fraction has lower protein content than the whole serum. These bands were identified as mostly higher abundant apolipoproteins, demonstrating the selectivity of Liposorb to capture apolipoproteins. ApoA-I was identified in several bands, possibly due to proteolytic

27 degradation or truncation of the protein, however the reason was not investigated. ApoM was not identified from this analysis likely due to its lower abundance.

Figure 3

Rec. ApoM Hm. Serum Liposorb Rec. ApoM Liposorb Hm. Serum Pellet Supernatant Pellet Supernatant MW kDa

-36 -22

Figure 3: Quantitative capture of apoM from human serum and recombinant protein using Liposorb

The quantitative capture of apoM was evaluated using the results of Western blot analysis. Human serum was incubated with Liposorb to selectively bind apolipoproteins.

The supernatant was then removed and both fractions were prepared for SDS-PAGE and

Western blot analysis of apoM. ApoM from human serum and human recombinant apoM were both captured by Liposorb with no detectable apoM remaining in the supernatant.

The quantitative capture of apoM was demonstrated using Western blot and later confirmed with MS analysis.

28 Detergent with Trypsin Digestion to Increase Recovery of Some Apolipoproteins

A MS-based assay for quantification of multiple apolipoproteins was developed in conjunction with this specific apoM assay. A few of the apolipoprotein B (apoB) and paraoxonase-1 (PON1)-derived tryptic peptides had a low recovery from human serum and their synthetic tryptic peptides were insoluble. An additional step in sample preparation was needed to keep select synthetic peptides in solution throughout preparation and during MS analysis and increase the recovery of these apolipoproteins from serum.

Detergent was evaluated for potential use in this assay to increase protein and peptide solubility and recovery from serum, although detergent is not typically used in

MS preparation due to its high ionization efficiency and dominating MS spectra. Several different detergents and concentrations were evaluated to determine the lowest concentration of detergent that yielded the highest recovery. Pierce Surfact-Amps NP40 and Surfact-Amps X100 (cat #28314) were evaluated at 0.01%, 0.05%, and 0.1% using the synthetic peptides of each apolipoprotein in the panel assay (unpublished assay).

These peptides were synthesized as tryptic peptides, so detergent was diluted into 100 mM ABC and added directly at these final concentrations. Synthetic peptides were analyzed using the targeted MS apolipoprotein panel assay and recovery was calculated as a percentage of control (no detergent). Detergent was also used to increase protein recovery from serum. Human serum was prepared using the method described above without detergent (control) and with the addition of detergent prior to trypsin digestion.

NP40 and Triton X100 were evaluated at 0.01%, 0.05%, and 0.1% and Waters RapiGest

SF Surfactant (cat #186001860) was used at 0.001%, 0.005%, and 0.01%.

29 The addition of TritonX100 or NP40 at any percentage prior to MS analysis was found to increase the peptide MS signal of apoB synthetic peptides more than 100-fold and PON-1 synthetic peptides more than 4-fold without detrimental effects to the MS signal of the other synthetic peptides in the panel of apolipoproteins (Figure 4A). The addition of 0.1% NP40 prior to digestion with trypsin increased the recovery of apoB from human serum by 10-fold and did not decrease the recovery of others (Figure 4B).

PON-1 was increased 5-fold with the addition of 0.001% RapiGest, but this surfactant should be neutralized with acid prior to MS analysis and did not increase the recovery of apoB, so was not used. The final selection was 0.1% NP40 added during trypsin digestion of serum and was included in synthetic peptide preparations to keep the peptides soluble, and use of one detergent and the same concentration keeps the preparation procedure simplified. Detergent is not commonly used in sample preparation prior to MS analysis, but since this is a targeted method, the detergent was eluted from the HPLC column at a high concentration of acetonitrile, after all of the target peptides were eluted and measured, and the detergent spray was diverted away from the MS source to the waste. This helped maintain a cleaner instrument and did not interfere with the measurement of the target tryptic peptides in this method.

ApoM was not included in these experiments because they were performed earlier in method development of other apolipoproteins and prior to the development of this assay, but the affect of detergent on apoM recovery was evaluated during the optimization of the apoM assay using the targeted MS assay and is later described in detail. The use of 0.1% NP40 during trypsin digestion was thus included in sample

30 preparation for apoM analysis to maintain consistency with the panel assay until further experiments were performed.

31 Figure 4

4A.

Detergent Increases Solubility of ApoB and PON-1 Synthetic Peptides 12000 10000 ctrl TX 0.01% 8000 TX 0.05% 6000 Ctrl TX 0.1% 4000 NP 0.01% 2000 NP 0.05%

Peptide Recovery, % Peptide Recovery, of 0 NP 0.1% A4 C1 C2 A2 B PON-1 Protein Name

4B.

Detergent Increases ApoB and PON-1-Derived Peptide Recovery from Human Serum ctrl 1200 TX 0.01% 1000 TX 0.05% 800 TX 0.1% NP 0.01% 600 NP 0.05% Control 400 NP 0.1% 200 RG 0.001%

Peptide Recovery, % of Peptide Recovery, 0 RG 0.005% A4 C1 C2 A2 B PON-1 RG 0.01% Protein Name

32 Figure 4: Evaluation of different concentrations of different detergents to increase apolipoprotein B and paraoxonase-1 recovery and solubility

Triton X100 (TX), NP40 (NP), and RapiGest (RG) were investigated to increase the solubility of apolipoprotein B (apoB) and paraoxonase-1 (PON1) synthetic peptides and apoB and PON-1 protein recovery from human serum. The control condition (ctrl) did not include detergent during trypsin digestion.

(4A) Different concentrations of detergent were added to synthetic peptides prior to MS analysis to increase solubility, reflected in an increase in synthetic peptide MS signal when compared against the detergent-free synthetic peptide MS signal, indicating an increase in solubility.

(4B) Different detergents and RapiGest were evaluated to increase apoB and PON-1- derived tryptic peptide recovery from human serum. The addition of 0.1% NP40 prior to digestion with trypsin resulted in the highest recovery of apoB peptides from human serum compared to control. This concentration of NP40 did not suppress the recovery of other apolipoprotein-derived tryptic peptides and was thus included in apolipoprotein sample preparation during trypsin digestion. 0.1% NP40 was also included during trypsin digestion in the sample preparation protocol for the targeted apoM assay.

33 Identification of ApoM-Derived Tryptic Peptides using Global Proteomics

Unbiased global proteomics studies were initially used to identify apoM in human and rat serum and to guide selection of tryptic peptides for development of a Multiple

Reaction Monitoring (MRM) method. Different aliquots of human and rat serum were treated with Liposorb and digested with trypsin prior to MS analysis. These samples were analyzed by LC-MS/ MS using a triple-play global proteomic method to identify unique peptides derived from Liposorb-bound proteins. Triple-play LC-MS/ MS performs three scans per peptide: full MS scan, zoom scan, and MS/ MS scan. The most abundant peptide present in the full MS scan was selected for zoom scan and then fragmented prior to full MS/ MS scan. The zoom scan estimated the charge state of the peptide and the full MS/ MS scan collected spectra from the product ions generated in the fragmentation of the precursor ion (tryptic peptide). The full MS/ MS spectra was used in database searches to obtain protein identifications (Experimental Section) and manual analysis of the apoM-derived tryptic peptide fragmentation spectra was used to build the targeted MS assay.

Three unique human apoM-derived tryptic peptides were identified from the global studies: AFLLTPR, WIYHLTEGSTDLR, and

EELATFDPVDNIVFNMAAGSAPMQHLR. Rat serum analysis also produced three unique apoM-derived tryptic peptides: KWTYHLTEGK, AFLVTPR, and FLLYNR.

Several criteria were used to evaluate whether or not these tryptic peptides are suitable to include in the targeted assay, i.e. the presence of potential modification sites such as the potential of methionine to be oxidized [19].

34 Elution of the tryptic peptides from the HPLC column by acetonitrile was recorded in the global study as retention time (RT). Human apoM-derived tryptic peptides AFLLTPR, WIYHLTEGSTDLR, and

EELATFDPVDNIVFNMAAGSAPMQHLR had retention times of 34.7, 39.6, and 74.5 minutes on the 142-minute gradient, respectively. Rat apoM-derived tryptic peptides

KWTYHLTEGK, AFLVTPR, and FLLYNR had retention times of 21.5, 28.6, and 33.2 minutes, respectively. The retention time of each apoM-derived tryptic peptide was used to estimate the percent of acetonitrile (ACN) needed to elute the peptides from the reverse-phase HPLC column based on the 142-minute gradient (Table 1) to facilitate development of a shortened gradient for the targeted human and rat apoM methods. To keep the HPLC gradient for the targeted method as short and simple as possible, the use of tryptic peptides that were extremely hydrophilic or hydrophobic based on the retention time in the global studies was avoided.

To develop a quantitative assay, it was important to avoid the selection of tryptic peptides that contained potential modifications sites. Peptide modifications such as methionine oxidation cause a shift in mass compared to the mass of the unmodified peptide. When this occurs, the modified peptide will have a different mass-to-charge ratio ( m/z ) than the unmodified peptide m/z , which is collected by the targeted method and so the unmodified peptide spectra will not be collected. Due to the presence of methionine in EELATFDPVDNIVFNMAAGSAPMQHLR, this human apoM-derived tryptic peptide was not included in the targeted method, since methionine can be readily oxidized. Thus, quantification of apoM was not based on a tryptic peptide that may become modified, to provide an accurate measurement of apoM. The other apoM-

35 derived tryptic peptides from global studies did not contain methionine or other potential modification sites and so were not discarded from use at this point.

The global study data was also analyzed for the presence of each apoM-derived tryptic peptide at more than one charge state. The presence of a tryptic peptide at multiple charge states splits the peptide MS signal which can result in multiple weaker peptide MS signals compared to a single MS signal if the peptide was present at one charge state. Low MS signal typically results in higher background noise and other interferences and lowers the quality of the peptide MS signal for integration.

The tryptic peptides identified from apoM in the global studies were evaluated for their presence at multiple charge states. Human tryptic peptide WIYHLTEGSTDLR and rat tryptic peptide KWTYHLTEGK were identified at both 2H + and 3H + charge states

(Table 2). Human peptide AFLLTPR and rat peptides FLLYNR and AFLVTPR were found at one charge state (2H +).

Although these two tryptic peptides were each identified at two different charge states, they were later evaluated using preliminary MRM studies for the consistency of the ratio of these two charge states and peptide MS signal intensity. Two human

(AFLLTPR and WIYHLTEGSTDLR) and three rat apoM-derived tryptic peptides

(AFLVTPR, FLLYNR, and KWTYHLTEGK) were all included in further evaluation for use in the MRM assay.

36 Table 1

Species RT (min) Est. %ACN ApoM-Derived Tryptic Peptide Human 34.7 18.5 AFLLTPR Human 39.5 23.5 WIYHLTEGSTDLR Human 74.5 27.0 EELATFDPVDNIVFNMAAGSAPMQLHLR Rat 21.5 18.3 KWTYHLTEGK Rat 28.6 15.9 AFLVTPR Rat 33.2 18.6 FLLYNR

Table 1: ApoM-derived tryptic peptides identified in global studies

The initial results from global profiling studies identified three human apoM-derived tryptic peptides (top) and three rat apoM-derived tryptic peptides (bottom). The triple- play MS method identified each unique tryptic peptide and the retention time (RT) based on the 142-minute HPLC gradient was captured. Retention time from the global studies was used to estimate the percent of acetonitrile needed to elute each peptide from the

HPLC column to estimate a starting gradient for the initial targeted method setup for human and rat apoM methods.

37 Table 2

Species Charge State ApoM Peptide Human 2 AFLLTPR Human 3 WIYHLTEGSTDLR Human 2 WIYHLTEGSTDLR Rat 2 KWTYHLTEGK Rat 3 KWTYHLTEGK Rat 2 AFLVTPR Rat 2 FLLYNR

Table 2: Charge states of apoM-derived tryptic peptides identified in the global proteomic studies

Tryptic peptides from apoM were evaluated for their presence at multiple charge states.

Human tryptic peptide WIYHLTEGSTDLR and rat tryptic peptide KWTYHLTEGK were identified at both 2H + and 3H + charge states. Human peptide AFLLTPR and rat peptides FLLYNR and AFLVTPR were found at one charge state (2H +).

38 Full MS/ MS Spectra of ApoM-Derived Tryptic Peptides Identified in Global

Studies

Full MS/ MS spectra of each tryptic peptide was collected from global studies and used to select the three most abundant fragments from each tryptic peptide. The tryptic peptide and its three fragment ions were needed to set up the MRM assay. Each tryptic peptide produces its unique fragmentation pattern that can be entered into the MRM method and used for identification and quantification of the target protein (apoM). This method is set up using the m/z ratio of the tryptic peptide (precursor ion) and m/z ratios of three fragment ions per tryptic peptide. In the targeted MRM method, the MS scans for these m/z values only.

The spectra collected in the full MS/ MS scan of the fragmented apoM-derived tryptic peptides (Figure 5) were manually evaluated to select the most abundant fragment ions per tryptic peptide. Each tryptic peptide ( m/z of the precursor ion) and three fragment selections ( m/z of each fragment) were: human AFLLTPR peptide was from m/z of 409.51 to m/z of 599.39 (y5, M+H + ion), m/z of 486.30 (y4, M+H + ion), and m/z of

373.22 (y3, M+H + ion); human WIYHLTEGSTDLR peptide (3H +) was from m/z of

531.26 to m/z of 646.32 (y11, M+2H +), m/z of 652.30 (b11, M+2H +), and m/z of 472.24

(b7, M+2H +); and human WIYHLTEGSTDLR peptide (2H +) was from m/z of 796.39 to m/z of 878.42 (y8, M+H +), m/z of 646.32 (y11, M+2H +), and m/z of 991.51 (y9, M+H +).

Rat FLLYNR peptide selections were from m/z of 413.50 to m/z of 565.31 (y4, M+H + ion), m/z of 261.16 (b2, M+H +), and m/z of 452.23 (y3, M+H + ion); rat AFLVTPR peptide was from m/z 402.49 to m/z of 585.37 (y5, M+H + ion), m/z of 472.29 (y4, M+H + ion), and m/z of 373.22 (y3, M+H + ion); rat KWTYHLTEGK (3H +) was from m/z of

39 421.82 to m/z of 567.78 (y9, M+2H +), m/z of 530.27 (b8, M+2H +), and m/z of 415.22 (b6,

M+2H +); and rat KWTYHLTEGK (2H +) was from m/z of 632.22 to m/z of 622.85 (b10,

M+2H +), m/z of 567.78 (y9, M+2H +), and m/z of 1134.56 (y9, M+H +) and were summarized in Table 3. These selections were used to set up a preliminary MRM method for further peptide evaluation.

40 Figure 5

Figure 5A

AFLLTPR M+2H+ Full MS/ MS

41 5B

WIYHLTEGSTDLR M+3H + Full MS/ MS

42 5C

WIYHLTEGSTDLR M+2H + Full MS/ MS

43 5D

AFLVTPR M+2H + Full MS/ MS

44 5E

FLLYNR M+2H + Full MS/ MS

45 5F

KWTYHLTEGK M+3H + Full MS/ MS

46 5G

KWTYHLTEGK M+2H + Full MS/ MS

Figure 5: Selection of Product Ions from Fragmentation Spectra of ApoM-Derived

Tryptic Peptides Collected from Global Studies

A. Human AFLLTPR peptide (2H +)

B. Human WIYHLTEGSTDLR peptide (3H +)

C. Human WIYHLTEGSTDLR peptide (2H +)

D. Rat AFLVTPR peptide (2H +)

E. Rat FLLYNR peptide (2H +)

F. Rat KWTYHLTEGK peptide (3H +)

47 G. Rat KWTYHLTEGK peptide (2H +)

Fragmentation spectra of all selected apoM-derived tryptic peptides were shown with y- and b-ions (top) and m/z annotations for these fragment ions (bottom). The preliminary transition selections that were used in MRM setup are circled. The most abundant fragment ions were initially selected for use in the MRM method based on these spectra.

The y- and b-ion annotations on the spectra were indicative of the fragmentation site on the tryptic peptide and the fragment that was measured in the global studies. The ions in gray were not recognized by the database search as measurements specific to the identified peptide and may or may not actually be derived from this peptide.

48 Table 3

Species AA Sequence Fragment Ion Selections Mono-A Ion-A Mono-B Ion-B Mono-C Ion-C Human AFLLTPR 599.39 y5, M+H + 486.30 y4, M+H + 373.22 y3, M+H + Human WIYHLTEGSTDLR 646.32 y11, M+2H + 652.30 b11, M+2H + 472.24 b7, M+2H + Human WIYHLTEGSTDLR 878.42 y8, M+H + 646.32 y11, M+2H + 991.51 y9, M+H + Rat AFLVTPR 585.37 y5, M+H + 472.29 y4, M+H + 373.22 y3, M+H + Rat FLLYNR 565.31 y4, M+H + 261.16 b2, M+H + 452.23 y3, M+H + Rat KWTYHLTEGK 567.78 y9, M+2H + 530.27 b8, M+2H + 415.22 b6, M+2H + + + + Rat KWTYHLTEGK 622.83 b10, M+2H 567.78 y9, M+2H 1134.56 y9, M+H

Table 3: Summary of apoM-derived tryptic peptides and the selected fragment ions

The results from the global study of human and rat serum produced several apoM-derived tryptic peptides that were used to set up a preliminary MRM method using the fragment ion selections shown in the table. The species and amino acid (AA) sequence of the tryptic peptides are shown with the selection of three fragment ions (mono). The fragmentation site on the tryptic peptide that produced the fragment ions is defined using b- and y-ions.

49 Preliminary Evaluation of Different Peptides by MRM

A preliminary MRM method was set up using the selected fragment ions to evaluate the two peptides that were each initially identified at two charge states. To set up the preliminary MRM method, average mass, not monoisotopic mass, was utilized as the center of the isolation window for the precursor ions as shown in Table 4. The selected fragment ions were reported in Table 4 using monoisotopic mass in the m/z ratio to assimilate with the labeled monoisotopic mass on the fragmentation MS/ MS spectra from the global studies and this mass is used to confirm the identification of the fragment ions in the MRM spectra. However, to setup the MRM method, the average mass of the precursor ion and fragment ions was used as the center of the selection window to capture the natural isotopic distributions of the precursor and fragment ions to confirm a positive tryptic peptide identification and evaluate the purity of the isolated peptide and resulting chromatographic peak [19].

Human and rat serum were prepared with Liposorb and digested with trypsin prior to MS analysis. Once the preliminary MRM method was set up using the fragment ion selections, human peptide WIYHLTEGSTDLR and rat peptide KWTYHLTEGK were measured from digested human and rat serum, respectively, to evaluate the peptide MS signals at two different charge states (2H + and 3H +). These peptide signals were measured along with AFLLTPR from human and AFLVTPR from rat to compare the split signal intensities and peptide peak shapes with these apoM-derived peptides that were identified in the global studies at only one charge state.

The split peptide MS signals from human peptide WIYHLTEGSTDLR (RT 3.93 min) and rat peptide KWTYHLTEGK (RT 3.29 min), each present at two charge states,

50 resulted in a lower MS signal for both charges and were subject to higher background noise when compared to the MS signal and purity of the peptide peak of AFLLTPR (RT

4.24 min) and AFLVTPR (RT 3.82 min), respectively (Figure 6). The MS peptide signal at either charge state of WIYHLTEGSTDLR was approximately 25% of the peptide signal from human AFLLTPR and the chromatographic peptide peak of

WIYHLTEGSTDLR is visually less clean than AFLLTPR. The isotopic distribution patterns measured by MRM of these two peptides also showed differences in cleanliness.

The MRM fragmentation pattern of human peptide AFLLTPR was clear and evenly distributed between the natural isotopes, with consistent spacing of one unit between isotopes for a single H+-charged species. The MRM fragmentation of human peptide

WIYHLTEGSTDLR at either charge state, especially 2H +, did not follow the expected pattern and the correct isotopes were not clearly present. This pattern occurred due to background noise or other interfering ions. The rat peptide KWTYHLTEGK also had lower MS peptide signals due to the split between two charge states and each charge state was compared to AFLVTPR peptide signal. The MS peptide signal of either charge state of KWTYHLTEGK was split evenly at approximately 40% of the peptide signal from

AFLVTPR. The chromatographic peaks from both charge states of peptide

KWTYHLTEGK had were subject to interferences and were too low in MS signal to be easily distinguishable from these interferences.

Thus, the human peptide WIYHLTEGSTDLR was discarded from this MRM method and was not used for further apoM measurement. The rat apoM-derived tryptic peptide KWTYHLTEGK was discarded from the MRM method for two reasons: the split

MS signal between two charge states and the presence of the N-terminal lysine in this

51 peptide. This peptide was formed as the result of a missed cleavage by trypsin so this peptide can not be assumed to be an accurate measurement of apoM, since trypsin may not create this peptide with a missed cleavage every time.

Another consideration to make in the selection of product ions was the m/z value.

The selection of a product ion that has a higher m/z value (>300) is more desirable to use in the MRM method because it is likely to give a cleaner spectrum. A product ion with a lower m/z has a greater chance of having the same m/z value as a random or unrelated sequence, since only a few amino acids need to combine to create a close value.

Therefore, a change was made in the selection of the final MRM method condition for

FLLYNR peptide. The selection of the b2-ion (M+H +) at m/z of 261.2 was changed to the selection of the next most abundant product ion, y5-ion (M+H +) at m/z of 678.4.

The final selection of human apoM-derived tryptic peptides after global studies and preliminary MRM analysis was narrowed to one candidate (AFLLTPR) and the rat tryptic peptide selections were FLLYNR and AFLVTPR. One additional tryptic peptide from human apoM was desired to have two tryptic peptides from each species in the

MRM method for apoM measurement.

One additional experiment performed using the preliminary MRM method was to confirm the Western blot analysis of the quantitative capture of apoM from serum using

Liposorb by MS analysis. Human serum was incubated with Liposorb and then spun down to produce a Liposorb pellet. The unbound protein-supernatant fraction was removed and both the Liposorb pellet and supernatant were digested with trypsin. Neat human serum that was not incubated with Liposorb was also digested with trypsin. The

52 digested Liposorb-bound fraction, supernatant fraction, and directly digested human serum were analyzed using the preliminary MRM method.

Human apoM-derived tryptic peptide AFLLTPR was measured in this analysis and a strong MS peptide signal was present in both the Liposorb-bound fraction and the whole human serum and the MS signal intensity measured from these samples was equivalent (Figure 7). There was not a measurable peptide signal for AFLLTPR in the supernatant fraction indicating that apoM was quantitatively captured using Liposorb.

The peptide MS signal measured in the Liposorb fraction was slightly higher (~30%) than the peptide MS signal from the neat serum, likely due to incomplete digestion or ion suppression during MS analysis since a purification method was not used to clean up the high concentration of protein in the neat serum prior to trypsin digestion. The results of the MS analysis strengthened the results from Western blot with the addition of the neat human serum analysis without Liposorb capture. The MS results confirmed the results of the Western blot and the same conclusion was drawn from two independent methods.

53 Table 4

Precursor and Fragment Ion Selections Species AA Seq Precursor m/z Width Ion-A center Width-A Ion-B Center Width-B Ion-C Center Width-C Human AFLLTPR 409.51 3 599.89 3 486.80 3 373.72 3 Human WIYHLTEGSTDLR 531.26 3 646.82 3 652.80 3 472.74 3 Human WIYHLTEGSTDLR 796.39 4 879.42 4 646.82 3 992.51 4 Rat AFLVTPR 402.49 3 585.87 3 472.79 3 373.72 3 Rat FLLYNR 413.50 3 565.81 3 452.73 3 261.66 3 Rat KWTYHLTEGK 421.82 3 568.28 3 530.77 3 415.72 3 Rat KWTYHLTEGK 632.22 3 623.33 3 568.28 3 1135.56 4

Table 4: Preliminary MRM method set-up using fragment ion selections from global studies

The average mass of the selected tryptic peptide fragments in the table was used as the center of the selection window in the MRM method to collect the isotopic distribution of each fragment instead of only the monoisotopic ion. The targeted MRM method scans for the m/z of the precursor ion, isolates this m/z and fragments the peptide prior to measurement of the selected fragment ions that are specific to that particular precursor ion.

54 Figure 6

6A

TIC MRM

4.24 NL: 2.01E4 599.30 100 TIC F: ITM S + c ESI SRM ms2 100 [email protected] [ 372.22-375.22, 486.22 80 485.30-488.30, 598.39-601.39] M S 80 B H 070514_M _8-hm 373.17 60 60

40 40 600.33 20 AFLLTPR 20 374.23 487.29

Relative Abundance 4.36 Relative Abundance 601.23 0 M+2H + 0 3.5 4.0 4.5 374 486 488 600 Time (min) m/z m/z m/z

3.94 NL: 5.45E3 646.46 100 TIC F: ITM S + c ESI SRM ms2 100 [email protected] [ 471.24-474.24, 80 645.32-648.32, 651.30-654.30] M S 80 B H 070514_M _8-hm 60 60 652.37 40 40 3.36 472.21 648.17 20 3.56 WIYHLTEGSTDLR 20 4.24 4.51 653.26 Relative Abundance + Relative Abundance 473.89 0 M+3H 0 3.5 4.0 4.5 472 474 646 648 652 654 Time (min) m/z m/z m/z

3.93 NL: 4.18E3 878.27 100 TIC F: ITM S + c ESI SRM ms2 100 [email protected] [ 645.32-648.32, 80 877.42-881.42, 990.51-994.51] M S 80 B H 070514_M _8-hm 991.36 60 60 646.34 879.32 648.12 40 40 992.36 3.31 3.81 4.17 WIYHLTEGSTDLR 20 4.19 20 880.31 993.39

Relative Abundance + Relative Abundance 0 M+2H 0 3.5 4.0 4.5 646 648 880 Time (min) m/z m/z m/z

55 6B TIC MRM

3.82 NL: 2.47E4 585.27 100 TIC F: ITM S + c ESI SRM ms2 100 [email protected] [ 372.22-375.22, 80 471.29-474.29, 584.37-587.37] MS 80 472.20 BH070514_r_M_12-r4 60 60 373.15 40 AFLVTPR 40 586.27 20 3.47 20 473.29 + 374.20 Relative Abundance 4.02 4.10 Relative Abundance 474.16 587.23 0 M+2H 0 3.5 4.0 4.5 374 472 474 586 Time (min) m/z m/z m/z

3.35 NL: 2.39E4 414.14 100 TIC F: ITM S + c ESI SRM ms2 100 [email protected] [ 413.72-417.72, 80 80 529.27-532.27, 566.78-569.78] MS 567.86 BH070514_r_M_12-r4 60 60 415.79 3.29 3.56 40 40 530.09 3.15 KWTYHLTEGK 20 20 530.80 568.97

Relative Abundance + Relative Abundance 416.59 531.68 0 M+3H 0 3.0 3.5 415 530 532 568 Time (min) m/z m/z m/z

3.44 NL: 1.13E4 567.80 100 TIC F: ITMS + c ESI SRM ms2 100 3.42 [email protected] [ 566.78-569.78, 80 947.49-951.49, 1133.56-1137.56] MS 80 3.35 BH070514_r_M_12-r4 60 60 948.35 1134.35 40 40 569.06 1135.36

20 3.29 3.79 KWTYHLTEGK 20 949.32 1136.38 Relative Abundance 3.06 + Relative Abundance 0 M+2H 0 3.0 3.5 568 950 1135 Time (min) m/z m/z m/z

Figure 6: Multiple charge states of human apoM-derived tryptic peptide

WIYHLTEGSTDLR and rat apoM-derived tryptic peptide KWTYHLTEGK

Using a preliminary MRM method, human peptide WIYHLTEGSTDLR (A) and rat peptide KWTYHLTEGK (B) were each evaluated at two charge states (2H + and 3H +) with human peptide AFLLTPR and rat peptide AFLVTPR at one charge state (2H +). The

TIC of each tryptic peptide is shown in the left column and the MRM fragmentation spectra of each peptide are shown in the right column. These peptides were discarded from further use based on low peptide MS signal and interfering ions in the MRM spectra.

56 Figure 7

RT: 3.50 - 6.00 5.39 NL: 5.82E4 100 5.37 TIC F: ITM S + c ESI SRM ms2 [email protected] [ 372.22-375.22, 80 Neat Serum 485.30-488.30, 598.39-601.39] M S BH071003_P1FA_B10 60

40 5.46

20

Relative Abundance 5.27 4.60 4.785.19 5.53 5.71 0 3.5 4.0 4.5 5.0 5.5 6.0 Time (min)

RT: 3.50 - 6.00 5.33 NL: 5.51E4 100 5.36 TIC F: ITM S + c ESI SRM ms2 5.29 [email protected] [ 372.22-375.22, 80 Liposorb 485.30-488.30, 598.39-601.39] M S BH071003_P1FA_B11 60 5.23 40 5.21 20 5.15

Relative Abundance 5.09 4.85 5.46 5.54 5.92 0 3.5 4.0 4.5 5.0 5.5 6.0 Time (min)

RT: 3.50 - 6.00 5.25 NL: 5.52E2 100 TIC F: ITM S + c ESI SRM ms2 5.33 [email protected] [ 372.22-375.22, 5.21 5.35 80 Supernatant 4.58 4.68 485.30-488.30, 598.39-601.39] M S 4.74 BH071003_P1FA_B12 60 4.56 5.41 5.07 4.78 40 5.57 5.72 5.95 20 Relative Abundance 0 3.5 4.0 4.5 5.0 5.5 6.0 Time (min)

57 Figure 7: Evaluation of the quantitative capture of apoM from human serum using

Liposorb

The quantitative capture of apoM from human serum using Liposorb was evaluated by

MS analysis to confirm a previous Western blot experiment. The MS signal intensity of

AFLLTPR peptide was compared between the total serum (no Liposorb treatment),

Liposorb fraction, and supernatant removed from the Liposorb sample after incubation.

The MS signal intensities of AFLLTPR in the total human serum and Liposorb fractions were equivalent, with an absent MS signal from AFLLTPR peptide in the supernatant sample. Liposorb was confirmed to quantitatively capture apoM from serum.

58 Human ApoM AA Sequence Evaluation for Additional Tryptic Peptide Selections

In addition to using global profiling studies for apoM protein identification and selection of the AFLLTPR tryptic peptide derived from human apoM, the AA sequence of human apoM was manually evaluated for the selection of additional tryptic peptides that were not identified in the global studies.

The amino acid sequence of human apoM was obtained from the NCBI protein database and was evaluated with the AA sequences of rat and mouse apoM (Figure 8) to identify unique and common tryptic peptides among these three species. The FLLYNR peptide that was initially identified from rat serum by the global profiling study was also identified in human apoM with manual AA sequence analysis. This peptide had already been evaluated for potential modification sites and other parameters and had been selected for inclusion in the targeted method for measurement of rat apoM, so FLLYNR peptide was also selected for inclusion in the human apoM targeted method.

The selection of a tryptic peptide that is common between human, mouse and rat apoM is beneficial under specific circumstances, such as the measurement of apoM in human transgenic animal models. The expression of human proteins in an animal model is becoming increasingly popular [24]. For example, human apoM-transgenic mice have been used to study the biological significance of the N-terminal signal peptide in human apoM using a mouse model [25]. The specific measurement of human apoM concentration in this mouse model may not be achieved using an antibody. Due to the high percentage of sequence homology between human and mouse apoM, the endogenous expression level of mouse apoM present in this model may interfere in an antibody-based measurement of human apoM. However, human and mouse apoM can

59 easily be distinguished by the MS using the targeted MRM assay to measure the species- specific tryptic peptides, AFLLTPR and AFLVTPR peptides, respectively. A single amino acid difference between these two peptides allows each tryptic peptide to be independently measured by MS, the specificity of which can not be achieved using an antibody-based method. The selection of an additional peptide FLLYNR can be used to confirm the species-specific apoM measurements of AFLLTPR and AFLVTPR peptides with the use of FLLYNR peptide for the measurement of total apoM. The final selection of tryptic peptides that were included in the MRM method to measure apoM are summarized in Table 5.

60 Figure 8

Human MFHQIWAALLYFYGIILNSI YQCPEHSQLTTLGVDGK EFPEVHLGQWYFIAGAAPTK EEL Rat MFHQVWAALLYLYGLLFNSMNQCPEHSQLMTLGMDDK ETPEPHLGLWYFIAGAAPTMEEL Mouse MFHQVWAALLSLYGLLFNSMNQCPEHSQLTALGMDDTETPEPHLGLWYFIAGAASTTEEL

ATFDPVDNIVFNMAAGSAPMQLHLR ATIR MKDGLCVPR KWIYHLTEGSTDLR TEGR PDMK ATFGQVDNIVFNMAAGSAPR QLQLR ATIR TK NGVCVPR KWTYHLTEGK GNTELR TEGR PD ATFDPVDNIVFNMAAGSAPR QLQLR ATIRTK SGVCVPR KWTYRLTEGK GNMELR TEGR PD

TELFSSSCPGGIMLNETGQGYQR FLLYNR SPHPPEK CVEEFK SLTSCLDSK MK TDLFSISCPGGIMLK ETGQGYQR FLLYNR SPHPPEECVEEFQSLTSCLDFK MK TDLFSSSCPGGIMLK ETGQGYQR FLLYNR SPHPPEK CVEEFQSLTSCLDFK

AFLLTPR NQEACELSNN AFLVTPR NQEACPLSSK AFLVTPR NQEACPLSSK

Figure 8: AA sequence of human, rat, and mouse apoM with tryptic peptide selections

The selected tryptic peptides of apoM amino acid sequences from human, rat, and mouse are shown above (highlighted). FLLYNR is common between human, rat, and mouse whereas AFLLTPR is human-specific and AFLVTPR is found in mouse and rat.

Although there is only one residue different between these two, the MS can distinguish these. This assay is both extremely species-specific and versatile across many species dependant on the selection of the specific apoM-derived tryptic peptide used for the quantification of apoM.

61 Table 5

AA Seq Precursor m/z width frg1 cntr width 1 frg2 cntr width 2 fgr3 cntr width 3 AFLLTPR 409.51 3 599.89 3 486.80 3 373.72 3 AFLVTPR 402.49 3 585.87 3 472.79 3 373.72 3 FLLYNR 413.50 3 565.81 3 452.73 3 678.89 3

Table 5: Final selection of apoM-derived tryptic peptides for MRM method set up

The final selections included apoM-derived AFLLTPR, AFLVTPR, and FLLYNR peptides. These peptides are shown in the table with the m/z value of the precursor ion and the isolation width used to collect the peptide in the MS method. Three centered m/z values of the fragment ions were used per precursor ion, with isolation widths of each m/z value that was used in the MRM method setup.

62 Confirmation of Full MS/ MS and MRM Spectra from Serum using Synthetic

ApoM Tryptic Peptides

Identification of the apoM-derived tryptic peptides in the global study was based upon comparison of MS/ MS spectra obtained from global profiling studies against a theoretical MS/ MS spectra in a protein database. Thus, the amino acid sequence and

MS/ MS spectra of the selected tryptic peptides from apoM need to be positively confirmed using MS/ MS spectra obtained from the tryptic digestion of purified apoM or synthetic apoM tryptic peptides.

Synthetic FLLYNR, AFLLTPR, and AFLVTPR peptides were diluted into 0.1%

NP40 in 100 mM ABC. Human, rat, and mouse serum were prepared with Liposorb and digested with trypsin prior to MS analysis. The synthetic peptides and digested serum samples were analyzed using the MRM method, with an additional full MS/ MS scan included for each parent ion. The full MS/ MS spectra and MRM spectra from the synthetic tryptic peptides were compared to corresponding b- and y-ions in the full MS/

MS and MRM spectra of AFLLTPR in human serum, AFLVTPR in rat and mouse sera, and FLLYNR in sera from all three species (Figure 9). The absence of AFLLTPR in mouse and rat serum and AFLVTPR in human serum was confirmed in this analysis as well. The full MS/ MS and MRM method included all three peptides so that each sample measured all three apoM-derived tryptic peptides and the presence and absences in respective sera were confirmed. The retention times of each synthetic peptide were also matched to the retention times of each peptide from serum as an additional confirmation of the peptides from serum and are summarized in Table 6.

63 Tryptic AFLLTPR, AFLVTPR, and FLLYNR peptides initially identified from global profiling studies in human and rat sera and the fragmentation MS/ MS spectra of each peptide was confirmed using the MS/ MS spectra of AFLLTPR, AFLVTPR, and

FLLYNR synthetic peptides so the development of the MRM method could be finalized and ready to use for the measurement of apoM in serum, but quantification of apoM was hindered without a calibration standard in place for this specific method.

64 Figure 9 Rat Serum Serum Serum Mouse Human Peptide Synthetic y5+ 599.58 599.53 599.55 y5+ 599.74 486.51 486.44 486.41 486.43 y4+ m/z y4+ MRM y3+ 374.17 373.35 y3+ 373.33 374.57 0 0 0 0 50 50 50 50 100 100 100 100 650 650 668.65 b6+ 643.47 b6+ 643.75 650 725.60 600 600 700 y5+ 599.53 599.52 y5+ 600 599.62 599.62 685.45 550 550 b5+ 546.35 562.68 562.68 546.43 b5+ 550 614.75 614.75 600 527.00 500 500 601.53 601.53 558.51 558.51 500 y4+ 486.41 486.41 486.42 y4+ 486.45 486.45 548.88 548.88 450 450 b4+ b4+ 445.22 445.22 445.25 450 500 459.98 459.98 514.47 514.47 417.31 400.43 400.43 400.47 400 400 b7++ b7++ 400 400.34 448.08 448.08 m/z y3+ 373.34 373.34 373.34 y3+ 350 350 400 401.38 350 351.82 351.82 b3+ 332.17 332.17 332.16 b3+ 372.40 372.40 FullMS MS/ 300 300 300 300.38 334.05 300.47 300.47 y5++ 300.41 300.41 y5++ 300 y2+ 250 250 288.29 272.31 250 227.14 227.14 257.99 257.99 b2+ 219.09 219.10 219.19 219.19 b2+ 200 200 200 200 191.15 191.15 201.26 201.26 191.12 171.10 171.10 150 150 150 133.05 133.05 120.09 120.05 0 0 0 0 50 50 50 50 100 100 100 100 6 6 6 6 5.90 5.90 5.93 5.93 5.91 5.71 5.71 5.63 5.33 5.33 5.55 5.33 5.29 5.31 5.20 5.20 5.21 5.21 5 5 5.13 5 5 5.12 5.00 5.00 4.97 4.97 4.84 4.75 4.75 4.58 4.58 4.60 4.57 TIC 4.39 4.39 4.35 4.16 4.16 4.33 4.30 4.86 4.86 3.97 4 4 4 4 Time Time (min) 4.00 3.99 3.96 3.87 3.57 3.72 3.72 3.71 3.71 3.76

0 0 0 0 0 0 0 0

100 100

100 100

100 100 100 100 Relative Abundance Abundance Relative Relative Relative Abundance Relative A.Peptide AFLLTPR A.

65 Rat Serum Serum Serum Mouse Human Peptide Synthetic y5+ 585.55 585.51 y5+ 585.54 585.52 y5+ y4+ 472.50 472.43 y4+ 472.42 472.40 y4+ m/z MRM y3+ 373.32 373.32 y3+ y3+ 373.35 373.34 0 0 0 0 50 50 50 50 100 100 100 100 650 650 650 650 632.40 629.40 603.46 600 600 602.49 600 600 585.39 y5+ y5+ 585.52 585.55 y5+ 585.50 577.42 550 550 550 550 554.42 b5+ 532.43 b5+ 532.39 525.07 525.07 526.93 500 500 500 500 516.09 y4+ y4+ y4+ 472.47 472.38 472.45 472.41 472.41 450 450 450 450 b4+ b4+ b4+ 431.29 431.25 431.28 431.30 431.30 400 400 400 400 b7++ 392.33 385.30 m/z y3+ 373.35 373.33 373.35 y3+ 373.34 y3+ 350 350 350 350 361.02 b3+ 332.21 b3+ b3+ 332.18 332.23 332.18 331.48 FullMS/MS 300 300 300 300 y5++ y5++ 293.38 293.38 293.36 288.35 y5++ 250 250 250 250 265.20 262.23 264.20 263.97 b2+ 225.16 b2+ b2+ 219.14 219.15 219.15 219.10 200 200 200 200 191.15 191.15 197.17 191.16 191.16 150 150 150 150 141.09 120.09 120.03 120.01 0 0 0 0 50 50 50 50 100 100 100 100 6 6 6 6 5.92 5.78 5.92 5.59 5.50 5.70 5.59 5.27 4.88 5 5 4.97 5 5 5.11 TIC 4.99 4.97 5.27 4.97 5.27 4.95 5.69 4.72 4.89 4.76 4.62 4.71 Time (min) Time 4.38 4.75 4.40 4.37 4.18 4.29 4.07 3.83 3.83 4.08 4.00 4 4 3.99

4 4 3.98 3.92

3.91

3.91 3.79 3.83 3.78 3.82 5.26

Relative Abundance Relative Relative Abundance Relative B. Peptide AFLVTPR 0 0 0 0 0 0 0 0 100 100 100 100 100 100 100 100 B.

66

Rat Serum Serum Serum Mouse Human Peptide Synthetic 678.61 y5+ 678.53 y5+ 678.50 678.40 y5+ 565.47 565.41 565.41 565.40 y4+ y4+ y4+ y4+ m/z MRM y3+ y3+ 452.39 y3+ 452.35 452.31 y3+ y5+ 452.34 0 0 0 0 50 50 50 50 100 100 100 100 650 650 650 650 645.38 634.44 619.31 613.49 600 600 600 604.13 600 599.54 y4+ 565.46 565.43 565.41 565.42 y4+ y4+ y4+ 550 550 550 550 537.42 b4+ 537.30 b4+ 505.48 500 500 500 500 509.58 509.40 477.60 483.40 483.40 450 y3+ 450 450 450 452.40 452.37 452.30 y3+ 452.31 y3+ y3+ 397.26 400 b6++ 400 400 400 394.47 b3+ 374.28 b3+ b3+ 374.19 374.21 374.21 m/z b3+ 350 350 350 350 338.34 329.21 404.48 329.23 515.50 515.50 FullMS/MS 300 300 300 300 301.23 301.21 289.23 289.24 404.53 289.24 404.53 b2+ 261.18 b2+ 261.15 261.10 261.12 261.12 250 b2+ b2+ 250 250 250 233.25 233.20 233.15 233.19 233.19 226.96 200 200 200 200 189.30 175.08 175.30 150 150 150 150 120.10 119.99 120.05 120.09 120.09 0 0 0 0 50 50 50 50 100 100 100 100 6 6 6 6 5.89 5.71 5.58 5.45 5.58 5.58 5.57 5.56 5.37 5.37 5.28 5.12 5.24 4.98 5.19 5 5 5 5.36 5 4.76 4.53 4.69 4.72 4.61 4.26 4.87 TIC 4.51 4.51 4.45 4.44 4.36 4.35 4.30 4.29 4.25 3.85 4 4.34 Time (min) Time 4 4 4 3.87 3.86 3.86 3.84 4.62 4.89 3.85 3.85 3.62 3.78 3.53 3.64

0 0 0 0 0 0 0 0

100 100

100 100 100 100 100 100 Relative Abundance Relative Relative Abundance Relative Peptide C.FLLYNR

C.

67 Figure 9: Full MS/ MS and MRM spectra of each synthetic peptide and apoM- derived tryptic peptide in serum

A. AFLLTPR peptide

B. AFLVTPR peptide

C. FLLYNR peptide

Tryptic peptides of apoM that were identified in human, rat, and mouse serum were confirmed with synthetic peptides with full MS/ MS and MRM spectra for validation of

AFLLTPR (human), AFLVTPR (rat and mouse), and FLLYNR (all). (A) Human apoM tryptic peptide AFLLTPR was confirmed with synthetic peptide by full MS/ MS of the precursor ion and targeted measurement (MRM) to confirm isotopic distribution and relative intensities of specific fragment ions. The fragmentation pattern seen in human serum was confirmed by the resulting fragmentations from the synthetic peptide.

Retention time of the peptide from human serum was also compared with synthetic

AFLLTPR. (B) ApoM tryptic peptide AFLVTPR from mouse serum (top) and rat serum

(middle) was confirmed with synthetic peptide AFLVTPR (bottom). More ions are present in full MSMS spectra from rat and mouse serum compared to synthetic due to non-specific ion interferences. The ‘y’ and ‘b’ ions specific to this peptide are present in the synthetic peptide. (C) FLLYNR peptide is conserved in apoM across many species, including human, mouse, and rat. This common apoM tryptic peptide measured in serum was confirmed using synthetic FLLYNR. Retention time, full MS/ MS spectra and isotopic distribution and charge state of fragment ions were criteria used to confirm apoM tryptic peptides to build this assay.

68 Table 6

Tryptic Peptide AFLLTPR Retention AFLVTPR Retention FLLYNR Retention

Source Time (min) Time (min) Time (min)

Mouse Serum - 3.85 4.35

Rat Serum - 3.79 4.30

Human Serum 5.09 - 4.25

Synthetic Peptide 5.21 3.87 4.43

Table 6: Comparison of HPLC retention times of synthetic peptide and apoM- derived tryptic peptides in serum

The HPLC retention time was used as another parameter to confirm the identification of apoM-derived tryptic peptides in serum using the retention time of the synthetic peptide to confirm the chromatographic location of the same peptide from serum.

69 Measurement of ApoM in Human Serum and Need for Calibration Standard

Typically, purified or recombinant proteins can be used as a standard to prepare a set of calibration standards for quantification of the protein of interest. For absolute quantification of apoM, two independent calibration standards were prepared and used to measure apoM in human serum. Two-fold serial dilutions of human recombinant apoM purified from E.coli were made into PBS to prepare a set of calibration standards. The recombinant apoM calibration standards and two aliquots of human serum were prepared with Liposorb and digested with trypsin prior to MS analysis. Serial dilutions of

FLLYNR and AFLLTPR synthetic peptides were prepared in 0.1% NP40 in 100 mM

ABC, the same final solution as the digested serum samples and all were analyzed for apoM using the MRM method. The serum samples and calibration standards were analyzed in tandem to keep variations in HPLC column conditions between the samples and standards to a minimum. The peptide MS signal intensities from human serum were integrated and compared with those of the synthetic peptides and recombinant apoM calibration samples to quantify apoM.

The concentration of apoM in human serum was 1.7 µM (46.4 µg/mL) using recombinant apoM as the calibration standard and 120 nM (5.3 µg/mL) measured by

FLLYNR and AFLLTPR synthetic peptides (Figure 10). There was a large discrepancy

(approximately 15-fold) in the measured concentration of apoM in human serum using between the two calibration standards. The recombinant protein measured apoM at a much higher concentration than the synthetic peptide measurement. Due to the discrepancy in the measurement of apoM concentration, these experiments were repeated twice and confirmed that the measurement of apoM in serum using recombinant apoM

70 and synthetic apoM peptides were inconsistent with one another. The concentration of the stock solutions of both standards was confirmed by nitrogen count to ensure that an inaccurate measurement of the initial stock concentrations was not a source of error.

The cause of the discrepancy in the measurement of apoM concentration between the recombinant protein and synthetic peptide standards was unclear and further experiments were performed to isolate the source of variation in apoM measurement in order to select and prepare a functional calibration standard.

71 Figure 10

Measured Concentration of ApoM in Serum Using Two Standards 100

80 46.4 µg/mL 60

40 5.3 µg/mL 20 Concentration of ApoM, µg/mL 0 Rec ApoM Syn Ptd

Figure 10: Measurement of apoM concentration in human serum using two different standards

The concentration of apoM in human serum was measured using two independent standards: human recombinant apoM and synthetic AFLLTPR and FLLYNR peptides.

The recombinant apoM measured 46.4 µg/mL of apoM in human serum whereas the synthetic peptides AFLLTPR and FLLYNR measured 5.3 µg/mL of apoM in the same human serum sample.

72 Measurement of Low Recovery of Recombinant Protein and ApoM from Serum

Initially, serial dilutions of human recombinant apoM purified from E. coli were prepared in PBS used as a set of calibration standards to quantify apoM in human serum.

Serial dilutions of synthetic tryptic peptides were prepared to cover the same concentration range as the recombinant apoM standard dilutions to confirm the measurement of apoM in human serum. The MS peak intensities of synthetic AFLLTPR peptide and recombinant apoM-derived AFLLTPR peptide were plotted against the known concentration (µM) of each calibration sample (Figure 11).

At the same molar concentration, the integrated peak area of recombinant apoM was about one-tenth of the peak area of the synthetic peptide. The area of the synthetic peptides was assumed to be 100% recovery, since no sample preparation was performed.

Thus, less than 10% of the recombinant apoM was recovered from the initial sample.

The average concentration of apoM in human serum was not available at this time, so a definitive conclusion on the percent of recovery of apoM from human serum could not be made. Theoretically, apoM in human serum should mimic the behavior of the recombinant protein in response to sample preparation and instrument analysis, so the recovery of apoM in human serum was considered to be less than 10% as well.

The low recovery of recombinant apoM provided one reason for the source of the discrepancy between recombinant apoM and synthetic AFLLTPR peptide in the measurement of apoM in human serum. The low recovery of recombinant apoM needed to be resolved prior to preparation of a calibration standard to accurately measure the concentration of apoM in experimental samples.

73 The initial step taken toward increasing the recovery of recombinant apoM was to identify the potential source of the protein loss. The recombinant protein may have been lost to the polypropylene labware (tubes, tips, etc.) during the preparation of serial dilutions due to the low protein concentration of recombinant apoM. There may also have been a loss of recombinant apoM during sample preparation prior to MS analysis or the recombinant protein may not accurately represent the behavior of apoM in human serum, distorting the measurement of apoM concentration in serum.

74 Figure 11

Synthetic Peptide and Recombinant ApoM Recoveries 250000

200000

150000 Syn Ptd Rec ApoM

Intensity 100000

50000 AFLLTPR Peptide, MS PeakMS Peptide, AFLLTPR 0 0 0.5 1 1.5 AFLLTPR Peptide Concentration, µM

Figure 11: Synthetic AFLLTPR peptide and recombinant apoM peak intensities across the same concentration range

Recombinant apoM from E. coli had a low MS signal compared to synthetic apoM tryptic peptide, AFLLTPR at the same molar concentrations. The cause of this discrepancy was investigated as potentially inaccurate stock concentration measurements or loss of during serial dilution or sample preparation. Synthetic peptide and recombinant apoM stock material concentrations were confirmed, so recombinant apoM (and assumedly apoM from serum) was considered to have a low recovery from sample preparation.

75 Serial Dilutions of Human Recombinant ApoM in PBS and BSA

Serial dilutions of standard proteins are typically made into PBS or other buffer in preparation of calibration standards used to measure the concentration of a target protein in experimental samples. Small losses protein can occur during dilution of the recombinant protein, due to a low concentration of total recombinant protein (µM or nM).

Any minute losses in these samples can result in a dramatic difference in actual protein concentration, whereas, for example, the concentration of neat serum after several dilutions into PBS may not be altered since the total protein concentration in serum is much higher (mg/mL) than the recombinant protein solution.

This may have been a potential source of recombinant apoM loss because the protein concentration in the recombinant apoM stock solution was 1.5 mg/mL and was diluted to 100 µg/mL, 20 µg/mL, and 4 µg/mL in PBS. To determine if the dilution of recombinant apoM into PBS were a source of the protein loss and to test if the loss of protein can be prevented, the recombinant apoM stock solution was diluted into bovine serum albumin (BSA). BSA was used as a dilution matrix to add a higher concentration of total protein.

1.5 mg/mL of recombinant human apoM stock solution was diluted into two different matrices: 12.5 mg/mL of BSA (Pierce, cat #17804) and PBS to 100 µg/mL, 20

µg/mL, and 4 µg/mL of recombinant apoM and were prepared and measured for apoM concentration as described previously. MS peak intensities from the diluted samples were expressed as a percent of the MS peak intensity from the initial stock solution of recombinant apoM (Figure 12). The use of 12.5 mg/mL of BSA as a dilution matrix helped minimize the loss of recombinant apoM. At the lowest concentration of

76 recombinant apoM more than 80% of the recombinant apoM was recovered when BSA was present compared to less than 20% recovery of recombinant apoM when diluted in

PBS alone. The error bars representing the difference in MS peak intensities between duplicate sample measurements was higher overall in the recombinant apoM samples diluted into PBS. The MS peak intensities between duplicate samples of the recombinant protein diluted into BSA were more consistent than the duplicates of recombinant protein diluted into PBS.

The recovery of recombinant apoM was improved when 12.5 mg/mL BSA was used as a dilution matrix instead of PBS, however, some loss of the protein still occurred

(~20%). The low recovery of recombinant apoM and apoM in human serum was also evaluated using a method independent from MS analysis, Western blot.

77 Figure 12

Recombinant Human ApoM Recovery in Different Dilution Matrices

140 120 100 80 12.5 mg/mL BSA 60 PBS 40

ApoM % Recovery, 20 0 4 20 100 1500 Recombinant ApoM concentration, µg/mL

Figure 12: Dilution of human recombinant apoM in PBS or 12.5 mg/mL BSA to increase protein recovery

Recombinant apoM recovery was expressed as a percent of initial recombinant stock solution (+/- SD). The error between duplicate samples of the recombinant protein diluted into PBS was larger than the error between duplicates of recombinant protein diluted into BSA. Recombinant human apoM was lost during dilution into PBS, but was partially recovered when the recombinant protein was diluted into a protein-containing dilution matrix, 12.5 mg/mL of BSA. More than 80% of recombinant apoM was recovered from dilution into BSA and this was attributed to the higher protein content to prevent recombinant protein losses to polypropylene labware during dilution in PBS.

78 Western Blot of Recombinant ApoM and ApoM in Human Serum

Western blot analysis was performed to confirm the loss of recombinant apoM during dilution into PBS and compare the amount of recombinant apoM present when diluted into PBS compared with the amount of recombinant apoM present when diluted into 12.5 mg/mL of BSA. The second analysis in this experiment was to evaluate the use of Liposorb to capture recombinant apoM and human apoM from serum.

Five-fold dilutions of human recombinant apoM were prepared in PBS and 12.5 mg/mL of BSA and these dilutions and human serum were prepared as described

(Experimental Section).

ApoM bands were present in the Liposorb fractions of human serum, 4 µg/mL, 20

µg/mL and 100 µg/mL of recombinant apoM diluted in 12.5 mg/mL of BSA, and 100

µg/mL of recombinant apoM diluted in PBS (Figure 13). 12.5 mg/mL BSA was loaded as a negative control and did not contain apoM. The supernatant fractions of each

Liposorb-bound sample did not contain any detectable amount of apoM. Recombinant apoM was detected in the 100 µg/mL dilution in PBS, whereas apoM was detected in 4

µg/mL, 20 µg/mL and 100 µg/mL dilutions in BSA, although the 4 µg/mL apoM band was very faint on the Western blot. A ladder of apoM bands at different molecular weights was present in the Western blot analysis of human recombinant apoM, whereas there was only one apoM band present in human serum. Liposorb captured recombinant apoM at all molecular weights, with no detectable apoM in the supernatant fractions.

The Western blot analysis showed that the loss of recombinant apoM occurred when the recombinant protein was diluted into PBS when compared to the dilution of recombinant protein into 12.5 mg/mL of BSA. The percent of recovery was not

79 calculated using Western blot, but the presence of recombinant apoM bands at a lower concentration with BSA matrix, combined with the absence of apoM bands in 4 µg/mL and 20 µg/mL recombinant apoM dilutions in PBS show that the BSA helps keep the concentration of recombinant apoM higher than the recombinant apoM at the same dilution in PBS.

The presence of apoM at many different molecular weights in the recombinant protein solution gave some explanation to the difference in the measurement of apoM in human serum between synthetic AFLLTPR peptide and recombinant apoM-derived

AFLLTPR peptide. Although Western blot is not quantitative, the results of this experiment were used to estimate the concentration of recombinant apoM. As confirmed by nitrogen count, the total protein concentration of the recombinant apoM stock solution was 1.5 mg/mL. However, this solution contained many forms of apoM whereas apoM in human serum was present at one molecular weight (~25 kDa). If the total recombinant apoM concentration was considered to include all forms of apoM, then a revised estimate of the concentration of apoM in serum was less than one-half of the measured concentration at ~20 µg/mL (770 nM) instead of 46 µg/mL (1.7 µM), based on visual analysis of the number of recombinant apoM bands compared to one apoM band in human serum in the Western blot. Even with the estimated correction factor applied, a discrepancy in the measured concentration of apoM in human serum was still present between synthetic AFLLTPR peptide and recombinant apoM (5.3 µg/mL and ~20

µg/mL, respectively). Human recombinant apoM purified from E.coli was present in different forms that were not found from apoM in human serum and it was unclear which form of the recombinant was measured using the targeted MS assay. If some of the

80 different molecular weights of the recombinant apoM are not measured by the MS in the targeted assay, then the recombinant protein can not be used to accurately represent apoM in serum. One additional experiment was performed using MS for a final evaluation of the use of recombinant apoM as a calibration standard to mimic the behavior and accurately measure apoM in human serum.

81 Figure 13

100 µg/mL 100 µg/mL 20 µg/mL 100 µg/mL 4 µg/mL 20 µg/mL 100 µg/mL 1.5 mg/mL Rec. in BSA Rec. in BSA Human Ser. Human Ser. MW BSA Rec. in PBS Rec. in PBS Rec. in BSA Rec. in BSA Rec. in BSA Rec. Stock + Liposorb Supernatant + Liposorb Supernatant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

kDa 150- 98- 64- 50- 36- 22-

Figure 13: Comparison of the recovery of recombinant apoM diluted into PBS and

12.5 mg/mL of BSA

Samples in wells 3-22 were prepared and loaded in duplicate. The same volume was loaded for each sample for direct comparison. Well #1: molecular weight marker, 2: 12.5 mg/mL BSA negative control, 3-4: 20 µg/mL Rec. apoM in PBS, 5-6: 100 µg/mL Rec. apoM in PBS, 7-8: 4 µg/mL Rec. apoM in BSA, 9-10: 20 µg/mL Rec. apoM in BSA, 11-

12: 100 µg/mL Rec. apoM in BSA, 13-14: 1.5 mg/mL Rec. apoM stock, 15-16: 100

µg/mL Rec. apoM in BSA + Liposorb, 17-18: Supernatant from #15-16, 19-20: Neat human serum + Liposorb, 21-22: Supernatant from #19-20. Mouse anti-apoM was used as a primary antibody for apoM in serum and recombinant protein.

82 Reduction-alkylation of recombinant protein and apoM in human serum to increase recovery

Typically, in protein solution preparation prior to MS analysis, disulfide bonds are reduced and alkylated to open the protein structure before trypsin digestion [26]. In this assay, reduction and alkylation (R/A) of disulfide bonds was not performed in this protocol because the initial preparation protocol was adapted from the apolipoprotein panel assay and most of these apolipoproteins do not contain cystine. However, apoM has six cystines and can form three disulfide bonds [27], so reduction and alkylation experiments were performed to further increase recombinant apoM recovery.

Human serum and three different concentrations of recombinant human apoM (4

µg/mL, 20 µg/mL and 100 µg/mL in 12.5 mg/mL BSA) were prepared with Liposorb, reduced and alkylated with dithiothreitol (DTT) and iodoacetamide, respectively, and then digested with trypsin. A R/A procedure using volatile compounds (acetonitrile, iodoethanol, and triethylphosphine) [26] was not used to avoid a drying step by speed vacuum to preserve the Liposorb beads. Human serum and recombinant apoM prepared with Liposorb and trypsin but not subject to reduction-alkylation prior served as the control. All tryptic digests were analyzed by MS using the MRM method for apoM analysis. The peak intensities of AFLLTPR and FLLYNR peptides measured in each sample were expressed as a percent of the control (100%) to evaluate the fold change in apoM with R/A.

ApoM recovery from human serum was increased by more than 6-fold, whereas recombinant human apoM recovery did not change at any concentration compared to control (Figure 14). The increase in recovery of apoM in human serum with R/A was

83 consistent with the presence of disulfide bonds in the protein structure. The recovery of recombinant apoM did not change with R/A, in conjunction with the results of the

Western blot analysis that revealed different molecular weights of recombinant apoM, lead to the conclusion that the recombinant protein may have a different secondary structure and does not accurately represent the behavior of apoM in human serum.

Therefore, recombinant apoM was not further evaluated or used as a calibration standard to quantify apoM in human serum and another source of calibration standard was evaluated.

84 Figure 14

Effect of R/A on Recovery of ApoM from Serum and Rec. Protein 1000 ApoM in Human Serum 800

600

FLLYNR 400 Recombinant ApoM AFLLTPR

200 ApoM Recovery, % control of Recovery, ApoM 0 Control R/A Control R/A Control R/A Control R/A 44 µg/mL µg/mL 2020 µg/mLµg/mL 100 100 µg/mLµg/mL

Figure 14: Effect of reduction-alkylation on the recovery of recombinant apoM and apoM in human serum

Human serum and recombinant apoM were prepared with (R/A) and without R/A

(Control) prior to MS analysis. ApoM recoveries were calculated using the MS signal: concentration ratio expressed as a percent of the control (+/-SD). The recovery of apoM in human serum was increased more than 6-fold when R/A was included in sample preparation compared to control (sample preparation without R/A). The recovery of recombinant apoM did not change in response to R/A compared to the recombinant control (no R/A).

85 Pooled Human Serum as a Calibration Standard

The recombinant protein did not mimic the behavior of apoM in human serum, possibly due to a difference in structure, but this hypothesis was not tested. Human recombinant apoM was no longer used as a calibration standard to measure apoM in human serum. A calibration standard that mimics the behavior of apoM from human serum is needed reliably measure the concentration of apoM. Synthetic apoM peptides can be used for quantification, but will not reflect variations in protein recovery from serum that may occur due to Liposorb binding and digestion with trypsin.

Therefore, pooled human and rat sera were used as a calibration standard to quantify apoM in human and rat serum, respectively. The serum calibration standards will go through the same sample preparation prior to MS analysis as the experimental samples, which can reduce differences in apoM behavior and recovery from serum between calibration standards and experimental samples. A serum dilution matrix was needed to prepare serial dilutions of the pooled human or rat sera for calibration standards. The use of a serum matrix maintained a consistent total volume of serum between each sample in the calibration series. Serum that could be used as a dilution matrix to create the calibration standards had to originate from a different species than the experimental samples so that the measurement of apoM in the experimental samples and calibration samples was not disrupted. The apoM-derived tryptic peptides in the matrix serum must contain different sequences than the target peptides used for quantification of apoM in this assay (i.e. AFLLTPR, AFLVTPR, and FLLYNR peptides).

86 Survey of Different Species to Select a Serum Dilution Matrix

Serum from many different species needs to be evaluated for the presence of apoM-derived tryptic peptides FLLYNR, AFLLTPR, and AFLVTPR to determine which species can be used as a dilution matrix for experimental samples from human, rat, and mouse origin, and the versatility of the assay can be expanded to include all species that share at least one of the selected tryptic peptides.

Aliquots of serum from human, rat, mouse, horse, rabbit, goat, sheep, dog, bovine, and pig were prepared and analyzed by MS. MS signal intensities from apoM- derived tryptic peptides: AFLLTPR, AFLVTPR, and FLLYNR were measured in each sample. In conjunction with MS analysis, the AA sequence of apoM was also manually evaluated for the presence or absence of AFLLTPR, AFLVTPR, and FLLYNR peptides when the sequence was available in the NCBI protein database.

The results of the survey of several different serum species were two-fold: (1) selection of background matrix, and (2) evaluation of assay versatility. The presence or absence of the three apoM-derived tryptic peptides in the evaluated species is summarized in Table 7. Based on the results of the experimental data, tryptic peptide

AFLLTPR was derived from human, horse, rabbit, goat, sheep, bovine, and pig apoM.

The presence of AFLLTPR peptide in apoM from these species was also evaluated manually using the AA sequence of apoM obtained from NBCI and confirmed to be present in the AA sequence of human, horse, bovine, and pig apoM. AA sequence analysis and the results of the MS analysis showed that AFLVTPR peptide was present in mouse and rat serum only. FLLYNR peptide was found experimentally in human, mouse, rat, horse, goat, sheep, dog, bovine, and pig apoM. Although monkey serum was

87 not evaluated in the MS experiments, the AA sequence of monkey apoM from NCBI contains FLLYNR. The AA sequences of rabbit, goat, and sheep apoM were not available from the NCBI protein database, so the presence of these apoM-derived tryptic peptides (AFLLTPR, AFLVTPR, and FLLYNR) was only determined by MS.

The results of this experiment were used to select a proper species for serum dilution matrices for each of the three selected apoM-derived tryptic peptides (Table 8).

The matrix serum species was selected if the tryptic peptide was absent either experimentally, from the AA sequence from the protein database, or both, and the MS signal intensity of noise and other interferences that eluted from the HPLC column at the same retention time as the target tryptic peptide(s) were minimal.

Rabbit was the only species that did not contain FLLYNR peptide, so it was evaluated for relatively low background noise and contaminant ions around the same retention time as FLLYNR in the targeted method (Figure 15A). Rabbit serum had a low background for FLLYNR and was selected as the species of background matrix serum.

Serum from human, horse, rabbit, goat, sheep, dog, monkey, bovine, and pig did not contain the tryptic peptide AFLVTPR. Of these species, horse serum had a low MS signal intensity of background noise compared to the MS signal intensity of AFLVTPR peptide from rat and mouse serum (Figure 15B) and was selected as the dilution matrix when AFLVTPR peptide is used to quantify apoM in rat or mouse experimental and calibration serum samples.

Rat, mouse, dog, and monkey serum did not contain the apoM-derived tryptic peptide AFLLTPR. Rat serum was selected as the best dilution matrix for the

88 measurement of AFLLTPR peptide in experimental and calibration standards (Figure

15C).

The selection of dilution matrices were rabbit serum for the measurement of

FLLYNR peptide in human, rat, or mouse serum. Horse serum was selected as the background matrix for the measurement of AFLVTPR peptide in rat and mouse serum and rat serum was initially selected as the dilution matrix for the measurement of

AFLLTPR peptide in human serum.

However, AFLLTPR was not used to measure apoM in human serum diluted into rat serum matrix. A relatively low MS peptide signal from AFLLTPR peptide was observed in human serum calibration samples diluted into rat serum, compared with the

MS peptide signal from FLLYNR peptide observed from human serum calibration samples diluted into rabbit serum at the same dilution factor (Figure 16). Thus, human serum experimental and calibration samples were diluted into rabbit serum and FLLYNR peptide was measured instead of AFLLTPR peptide measurement in human serum diluted in rat serum as the human-specific peptide in these experiments. Final evaluations of the selected species for dilution matrices were made (Table 9) and the final selections were horse serum for the measurement of AFLVTPR peptide in rat and mouse serum and rabbit serum for the measurement of FLLYNR peptide in human, rat, and mouse serum.

Using the experimental results that were discussed above, the versatility of the assay for quantification of apoM in human, rat, and mouse serum was expanded to include the quantification of apoM in many additional species without additional tryptic peptide selections or changes in the HPLC gradient and MS method.

89 The unique tryptic peptides from apoM that were included in the final assay can be used to measure apoM in any species that contains at least one of these tryptic peptides in their apoM AA sequence. The selection of a new dilution matrix is the only additional step in converting this assay to another species from human, rat, and mouse. For example, using the data collected in Tables 7 and 8, the measurement of apoM in horse serum samples can be made using FLLYNR peptide (present in horse) in rabbit serum dilution matrix for experimental and calibration standards, since rabbit serum does not contain FLLYNR peptide. Thus, measurement of apoM in the species that were evaluated in this experiment can be achieved in a quick, straightforward manner. The

FLLYNR peptide was conserved among multiple species except rabbit. Therefore, any other species that was surveyed in this experiment can have apoM quantified by

FLLYNR peptide and the use of rabbit serum as the dilution matrix. New calibration samples should be prepared for apoM quantification in each new species and experimental serum samples should be diluted into the same matrix as the calibration samples. This yielded a valuable and robust MS assay with broad application, measuring apoM in many pre-clinical species with the potential for seamless crossover to clinical measurements via the same assay, or for use in transgenic animal models where apoM from more than one species may be present.

90 Table 7

Peptide Human Mouse Rat Horse Rabbit Goat Sheep Dog Monkey Bovine Pig Sequence

FLLYNR +* +* +* +* - * * +* + +* +*

AFLLTPR +* - - +* * * * - - +* +* AFLVTPR - +* +* ------

Table 7: Presence or absence of the AA sequences of FLLYNR, AFLLTPR, and/ or

AFLVTPR peptides in several different species

* Presence of tryptic peptide was determined experimentally using the MS-based assay

+ Tryptic peptide was present in NCBI AA sequence of apoM in protein database

- Tryptic peptide was not present in AA sequence and/ or was not found experimentally

The survey of different serum matrices lead to the selection of a dilution matrix that did not contain the same apoM-derived tryptic peptide as apoM from the target species.

FLLYNR was present in all species except rabbit, AFLLTPR was not present in rat, mouse, dog, or monkey serum, and AFLVTPR was absent in all species that were tested except rat and mouse.

91 Table 8

Peptide Species for Dilution Matrix Sequence Human Mouse Rat Horse Rabbit Goat Sheep Dog Monkey Bovine Pig

FLLYNR x x x x yes x x x x x x

AFLLTPR x yes yes x x x x yes yes x x

AFLVTPR yes x x yes yes yes yes yes yes yes yes

Table 8: Suitable dilution matrices to use when measuring apoM-derived FLLYNR,

AFLLTPR, or AFLVTPR tryptic peptides

The selection of suitable dilution matrix was based on the absence of the apoM-derived peptide of interest in serum from the species used as a dilution matrix. The ‘x’ indicates that the selected apoM-derived peptide is found in that particular species and therefore the species is not suitable to use as a dilution matrix. A ‘yes’ in the species column indicates that this species is suitable to use as a dilution matrix when the selected apoM- derived tryptic peptide is measured because that particular peptide was not present in the dilution matrix species.

92 Figure 15

A.

4.32 NL: 7.93E3 100 TIC F: ITMS + c ESI SRM 90 ms2 [email protected] [ 451.23-454.23, 80 564.31-567.31, 70 677.39-680.39] MS BH071114_P1B_G11 60

50 40 Human

Relative Abundance Abundance 30 Serum 20 10 3.67 4.49 0 4 5

3.92 NL: 5.96E2 100 TIC F: ITMS + c ESI SRM 90 ms2 [email protected] [ 451.23-454.23, 80 564.31-567.31, 677.39-680.39] MS Relative Relative Abundance 70 BH071114_P1B_H01 60

50 40 Rabbit Relative Abundance 30 Serum 3.40 20 4.29 10 4.62

0 4 5 Time (min)

93 B.

3.83 NL: 2.31E4 100 TIC F: ITM S + c ESI SRM ms2 [email protected] [ 372.22-375.22, 80 471.29-474.29, 584.37-587.37] M S BH070514_r_M _11-r3 60

40

20 3.37 Rat

Relative Abundance 4.03 0 Serum 3.0 3.5 4.0 4.5

RT: 3.00 - 4.50 3.83 NL: 2.63E4 100 TIC F: ITM S + c ESI SRM ms2 [email protected] [ 372.22-375.22, 80 471.29-474.29, 584.37-587.37] M S 001_0042_P_060408_B01 60 40 Mouse 20 3.07

Relative Abundance 3.33 4.00 0 Serum 3.0 3.5 4.0 4.5

Relative Abundance Relative Time (min)

3.25 NL: 1.70E3 100 TIC F: ITM S + c ESI SRM ms2 [email protected] [ 372.22-375.22, 80 471.29-474.29, 584.37-587.37] M S 3.28 BH070514_r_M _2-hrs 60 3.57

40 3.67 3.96 20 Horse Relative Abundance 0 Serum 3.0 3.5 4.0 4.5 Time (min)

94 C.

4.24 NL: 2.01E4 100 TIC F: ITMS + c ESI SRM 90 ms2 [email protected] [ 372.22-375.22, 80 485.30-488.30, 70 598.39-601.39] MS BH070514_M_8-hm 60

50 40 Human

Relative Abundance 30 Serum 20 10 4.12 4.36 4.42 0 4.2 4.4 Time (min)

4.12 NL: 1.28E2 100 TIC F: ITMS + c ESI SRM 90 ms2 [email protected] [ 4.43 372.22-375.22, 80 4.38 485.30-488.30, Relative Abundance Relative 70 598.39-601.39] MS 4.20 4.34 BH070514_M_11-r3 60 4.31 50

40 4.24 Rat

Relative Abundance 30 Serum 20

10

0 4.2 4.4 Time (min)

Figure 15: Selection of dilution matrix for FLLYNR and AFLVTPR peptides

A. FLLYNR peptide was used to measure apoM in human, rat, or mouse serum and was not present in rabbit serum, leading to the selection of rabbit serum as the dilution matrix

95 when FLLYNR was present in the experimental samples and used for apoM quantification.

B. AFLVTPR peptide was used to measure apoM in rat and mouse serum and was not present in horse serum.

C. AFLLTPR peptide was used to measure apoM in human serum and was not present in rat serum. Therefore, rat serum was used as a dilution matrix for the measurement of

AFLLTPR peptide in human serum.

Selection of the dilution matrix was based on the absence of the target apoM peptide in digested serum from the dilution species. FLLYNR peptide was not present in rabbit serum and has a relatively strong MS peptide signal from human serum and was selected as the dilution matrix. AFLVTPR peptide was not present in horse serum and the background noise was low compared to the peptide MS signal intensity of AFLVTPR from rat and mouse serum. AFLLTPR peptide was measured from human serum diluted into rat serum, which also had relatively low background noise levels.

96 Figure 16

4.34 NL: 1.69E4 100 TIC F: ITMS + c ESI SRM 90 ms2 [email protected] [ 451.23-454.23, 80 564.31-567.31, 70 677.39-680.39] MS BH071114_P1A_G11 60 50 40 Human FLLYNR Peptide 30 in Rabbit Serum Matrix 20

10 2.56 2.82 5.01 0 0 2 4

3.95 NL: 5.63E3 100 TIC F: ITMS + c ESI SRM ms2 90 [email protected] [ 372.22-375.22, 485.30-488.30, 598.39-601.39] 80 Relative Relative Abundance MS 70 LTQ1_001_0042_P_060707_BH0 70523_M_P1Fa_E12 60 50 40 Human AFLLPTR Peptide

Relative Abundance 30 in Rat Serum Matrix 20 3.81 10 5.21 0 0 2 4 6 Time (min)

Figure 16: Comparison of AFLLTPR and FLLYNR peptide MS signals in different dilution matrices

AFLLTPR peptide MS signal from human serum diluted into rat serum matrix was low compared to the MS signal from FLLYNR peptide from human serum diluted into rabbit serum matrix. FLLYNR peptide was used to measure apoM in human serum diluted in rabbit serum matrix to obtain a higher peptide MS signal to use for apoM quantification in human serum .

97 Table 9 matrix had a stronger had MS in rat in serum dilution matrixto Comments measure measure AFLVTPR The The measurement of rat AFLLTPR was serum using low signal diluted signal rabbit than in diluted AFLLTPR when Horse had serum cleanest the as background-used the Used Used formeasurement apoMserum- of human in FLLYNR rabbit DilutionMatrix mouse,rat, monkey dog, dog, monkey, bovine, dog, monkey, pig horse,rabbit, goat, sheep, Peptide Analyte FLLYNR AFLLTPR AFLVTPR mouse Human Rat/Mouse Human, rat,Human,

Species Species

98 Table 9: Detailed evaluation of different species to use as a dilution matrix

ApoM-derived tryptic peptides FLLYNR, AFLLTPR, and AFLVTPR were evaluated to ensure the optimal measurement of apoM was obtained using these peptides. The peptide

MS signal from AFLLTPR peptide was low from the dilution of human serum into a rat serum matrix, so the measurement of human apoM was changed to measure FLLYNR peptide from human serum diluted into rabbit serum.

99 Use of External Calibration Standard in Quantitative Assay

The selections of proper dilution matrices for experimental and calibration samples were made for the measurement of apoM in human, rat, and mouse serum.

Serial dilutions of human serum were prepared in a rabbit serum matrix for the measurement of FLLYNR peptide. Rat and mouse serum were diluted into the horse serum matrix for measurement of AFLVTPR peptide (see methods). A species-specific set of external calibration standards was included in each 96-well plate of experimental samples.

Typically in human serum preparation, 10 µL of human serum from an experimental sample was diluted into 20 µL of rabbit serum and 120 µL of PBS. The total serum-to-PBS ratio was fixed at 1:4 for the experimental samples which is the same fixed ratio as the total serum-to-PBS ratio in the calibration samples. A 50 µL volume of both the diluted experimental sample and calibration samples was used. The diluted experimental samples and calibration samples were then mixed with 200 µL of Liposorb prior to digestion using 2 µg of modified trypsin.

Serial dilutions of synthetic AFLLTPR and FLLYNR peptides were prepared and used as a set of calibration standards to measure the concentration of apoM in the human serum used to prepare the human calibration standards (Figure 17) and the apoM concentration in undiluted human serum was measured at 401 nM (10.4 µg/mL). The molar concentration of apoM in the serial dilutions of calibration samples was then calculated using this measurement and used to create a standard curve (Figure 18). Thus, the absolute quantification of apoM in human serum could be measured in experimental samples.

100 Figure 17

Measurement of Human ApoM Concentration Using Synthetic Peptides FLLYNR and AFLLTPR

1400000 1200000 1000000 Syn Ptd FLLYNR 800000 Human ApoM 600000 Syn Ptd AFLLTPR 400000 200000 0 Peptide MS Signal (AUC) 0 500 1000 1500 2000 ApoM Concentration, nM

Figure 17: Measurement of apoM in human serum using serial dilutions of synthetic peptides AFLLTPR and FLLYNR

Using both apoM peptides for quantification, apoM in human serum was 401 nM (10.4

µg/mL +/- 1.2 SD). Aliquots of this serum were processed with each experiment to use as a control, allowing comparison of apoM measurements across different experiments.

101 Figure 18

Human Calibration Set G: Standard Curve

300000

250000

200000

150000

100000

MS Peptide Signal 50000

0 0 100 200 300 400 500 600 700 ApoM Concentration, nM

Figure 18: Human calibration set G standard curve

Standard curve generated from serial dilutions of human serum into rabbit serum matrix.

102 Optimization of Sample Preparation to Maximize ApoM Recovery from Serum

Prior to the use of the calibration standards to quantify apoM in experimental samples, the sample preparation protocol was optimized to ensure maximum recovery of apoM from serum. A few steps in this sample preparation protocol could result in the loss of apoM, including: Liposorb binding, washing the Liposorb pellet, and digestion with trypsin, all of which were evaluated experimentally for the percent of apoM recovery and optimized to obtain the maximum apoM-derived peptide MS signals from

FLLYNR and AFLLTPR peptides from human serum.

The saturation level of Liposorb was evaluated to confirm the quantitative capture of apoM from serum, by evaluating the percentage of apoM bound with Liposorb compared to the amount of apoM remaining in the supernatant after incubation of human serum with Liposorb. 10 µL of human serum was incubated with different volumes of

Liposorb stock solution (20- 400 µL) and both the supernatant and Liposorb pellet were digested with trypsin. Both fractions were analyzed by MS for apoM-derived tryptic peptides using the targeted assay to evaluate the saturation level of Liposorb with 10 µL of human serum.

It was found that 20 µL and 50 µL of Liposorb did not completely capture apoM from serum, resulting in lower apoM-derived peptide intensities from MS compared to the signal intensities from the same peptide in the samples containing 100 µL and 200 µL of Liposorb. The larger volume of Liposorb gave higher apoM-derived tryptic peptide signal intensities from MS. At 400 µL of Liposorb, the peptide MS signals did not further increase compared to 100 µL and 200 µL of Liposorb. Thus, 200 µL of Liposorb stock solution was used as the standard volume per 10 µL of serum to avoid saturation of

103 the Liposorb while keeping the sample volume as low as possible. 100 µL of Liposorb was not used as the standard volume because it may have been close to saturation since

50 µL of Liposorb was saturated with a positive MS signal from apoM-derived tryptic peptides measured in the supernatant. The Liposorb saturation level was not a source of low recovery since apoM was not found in the supernatant of 100 µL of Liposorb and higher and 100 µL of Liposorb was used in the previous experiments to capture apolipoproteins from 10 µL of human serum.

The length of the serum and Liposorb incubation period was evaluated to find the optimal incubation time for the highest MS signal from apoM-derived tryptic peptides, indicating the highest apoM recovery from serum. The current incubation time, 30 minutes, and one hour were tested. Aliquots of 10 µL of human serum were incubated with 200 µL of Liposorb for 30 minutes or one hour at 4°C with shaking. MS signal did not increase significantly (<10%) after one hour incubation compared to 30 minutes

(Figure 19). Therefore, 30 minutes was used as the Liposorb incubation time to keep the protocol length as short as possible to increase the throughput of the assay.

The number of Liposorb washing steps was optimized and evaluated for the highest apoM recovery. In a previous experiment, it was established that apoM was captured by Liposorb in a quantitative manner, but the binding strength between Liposorb and apoM was not evaluated. Prior to the optimization of the protocol, Liposorb was washed three times with 100 mM ABC prior to digestion with trypsin. In this experiment, aliquots of 10 µL of human serum were incubated with 200 µL of Liposorb.

The supernatant was aspirated and different repetitions of washes were performed: zero, one, two, and three washes with 100 mM ABC. One wash was sufficient to increase the

104 MS peptide signal of FLLYNR peptide approximately 4-fold compared to zero washes

(Figure 20) and AFLLTPR peptide signal remained unchanged. However, without a wash step in the protocol, proteins or other contaminants binding to the Liposorb pellet may cause ion suppression in the MS measurement. After one wash, subsequent washes did not change the MS peptide signal, or result in a cleaner background. The increase in

MS signal for FLLYNR peptide and no change in MS signal for AFLLTPR peptide after multiple wash steps confirmed that the binding affinity between apoM and Liposorb was strong. The reduction of the protocol to include only one wash step after a 30-minute

Liposorb incubation time increased the throughput of this assay.

The use of detergent during digestion with trypsin was previously evaluated to increase the recovery of apoB and PON-1-derived tryptic peptides from serum and synthetic peptides. However, apoM was not included in these initial experiments.

Aliquots of 10 µL of human serum were prepared with 200 µL of Liposorb prior to digestion. One half of the samples were digested with trypsin in 0.1% NP40 in 100 mM

ABC and the other half were digested in 100 mM ABC alone, and 0.1% NP40 was added after overnight digestion. All samples were filtered and analyzed.

The MS signal from apoM-derived tryptic peptides did not change significantly with addition of detergent (Figure 21). Therefore, apoM recovery did not change with the addition of NP40 during digestion, but the error bars between duplicate measurements were smaller (<5% error) with the addition of NP40 at the start of trypsin digestion compared to duplicate errors with detergent addition after digestion was complete (15-

20%). The recovery of apoM was relatively unchanged and more consistent with detergent, so 0.1% NP40 was added at the beginning of digestion with trypsin.

105 In summary, the incubation time of serum in Liposorb was determined to 30 minutes in 200 µL of Liposorb per 10 µL of serum from the experimental samples and the number of Liposorb washing steps was reduced to one. While these improvements helped to maximize the peptide MS signal from apoM-derived tryptic peptides, this sample preparation protocol must also be optimized to maximum peptide MS signals from apolipoproteins in the targeted apolipoprotein panel assay.

106 Figure 19

Liposorb Binding Complete After 30 Minutes

120

100

80 30 min Control 60 1 hr 40

20 Percent of 30 min Control, % 30 min Control, of Percent 0 FLLYNR AFLLTPR

Figure 19: Optimization of Liposorb-serum incubation time

Human serum was incubated with Liposorb at 4°C for 30 minutes or one hour. ApoM recoveries were calculated as a percent of 30 minutes as the control (+/-SD).

107 Figure 20

Effect of Consecutive Liposorb Washes on ApoM Recovery

100000 80000

60000 FLLYNR 40000 AFLLTPR 20000 0

MS Peptide Signal, AUC Signal, MSPeptide no wash 1x wash 2x wash 3x wash

Figure 20: Evaluation of consecutive Liposorb washing steps on apoM-derived peptide MS signal

The AUC of peptides expressed as arbitrary relative units and is plotted as a function of number of washes.

108 Figure 21

0.1% NP-40 Does Not Significantly Affect Digestion and Recovery of ApoM

140 120 100 80 60 40 During Trypsin 20 After Trypsin 0 Peptide Recovery, % of Control of Recovery,% Peptide FLLYNR AFLLTPR

Figure 21: Addition of 0.1% NP40 during trypsin digestion does not affect apoM recovery

The addition of 0.1% NP40 did not change the recovery of apoM from serum. Error is expressed as +/- SD.

109 Optimization of Sample Preparation Protocol for ApoM and the Apolipoproteins in the Panel Assay

Thus far, the serum preparation protocol was optimized to obtain the highest MS peptide signals from apoM-derived tryptic peptides in serum. However, its effect on the panel of apolipoproteins needs to be determined. Since R/A dramatically increased apoM recovery from serum (>6-fold) this treatment should be applied to the panel assay to evaluate its effect on the recovery of other apolipoproteins. The following conditions were tried to maximize recovery of apoM and other apolipoproteins in the panel assay, the use of 8 M urea, 0.1% NP40, heat (55°C), R/A, and different concentrations of modified trypsin were tested individually and in different combinations. Duplicate samples were prepared per treatment per trypsin concentration (Table 10).

Liposorb pellets were prepared in either 0.1% NP40, 10 mM DTT, 8 M urea, or directly in trypsin-NP40 solution (control only). Samples receiving R/A treatment were reduced with 10 mM DTT for 45 minutes at 37°C and then alkylated with 60 mM iodoacetamide for 30 minutes at room temperature in the dark, since iodoacetamide is light-sensitive. A non-volatile R/A protocol was chosen to avoid a drying step with the

Liposorb beads, which is a necessary drying step after a R/A reaction using volatile reagents [26]. All incubation periods involved continuous shaking of the samples to keep the Liposorb beads suspended in solution for even exposure of the apolipoprotein- containing Liposorb beads to the reactants in the solution. Samples were heated at 55°C and then cooled to room temperature before further treatments. All samples were prepared in a final trypsin solution of 0.1% NP40, with different amounts of trypsin added. One full set of samples (duplicate samples from treatment) was digested

110 overnight at 37°C with 0.1, 0.5, 1.0, or 2.0 µg of modified trypsin, while the other set was digested for 2 hours at 37°C with the same concentrations of modified trypsin.

Heating and/ or addition of NP40 to the Liposorb pellet did not increase the MS signal from apoM-derived tryptic peptides and was not used in the sample preparation protocol. R/A gave the highest recovery of apoM, confirming previous observations

(Figure 22). However, R/A did not increase or was detrimental to the recovery of the panel of apolipoproteins from serum. The addition of 8 M urea prior to digestion also increased apoM recovery (~200%) and this condition resulted in satisfactory recoveries of the apolipoprotein in the panel assay (Table 11). Higher increases in apoM recovery in response to R/A were measured after addition of all sample prep optimizations were included. Use of 8 M urea prior to R/A did not further increase apolipoprotein recoveries, including apoM (data not shown). Thus, two serum preparation protocols were chosen: R/A and 8 M urea to maximize MS signal from apoM-derived tryptic peptides and the other apolipoprotein peptides in the panel assay. The sample preparation procedure was further optimized to increase throughput using a robotics system for liquid handling steps and in resuspension of the Liposorb pellets for 96 samples simultaneously.

111 Table 10

Sample NP40 + Heat Reduction & 8 M Urea Trypsin (0.1, 0.5,

Preparation Alkylation 1, and 2 µg)

Procedure

Treatment 1 + +

Treatment 2 + +

Treatment 3 + +

Treatment 4 + + +

Treatment 5 + + +

Treatment 6 + + +

Treatment 7 + + + +

Treatment 8 +

(control)

Table 10: Combinations of different sample preparation protocols were evaluated to maximize apoM and apolipoprotein recovery from serum

A plus sign (+) indicates the treatment used per sample, and each treatment condition was digested at four different concentrations of trypsin. Protein recoveries in all treatments were compared to the control condition.

112 Table 11

Digest Time 2hr 2hr 2hr O/N O/N O/N Trypsin Conc 0.5 µg 1 µg 2 µg 0.5 µg 1 µg 2 µg Treatment RA RA RA Urea Urea Urea M-144 306 310 294 211 212 206 M-172 248 257 241 107 162 193 A1-231 35 134 257 219 204 184 A1-52 21 61 82 32 41 69 A2-54 13 26 40 28 34 60 A2-52 121 191 176 216 208 173 A4-222 19 74 104 48 75 93 A4-135 58 125 142 124 114 113 B-3869 68 111 120 126 116 120 B-3847 40 86 92 113 95 93 C1-27 1 10 46 19 38 81 C1-11 1 23 84 41 94 150 C2-62 48 107 112 96 95 99 C2-42 80 114 106 103 94 93 C3-45 57 90 100 90 92 94 C3-61 3 16 54 34 57 90 E-57 44 89 111 106 106 96 E-199 91 113 120 97 96 101 F-296 64 76 91 76 81 94 F-233 26 78 97 60 77 95 P1-234 1 16 51 43 75 96 P1-291 19 44 64 99 96 100 P-341 35 59 91 135 133 129 PL-252 12 40 73 80 95 110 PL-41 63 82 74 106 94 102

Table 11: Percent recovery of all apolipoproteins with various sample preparations

All recoveries expressed as a percent of individual peptide’s control sample (100%).

Peptides are displayed as protein name- AA number in whole protein sequence. For example, M-144 is the apoM-derived tryptic peptide with the N-terminal AA at residue

144 in the AA sequence of human apoM. R/A resulted in the highest recovery of apoM, whereas this condition was not suitable for the expanded panel. Some peptides, such as apoC-1 were decreased with R/A. Therefore, the R/A protocol was used when measuring apoM concentration only and the 8 M urea method was used for the measurement of the apolipoproteins in the panel assay, including apoM. This protocol gave the next-highest recovery for apoM while increasing or maintaining the recovery of other apolipoproteins from human serum.

113 Figure 22

Percent Increase of ApoM Peptide Signal With Different Preparations vs. Control 400

300

200 FLLYNR AFLLTPR

100

% of Control, 2ug O/N 0 R/A R/A R/A Urea Urea Urea Control 2ug 0.5ug 1ug 2ug 0.5ug 1ug 2ug Trypsin Conc. O/N 2hr 2hr 2hr O/N O/N O/N Digestion Time

Figure 22: Reduction-alkylation and urea sample preparation methods were evaluated to maximize apoM recovery

Reduction-alkylation (R/A) was the protocol chosen to give the highest recovery of apoM based on the highest MS signal from human apoM-derived tryptic peptides, FLLYNR and AFLLTPR. The MS signals of these peptides in each treatment were expressed as a percent of the control treatment (+/-SD). R/A increased apoM >300% compared to the control preparation. Addition of 8 M urea was also satisfactory to increase apoM recovery >200% and was used as an alternative protocol.

114 Use of a Robotic System during Sample Preparation to Increase the Throughput of the Assay

A robotic system was used to automate some pipetting and mixing steps in the sample preparation protocol. A Biomek FX from Beckman Coulter was used, and a custom robotic program was created using the Biomek software. The robot has two heads used for pipetting: a 96-tip head and an 8-tip head (Span-8). A program was created using the 96-tip head to transfer the internal standard-PBS solution (pre-mixed before using robot) to the Varian filter plates in duplicate. Using new tips the 96-tip head then mixed 10 µL of the serum samples with 140 µL premixed matrix serum and PBS to create a 1:2 dilution of the experimental samples. The matrix serum was mixed with PBS at a 1:6 ratio before placed on the robot. The sample and premix was then transferred to the filter plate containing the internal standard solution, in duplicate. Robotic optimization included ensuring all samples were treated equally during pipetting, mixing, and transferring. Parameters were adjusted to remove excess droplets from the end of the tips and mixing heights were adjusted to thoroughly mix each well. Tips were not reused or pre-wet to prevent contamination and ensure that each liquid transfer was constant. If tips were used twice between plates, the volume of the first transfer would differ slightly from the second transfer, since some liquid would be left in the tips after the first pipetting step.

After the sample-containing matrix was added to the filter plate, the 96-tip head transferred the Liposorb to each well of the filter plates and mixed thoroughly. The filter plates were then removed from the robot for incubation. After the Liposorb washing steps, the robot was again used in the sample prep. For the urea-containing protocol, the

115 96-tip head added 8 M urea to each well of the filter plate. The Span-8 (8-channel) arm was used at this step to resuspend the Liposorb in the urea. For the R/A protocol, 100 mM ABC was added instead. The same robot method was used for both protocols. The

Span-8 was optimized to move in a circular fashion within each well to fully resuspend the Liposorb pellet. As the Span-8 pipetting the solution up and down within each well, the arm was moving. The arm moved back and forth, and then moved at a fixed degree to the next spot in the well as close to a circular shape as was possible. The tips of the

Span-8 arm are fixed so they were set to actually scrape the bottom of the filter plate to disturb the pellet. The tips were washed with water between each well to prevent contamination. The strength of the filters was tested by scraping with a pipet tip, and it was found that they were sturdy enough to handle the scraping by the Span-8 tips without puncture and leaking of the samples. The loss of sample was also prevented at all steps on the robot because the bottom cap mat and holder plate were attached at all times. The remaining steps of the sample prep were completed manually; with remaining reagent additions were completed using a MultiDrop dispenser (Thermo).

Measurement of ApoM in Human, Rat, and Mouse Serum using Optimized Assay

The measurement of apoM in human, rat, and mouse serum was made using the optimized assay to obtain an average concentration of apoM in serum from each species to use as a reference point for apoM measured in experimental samples. Aliquots of 10

µL of human, rat, and mouse sera were prepared using the optimized R/A protocol.

Serial dilutions of apoM synthetic peptides AFLLTPR, AFLVTPR, and FLLYNR were used as a calibration standard to measure the absolute concentration of apoM in these

116 serum samples. The synthetic peptides were each diluted to the same molar concentration (60 µM) and 2-fold dilutions were made into 0.1% NP40 to mimic the final solution of digested serum samples. Synthetic AFLLTPR and FLLYNR peptides were previously used to measure apoM concentration in the human serum used to prepare the human calibration standards. The measured concentration of apoM in this human serum was 401 nM (10.4 µg/mL +/-1.2 SD).

In this experiment, human apoM concentration was measured in purchased human serum at 272 nM (7.1 µg/mL +/- 0.70 SD). ApoM was measured in purchased rat serum at 35 nM (0.9 µg/mL +/-0.11 SD) and in-house lean Long-Evans at 113 nM (2.9 µg/mL

+/- 0.33 SD). Differences in endogenous apoM concentration between two rat serum sources demonstrated the importance of using experiment-specific controls originating from the same rodent strain as the serum in the experimental samples. Mouse apoM concentration measured in serum was 118 nM (3.1 µg/mL +/- 0.01 SD). Synthetic peptide chromatographic AUCs were compared between equimolar concentrations of the synthetic peptides AFLLTPR, AFLVTPR, and AFLVTPR (Figure 23) to ensure the same concentration of each peptide was in agreement with the signal intensity levels of the other peptides. Comparable AFLVTPR, AFLLTPR, and FLLYNR peptide MS signal intensities at the same molar concentration confirmed the concentration measurement between peptides was in agreement and so AFLVTPR or AFLLTPR can be used for quantification interchangeably with FLLYNR within a specific species.

Aliquots of human, rat, or mouse serum used in this experiment to measure apoM were included in every apoM experiment as a control for changes in apoM concentration between experiments. The concentration of apoM was measured in the control samples

117 in each experiment and the concentration of apoM in the experimental samples was expressed as a percent of the control serum to directly compare this percentage to the apoM concentration expressed as a percentage of the control in previous experiments.

118 Figure 23

RT: 3.00 - 5.00 RT: 4.00 - 6.00 RT: 3.50 - 5.50 RT: 3.91 NL: 3.03E5 RT: 5.31 NL: 2.00E5 RT: 4.45 NL: 1.76E5 AA: 1107037 TIC F: ITMS + c ESI SRM AA: 1200993 TIC F: ITMS + c ESI SRM AA: 712692 TIC F: ITMS + c ESI SRM 100 100 100 ms2 [email protected] [ ms2 [email protected] [ ms2 [email protected] [ 372.22-375.22, 372.22-375.22, 451.23-454.23, 90 471.29-474.29, 90 485.30-488.30, 90 564.31-567.31, 584.37-587.37] MS ICIS 598.39-601.39] MS ICIS 677.39-680.39] MS ICIS 80 Syn AFLVTPR 80 Syn AFLLTPR 80 Syn FLLYNR

70 70 70

60 AFLVTPR60 AFLLTPR60 FLLYNR

50 50 50

40 40 40 Relative Abundance Relative Abundance Relative Abundance 30 30 30

20 20 20

10 10 10

0 0 0 3 4 5 4 5 6 4 5 Time (min) Time (min) Time (min)

Figure 23: Equimolar concentrations of apoM synthetic peptides

Equimolar concentrations of synthetic apoM peptides were measured to compare peptide

MS signal intensities. All three peptides produced similar MS signal intensities, with

AFLVTPR at 3.03E5, AFLLTPR at 2.00E5, and FLLYNR 1.76E5. Variations in peptide

MS signals may be due to a difference in behavior of the peptide (i.e. peak width) through the HPLC column and instrument analysis, so it is important to have a synthetic peptide for each tryptic peptide measured in serum.

119 15 N-Labeled Human Apolipoprotein A-IV as Internal Standard for Normalization

An intact heavy isotope-labeled apolipoprotein was used as an internal standard to normalize variations in apoM recovery between samples that can occur during sample preparation (i.e. trypsin digestion) and instrument analysis. The standard apolipoprotein was spiked into each experimental and calibration sample at the beginning of sample preparation and was exposed to the same preparation procedure and instrument analysis as the proteins in serum.

An experiment was performed by Dr. Bomie Han to assess the effectiveness of

Liposorb capture of 15 N15-Apo AI-V. 15 N-labeled human apolipoprotein A-IV was incubated with Liposorb and then spun down to create a Liposorb pellet. The supernatant was separated from the Liposorb and the Liposorb pellet and supernatant were digested with trypsin prior to MS analysis. The MS peptide signals from two different peptides derived from the standard protein were measured in the Liposorb and supernatant fractions.

The MS peptide signals from two 15 N-Apo A-IV-derived tryptic peptides were strong in the Liposorb fraction and weak in the supernatant fraction (Figure 24). Based on the ratio of the peptide MS signal from the Liposorb pellet to the MS signal from the supernatant, more than 95% of the standard protein was recovered in the Liposorb pellet, so the internal standard protein was used to represent the capturing efficiency of Liposorb in every sample.

In order to accurately represent the capturing efficiency of Liposorb for apolipoproteins from each serum sample, 15 N-labeled Apo A-IV was added to each sample at a constant concentration (250 ng) prior to Liposorb addition and digestion with

120 trypsin. The 15 N-Apo A-IV-derived tryptic peptide (LEPYADQLR) was used to normalize the apoM-derived peptides in each sample. Possible variations that can occur during sample prep or the LC-MS analysis will be reflected in the recovery of the internal standard and used to correct the corresponding variations in apoM-derived tryptic peptide recovery.

121 Figure 24

RT: 0.00 - 15.00 NL: 3.00E5 RT: 10.56 m/z= 403.46-407.46 F: 100 AA: 2836401 ITMS + c ESI Full ms2 80 Liposorb [email protected] [ 200.00-1500.00] MS 60 ICIS BH060120B-Apo-03 40 20 0 NL: 3.00E4 100 m/z= 403.46-407.46 F: ITMS + c ESI Full ms2 80 Supernatant [email protected] [ 200.00-1500.00] MS 60 ICIS BH060120B-Apo-09 40 RT: 10.60 AA: 73754 20

Relative Relative Abundance 0 NL: 4.50E4 RT: 4.95 m/z= 544.57-548.57 F: 100 AA: 1136945 ITMS + c ESI Full ms2 80 Liposorb [email protected] [ 185.00-1500.00] MS 60 ICIS BH060120B-Apo-03 40 20 RT: 9.89 AA: 16186 0 NL: 4.50E3 m/z= 544.57-548.57 F: 100 ITMS + c ESI Full ms2 80 Supernatant [email protected] [ 185.00-1500.00] MS 60 ICIS BH060120B-Apo-09 40 RT: 5.06 AA: 22355 20 0 0 2 4 6 8 10 12 14 Time (min) *Data and Figure were produced by Dr. Bomie Han

Figure 24: Extraction Efficiency of 15 N-Apo A-IV by Liposorb

Liposorb capture of 15 N-labeled Apo A-IV recovered more than 95% of the initial standard protein amount. Less than 5% of the standard protein remained in the supernatant fraction so the internal standard could be used to accurately represent the capturing efficiency of Liposorb in every experimental and calibration standard.

122 Statistical Validation of the Assay by Repeated Spike-Recovery Measurements

Statistical analysis to validate the accuracy and reproducibility of apoM quantification using the MRM assay was performed on MS data that was generated from a series of spike-recovery experiments. Statistical analyses were performed on data generated from triplicates within the same experiment and repeated preparations of the same experiment at three different times.

Three sets of human calibration standards in rabbit serum matrix (Cal-G and Val-

H) were prepared for MS analysis using the urea protocol and three more sets were prepared using the R/A protocol (Experimental Section). To quantify apoM in these samples, one set of calibration samples (Val-H) was treated as a set of experimental samples and the other set of calibration samples (Cal-G) was used as the calibration standards to measure the concentration of apoM in the Val-H samples, and vice versa.

The MS peptide signal from 15 N-Apo A-IV-derived tryptic peptide LEPYADQLR was used to normalize the intensity of the MS peptide signal from apoM-derived tryptic peptide FLLYNR prior to quantification of apoM. The MS peptide signal from FLLYNR in the calibration standards was compared to the MS peptide signal from FLLYNR in the experimental samples to quantify apoM.

The coefficient of variation (CV) and percentage of relative error (RE) between the known concentration of apoM in the calibration standards and the measured concentration of apoM in the calibration samples that were treated as experimental samples was calculated with and without the normalization of the MS peptide signal from

FLLYNR using the MS signal from LEPYADQLR prior to quantification of apoM. The

% CV and % RE between the measured and known concentrations of apoM were

123 calculated within each experiment and across three separate experiments. The acceptance criteria used to define the validated working range of the assay were: (CV) <20%, (RE)

<20% and total error (% CV + % RE) <30%. The validated working range of the assay was defined as the range of consecutive calibration samples that meet all of these criteria, or the lowest quantification limit (LQL) to the highest quantification limit (HQL).

The validated working ranges of the apoM assay were 0.29-13.0 µg/mL (11.2-500 nM) in the urea protocol and 0.23-13.0 µg/mL (8.8-500 nM) in the R/A protocol (Figure

25). In the statistical evaluation of normalized MS signals and raw MS signals, the overall variations between triplicate measurements across the entire apoM concentration range of the calibration samples were lower in the normalized group, especially at the low and high concentrations of apoM (Figure 26). Therefore, the necessity of this internal standard for apoM-derived tryptic peptide normalization was validated and used in every experimental and calibration sample to normalize tryptic peptide recovery across the entire experiment. The use of the internal standard also increased the concentration span of the validated the working range of this assay.

124 Figure 25

3-day spike-recovery of hApoM in rabbit serum: R/A protocol 3-day spike-recovery of hApoM in rabbit serum: urea protocol

80 100 tot Error tot Error %RE 60 80 %RE %CV inter %CV inter %CV intra 40 60 %CV intra |%RE| |%RE| 40 20 20 0 % Error % Error % 0 -20 -20

-40 -40

-60 -60 0.1 0.2 0.4 0.6 0.9 1.4 2.2 3.4 5.3 8.3 0.1 0.2 0.4 0.6 0.9 1.4 2.2 3.4 5.3 8.3 13.0 13.0

ApoM conc, ug/ml ApoM conc, ug/ml

Figure 25: Results from three day spike recovery experiments for each sample preparation protocol: urea or R/A

Statistical validation of the accuracy and reproducibility of apoM quantification using the

MRM assay was performed on MS data that was generated from a series of spike- recovery experiments. Statistical analyses were performed on data generated from triplicates within the same experiment and repeated preparations of the same experiment at three different times. The urea protocol has a validated working range of 0.29-13.0

µg/mL (11.2-500 nM) and R/A has a validated working range of 0.23-13.0 µg/mL (8.8-

500 nM).

125 Figure 26

Variation in Calibration Standards With and Without N15- 15 N-ApoApoA4 A-IV Normalization Normalization 40 30 20 10 0 -10 -20 Coefficient of Variation, % Variation, of Coefficient

.1 .2 .7 .1 .7 .7 0.4 6.3 0 0 0.3 0.5 0 1 1 2.7 4.3 6 1 1 ApoM Concentration, ug/mlµg/mL WithoutWithout 15N15-A4N-AIV WithWith N15-A415 N-AIV

Figure 26: Variability in measured apoM concentration with and without the use of

15 N-Apo A-IV for normalization

The use of internal protein standard 15 N-Apo A-IV in each sample and calibration standard minimized variability between samples due to sample preparation, instrument conditions, or other interferences. A standard protein was used to undergo the same sample preparation (Liposorb binding, trypsin digestion, etc.) and instrument analysis as all experimental and calibration samples to accurately capture the percent recovery of apoM from serum. The human apoM standard curve was prepared and analyzed in triplicates. Coefficient of variation (% CV) was calculated between triplicate measurements, with and without internal standard normalization. The calibration curve was more variable and outside acceptance range without use of the internal standard. The internal standard kept the variation low and the calibration curve had a wider working range.

126 Orthogonal Validation of the MS Assay

Human serum samples measured with the MS apoM assay were compared with the results of a more conventional method, Western blot to further validate the measurement of apoM concentration using this MS assay. Aliquots of the same human serum samples were prepared for MS and Western blot analysis.

Human apoM was initially measured in these serum samples using the MS assay at 4.6, 5.5, 5.5, 5.6, 9.4, 9.4, 9.4, 9.5, 19.5, 16.1, 15.7, and 15.6 µg/mL. The same samples were prepared and analyzed by Western blot. A visual increase in the apoM band intensities was observed in the Western blot that was complementary to the concentration increases measured by MS (Figure 27). Well 15 (9*) contained the negative control to validate the apoM band intensities of the experimental samples.

Primary anti-apoM antibody (Ab) and recombinant apoM binding was intended to prevent the primary antibody from binding apoM in the human serum sample. There was a faint band still present, but the intensity was very weak compared to the sample without recombinant apoM competition (well 9). Although the Western blot is not highly quantitative and the concentration variations of apoM in these samples may not result in a clear difference in band intensities, it was used to provide a visual comparison of the results of the MS assay with a more conventional proteomic method and demonstrates the value of the MS assay for absolute quantification of apoM.

127 Figure 27

Concentration of ApoM in Human Serum by LC-MS/MS

30 19.5 25 15.7 16.1 15.6 20

15 ApoM 9.4 9.4 9.4 9.5 10 4.9 5.5 5.5 5.6 5 ApoM Concentration, ug/ml Concentration, ApoM

0 1 2 3 4 5 6 7 8 9 1011 12 Sample Number

M W 1 2 3 4 5 6 7 8 9 10 11 12 M W 9 *

6 2 4 9 3 8 2 8 1 7 1 4

Figure 27: Orthogonal validation of the MS assay with Western blot analysis of the apoM in human serum

ApoM levels in human serum were measured by MS and Western blot. ApoM concentration (+/-SD) measured by MS, ranges from 4.9-5.6 µg/mL (low), 9.4-9.5 µg/mL

(medium), and 15.6-19.5 µg/mL (high). The increase in apoM from the MS data (top graph) was visualized as increasing band intensities in the Western blot. The far-right well (9*) of the Western blot was the negative control for the primary antibody.

128 Determination of the Lowest Detection Limit (LDL) of ApoM by MS

The lower detection limit (LDL) of this assay was evaluated to determine the sensitivity limit for apoM identification in the MS assay. The LDL was determined by manual analysis of each MS chromatogram for the presence of FLLYNR peptide. The human serum calibration standards from the validation experiment were used to determine the LDL. The negative control was digested rabbit serum only and did not contain human serum. The rabbit AA sequence differs from human apoM and does not contain peptide FLLYNR. The samples were analyzed using both apoM prep methods

(urea and R/A) as part of the validation study. Samples were blinded and randomized, then manually evaluated for the presence of FLLYNR. This was repeated three times for each protocol. The criteria used to distinguish the peak were the correct retention time of the chromatographic peak and the presence of all three transitions to clearly identify the peptide.

The lowest limit of detection was determined to be 0.15 µg/mL (5.8 nM) for both the R/A and urea methods (Figure 28). Two of three trials in evaluation of R/A data resulted in this conclusion, producing 66% precision in the LDL determination. A lower detection limit (0.12 µg/mL) was determined in one trial, so the higher of the two detection limits (and chosen in two-of-three trials) was determined to be the LDL when using the R/A prep method. The urea method LDL was determined with 100% precision.

The negative control was determined to be without human serum in 3-of-3 trials, too.

The lowest detection limit of this assay is only slightly lower than the LQL, indicating that this assay can accurately measure apoM at near-lowest detection limit concentrations.

129 Figure 28

3.17 100 565.37 100 90 LDL 90 80 0.15 µg/mL 80 UREA 70 70 60 60 50 50 40 3.22 40

Relative Abundance 30 452.25 566.41 Relative Abundance 30

20 3.89 20 3.25 2.58 678.56 10 3.94 4.29 453.43 2.99 3.78 10 4.74 5.02 5.53 567.29 679.43 680.20 0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 452 453 454 565 566 567 678 679 680 Time (min) m/z m/z m/z

RT: 0.00 - 6.00 2 #628-675 RT: 4.53-4.68 AV: 16 NL: 2.89 3.19 F: ITMS + c ESI SRM ms2 [email protected] [ 451.23-454.23, 564.31-567.31, 677.39-680.39] 100 678.83 100 90 Negative 90 80 Control 80 70 70 565.41 60 60 680.14 50 50 40 40

Relative Abundance 30 3.92 Relative Abundance 30 20 3.95 20 2.58 10 3.29 10 4.29 5.29 4.32 5.63 564.85566.24 566.98 0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 452 453 454 565 566 567 678 679 680 Time (min) m/z m/z m/z

Figure 28: Lowest detection limit (LDL) of apoM in the MS assay

The lowest detection limit (LDL) of this assay was the same in urea and R/A sample preparation methods. Shown is the LDL of each preparation method compared to the negative control (no FLLYNR peptide). The negative control was digested rabbit serum which has a different AA sequence than human apoM. Serial dilutions were used from the validation data set. The MS data was blinded and evaluated in triplicates to determine the LDL for each prep method. Both methods have an LDL of 0.15 µg/mL (5.8 nM) apoM concentration.

130 Stability of ApoM in Human Serum during Multiple Freeze-Thaw Cycles

Stability of native apoM in human serum was evaluated using different serum storage conditions and repeated freeze-thaw cycles. Aliquots of the same human serum were stored under different conditions and compared to control storage conditions (frozen at -80°C until first use). Overnight storage of human serum at 4°C and room temperature did not significantly affect the recovery of apoM in human serum, indicating that apoM is stable under these conditions. Repeated freeze-thaw cycles at were tested at two freezing speeds. A -80°C freezer was used to slowly freeze the serum and dry ice was used for a quick freeze. All samples were thawed in a 30°C water bath then placed on ice until sample prep. Several slow freeze-thaw (FT) cycles slightly decreased apoM recovery.

After six slow FT cycles, the percent decrease was less than 20% from the control, but the means were statistically different from control conditions (p<0.01). Serum samples frozen using quick freezing were not different than the control (Figure 29). Thus, this is the method of choice when re-freezing used human serum samples. Slow freezing should not be used if possible since a slight significant decrease was measured compared to the control.

131 Figure 29

ApoM Stability in Serum After Different Storage and Freeze- Thaw Conditions 140 ** ** 120 *

100

80

60 ApoM

40 Percent of Control, % Control, Percent of

20

0 Control 4C O/N RT O/N slow slow slow quick quick quick FT 2x FT 4x FT 6x FT 2x FT 4x FT 6x

Figure 29: Stability of apoM in human serum under different storage conditions

Measured apoM concentrations (+/-SD) are shown here as a percentage of control storage conditions (frozen at -80°C until initial thaw and use).

132 Distribution of ApoM Concentration in Human Population

ApoM concentrations were measured in a sample population of human serum

(n=105) to evaluate variation among the human population. Human serum was drawn in a clinical study prior to drug treatment, so this measurement represents apoM concentration in normal human serum. This population consisted of 47.5% males with a mean age of 54.1 years (+/- 11.1 yr). Participants had no known diabetes or clinical diagnosis of coronary heart disease, providing a good representation of an average adult population. The average concentration of apoM across these samples was 370 nM (9.6

µg/mL +/-2.4 SD), resulting in an average human apoM concentration range of 280-460 nM (7.4-12.0 µg/mL). The lowest measured concentration was 6.0 µg/mL and the highest was 19.5 µg/mL (Figure 30). The lowest measurement was 64% of the average concentration, whereas the highest concentration was almost double (+179%). An overall three-fold variation in human apoM concentrations was present across the normal population.

133 Figure 30

Variation in Human Serum ApoM Concentrations Across Sample Population (n=105) 30

25

20 ApoM 15

10

ApoM Conc, ug/ml 5

0 0 20 40 60 80 100 120 Sample Number

ApoM Concentration Across Human Serum Samples

Average Average +/- SD Minimum Maximum Median

(nM) (µg/mL) (µg/mL) (µg/mL) (µg/mL)

370 9.6 +/- 2.4 6.0 19.5 9.4

Figure 30: ApoM concentration measured in a human sample population

Variation in human apoM concentration in serum (n=105) was measured using this assay.

Human apoM concentration ranged from 6.0 µg/mL to 19.5 µg/mL, averaged at 9.6

µg/mL (+/-2.4) or 370 nM. Median apoM measurement was 9.4 µg/mL. ApoM measurements are shown with +/-SD error bars.

134 APPLICATION OF MOUSE APOM ASSAY TO MEASURE APOM IN

PRE-CLINICAL STUDY

Microsomal triglyceride transfer protein (MTTP) is present in the endoplasmic reticulum (ER) and enhances the transfer of triglycerides, phospholipids, and cholesterol esters between vesicles [28]. MTTP is necessary for the assembly and secretion of apoB- containing lipoproteins, specifically very low density lipoprotein (VLDL) particles [29].

ApoB is a structural apolipoprotein bound to the surface of VLDL [29]. MTTP-apoB binding during translation and translocation of apoB prevents its degradation [28].

VLDL assembly occurs in two steps [29].

The first step of VLDL assembly is facilitated by MTTP in the rough endoplasmic reticulum, where triglycerides form a complex with apoB by MTTP lipid transfer activity

[1, 28]. In this step, a VLDL lipid droplet is formed without the presence of apoB [30].

The pre-VLDL and lipid droplet fuse prior to leaving the ER. The second step in VLDL assembly does not require MTTP. The pre-VLDL complex is transported out of the ER and stored triglycerides are added form the mature VLDL particle [29].

In the absence of MTTP, apoB particles are tagged for proteasomal degradation

[30], and individuals lacking MTTP also lack apoB-containing lipoproteins in circulation

(abeta-lipoproteinemia) [29]. The addition of MTTP inhibitors to liver cells has been shown to prevent the assembly of VLDL particles [30].

ApoE also has an important role in regulation of VLDL assembly and secretion.

It has been shown that in the absence of apoE, smaller and less TG-rich VLDL particles were formed [29]. Increases in hepatic VLDL production are associated with an increase

135 in atherogenic lipoproteins in circulation, specifically apoB and apoE [29]. Thus, the

VLDL assembly and secretion pathway serves as a drug target in an effort to lower the risk of coronary heart disease (CHD) and other cardiac events. Several drugs to lower

LDL levels in circulation are currently marketed; however an alternative approach to lowering LDL is lowering the production of its precursor, VLDL. One way to accomplish this is to inhibit MTTP to prevent the assembly and secretion of VLDL, however hepatic triglyceride concentrations can increase [1]. Circulation levels of apoB, apoE, apoAI, and apoM in mice treated with different doses of an MTTP inhibitor were measured in the mouse serum to evaluate relative changes in the apolipoproteins in response to MTTP inhibition. The secretion of apoB was hypothesized to decrease with these treatments, but was decrease in HDL-associated lipoproteins apoAI and apoM was not certain. MTTP inhibitors should inhibit the assembly of VLDL and HDL and its associated apolipoproteins may not be directly involved in VLDL assembly.

Pre-Clinical Study Design

Mice were treated with different doses of an MTTP-inhibitor at 1, 3, 10, and 30 mpk, or a control vehicle solution.

Previously Measured Effects of MTTP Inhibition on Lipoprotein Levels

Changes in the levels of lipoproteins from mice treated with the control vehicle or the MTTP inhibitor were previously separated by fast performance liquid chromatography (FPLC) and the fractions were measured for cholesterol content to

136 determine the amount of HDL, LDL, VLDL, and total cholesterol. Lipoprotein particle concentrations were converted to percent of the control vehicle.

As the concentration of the MTTP-inhibitor was increased, measured lipoprotein levels in serum were decreased (Figure 31A). The most dramatic decrease was observed in VLDL, then LDL, total cholesterol, and HDL. These results are consistent with the expected results of MTTP-inhibition to prevent the intracellular assembly of VLDL.

VLDL in circulation was decreased ~95% with the lowest dose (1 mpk) and had non- detectable levels at the remaining concentrations of MTTP-inhibitor. As a result of a dramatic decrease in its precursor particle, LDL was decreased by ~80% at the lowest dose to below detection levels at 10 and 30 mpk treatments. Total cholesterol was decreased by ~70% with the lowest dose to ~95% decrease at the highest dose. HDL levels were decreased by ~35% with the lowest dose and dose-dependently thereafter by

~80% lower than control at the highest dose (30 mpk).

Effects of MTTP Inhibition on Apolipoproteins

The changes in apolipoproteins apoB, apoE, apoAI, and apoM were measured using a modified method of the targeted assay for apoM to include the measurement of addition tryptic peptides from these apolipoproteins, with adjustments to the HPLC gradient and MS method made accordingly. The apolipoproteins were measured as part of the panel assay, briefly described in assay optimization. Apolipoproteins B, E, AI, and

M were measured using the modified MRM assay and concentrations of each were reported as a percent of control vehicle solution concentration.

137 ApoB showed the most dramatic decrease in circulation levels by ~80% decrease with the lowest dose of MTTP-inhibitor, then ~95% decrease at the second dose and non- detectable at higher doses (Figure 31B). The decreases in apoB were consistent with the decreases in LDL and the same dose-dependent response was observed in apoE levels, to the same extent as apoB.

ApoAI decreased to ~60% of the control with the lowest dose and the percent decrease was the same in HDL particles. ApoAI levels at each treatment were closely correlated with HDL levels and apoM levels also followed a decreasing trend, but at a slower rate than apoAI and HDL particles. The decreases observed in HDL and HDL- associated apolipoproteins AI and M may be due to elevated levels of hepatic triglyceride as a result of MTTP-inhibited assembly and secretion of triglycerides in VLDL particles, decreasing the function of HDL particles to return cholesterol to the liver from the periphery.

138 Figure 31

A.

Effect of MTTP-I on Lipoproteins measured by FPLC/Cholesterol

120

100

80 VLDL LDL 60 HDL 40 total Conc, % Vehicle % Conc, 20

0 Veh 1 mpk 3 mpk 10 mpk 30 mpk

B.

Mouse Apolipoproteins with MTP-Inhibitor

120.0

100.0

80.0 ApoA1 ApoB 60.0 ApoE ApoM 40.0 Conc, % Vehicle group

20.0

0.0 Veh 1 mpk 3 mpk 10 mpk 30 mpk

139 Figure 31: The effect an MTTP inhibitor at different doses on the concentration of lipoprotein particles and apolipoprotein levels

The addition of MTTP-inhibitor decreased all lipoproteins (A) and measured apolipoproteins: apoB, E, AI, and M (B). MTTP inhibitors prevent the assembly and secretion of apoB-containing VLDL particles. A decrease in VLDL secretion lowered

HDL proteins apoAI and apoM, possibly due to an increase in TG in the liver.

140 APPLICATION OF MS ASSAY TO MEASURE HUMAN APOM FROM A

CLINICAL STUDY

Human apoM plays an important role in lipid transfer and cholesterol efflux from peripheral tissues. ApoM has been largely implicated as a necessary component to pre β-

HDL formation, is found mainly associated with HDL particles [2] and to a lesser extent chlyomicrons, VLDL, and LDL particles [5]. Lowering LDL and total cholesterol levels is a major goal of cardiovascular or cholesterol-lowering drug therapies including statins, bile-acid sequestrating agents, squalene synthase inhibitors, and ezetimibe, all of which lower serum cholesterol levels [1]. Statins are fungal metabolites that inhibit 3-Hydroxy-

3-methyl-glutaryl-CoA (HMG-CoA) reductase, the enzyme that catalyzes the rate- limiting step in cholesterol biosynthesis: the conversion of HMG-CoA to mevalonic acid

[31]. Normally, cholesterol obtained from the diet enters the liver and suppresses HMG-

CoA reductase [32]. Statins are targeted to the liver to inhibit this enzyme, reducing the cholesterol produced by the body and thus lowering hepatic cholesterol levels. Low hepatic LDL levels stimulate increased hepatic LDL receptor (LDLR) expression, which in turn increases hepatic uptake of circulating LDL, lowering plasma levels [1]. LDL cholesterol taken up by LDLR can be cleared through the liver and degraded. Lowering

LDL cholesterol from circulation contributes to the anti-atherogenic properties of statins

[32]. The enzyme cholesterol ester transfer protein (CETP) aids in cholesterol transfer from HDL to VLDL, eventually becoming LDL particles. In this way the cholesterol is returned to the liver from the tissues when LDL is taken up by hepatic LDL receptors [1].

141 HDL also transports cholesterol from the tissues to the liver directly during recirculation of HDL where cholesterol can be unloaded into the liver cells [33].

Stains have strong anti-oxidative properties, helping prevent oxidation of circulating LDL and foam cell formation that leads to atherosclerosis [34]. Theoretically, statins may also induce a slight increase in HDL particle uptake by the liver, lowering circulating levels of HDL [1]. However, the data surrounding this theory is conflicting.

This is not desirable since HDL particles have anti-atherogenic properties as discussed.

However, due to other mechanisms, no change or a slight increase in HDL levels has typically been observed after statin treatment [32]. With the main focus of these treatments on decreasing LDL plasma levels, changes in HDL in response to statin treatments have not been thoroughly studied. Measurement of the apoM-containing sub population of HDL in statin-treated samples gives deeper insight into the specifics of

HDL changes and may help to better understand the underlying mechanisms. Increases in apoM-containing HDL may add additional benefit to treatments with HDL increase or

LDL decrease, or both.

Another major therapeutic target in lipoprotein regulation is nuclear receptors, specifically peroxisome proliferator-activated receptors (PPARs) [35]. These ligand- activated transcription factors have been implicated in lipoprotein metabolism regulation.

PPARs mediate the effect of dietary fatty acids on lipoproteins in the plasma [36]. The three PPAR isotypes, α, β/δ, and γ, have different tissue expression sites and activators

[37]. PPAR-α was a drug target in this study.

PPAR α is expressed in liver, heart, and small intestine and regulates different genes involved in cellular lipid and glucose metabolism. Fibrates (fibric acid

142 derivatives), which have long been used to treat lipid disorders [23], act as a ligand for

PPARs. PPAR-α activation has been shown to significantly lower human plasma triglyceride (TG) levels and slightly increase HDL levels [23, 36]. In mice, PPAR-α activation via fibrates has been shown to lower hepatic TG levels, which lowers hepatic

VLDL production and secretion. The same experiments in humans resulted in conflicting and inconclusive data. However, fibrates increase HDL-C plasma levels approximately

10%, equivalent to a 25% reduction in the risk of coronary complications [36]. Fibrates have been shown to increase plasma and hepatic levels of the major protein component of

HDL, apolipoprotein AI (apoAI). Apolipoprotein AII (apoAII), an apolipoprotein present in <50% of HDL particles, may also increase with PPAR-α activation, since its levels have been strongly correlated to circulating HDL levels [38]. The positive or negative influences of apoAII levels remain a source of ambiguity. ApoAII was recently inversely associated with CHD risk [38], but has also been positively associated with high VLDL triglyceride levels in plasma [39], the latter of which was not observed in this study.

The coupling of a PPAR-α agonist with statin treatment may aid to increase plasma HDL and further lower LDL concentrations [23]. Apolipoprotein B (apoB), the main apolipoprotein component of LDL particles and triglycerides, decreases significantly with statin treatment. This same statin treatment also resulted in a slight increase in plasma HDL levels [31]. Measurement of human apoM concentration in patients undergoing these drug treatments increases the understanding of the biology and significance of apoM in human cholesterol metabolism and its association with drug- induced changes in HDL. The behavior of apoM in response to statin or PPAR-α agonist

143 treatment was unknown prior to this evaluation. Since apoM is necessary for the formation of lipid-poor pre β-HDL [2], a change in plasma apoM concentration may be a strong indication of change in capacity to remove cholesterol from tissues. Typically,

PPAR-α agonists increase HDL concentrations in plasma ~10-11% [36]. HDL concentrations reflect plasma levels of total HDL particles in circulation, but this assay can measure its apoM-containing sub population. This assay was used to gain a deeper understanding of these drug actions on cholesterol, through the measurement of apoM.

Drug-induced increase in plasma HDL levels may be due to a much larger increase in the small apoM-containing HDL population. This hypothesis was explored with the measurement of apoM in clinical human serum samples using this assay. ApoM response to LY518674 (PPAR-α agonist) was hypothesized to follow the increase in plasma HDL levels (12-15% in this study [23]) at a much larger percent increase.

Additional benefits or drawbacks to these treatments may be revealed by this deeper evaluation. ApoM concentration has been previously correlated to HDL levels [14] and was hypothesized to have a strong correlation with HDL in this study. Weak or no correlation with LDL was expected in this study due to apoM’s small association with

LDL [10].

Human Clinical Study Design

A statin drug and PPAR-α agonist were previously administered individually and in combination to measure changes in human lipid and lipoprotein profiles [23]. These human serum samples were used in this experiment to measure human apoM. The statin used in this study was atorvastatin, a hydrophobic [32] and purely synthetic statin [1].

144 Low doses of atorvastatin (10 mg) have been shown in several studies to have a significant effect on lowering total cholesterol and LDL cholesterol levels (-40%) in human serum [31, 40, 41]. The PPAR-α agonist used in this study, LY518674, was administered at 10 µg and 50 µg doses alone and in combination with two different doses of atorvastatin (10 mg and 40 mg).

The study participants were grouped into four categories: placebo, atorvastatin,

LY518674, and atorvastatin plus LY518674. This human clinical study was designed and executed with results were published by Nissen et al [23] prior to our involvement and measurement of apoM. Their initial sample size was n=304 [23], with a subset of this sample size available for apoM measurement (n=105). Participants were initially placed in three groups: placebo, 10 mg atorvastatin, or 40 mg atorvastatin for four weeks.

Then groups were divided further into placebo, 10 µg LY, 50 µg LY, 10 mg atorvastatin,

40 mg atorvastatin, and AT/LY combinations of 10 mg/10 µg, 10 mg/50 µg, 40 mg/10

µg, and 40 mg/50 µg (Figure 32). In these groups, participants received placebo, 10 µg

LY, or 50 µg LY for twelve weeks to generate the different combinations listed above

[23, 42].

Serum was drawn before treatment (baseline), after four weeks (1 st measurement), and 16 weeks (2 nd measurement) from the start of the study. Triglycerides, LDL cholesterol, HDL cholesterol, and total cholesterol, apoB, apoAI, and apoAII were measured in mg/dL previously. Human apolipoprotein M was measured using the described MS-based assay. The results of other lipid measurements are reported here to compare with changes in apoM concentration.

145 ApoM measurements were normalized per patient as percent of baseline. ApoM percent difference and statistical significance were calculated from baseline to directly compare all treatment groups. The mean of each treatment group was also compared to placebo using analysis of variance (ANOVA) with significance at p<0.05.

Figure 32

1st Measurement: Pre-Study

10mg 40mg Placebo AT AT

2nd Measurement: 4 Weeks

Placebo 10µg LY 50µg LY Placebo 10µg LY 50µg LY Placebo 10µg LY 50µg LY

3rd Measurement: 16 Weeks

Figure 32: Human clinical study design

Nissen et al [23] study design for human clinical study of statin drug atorvastatin (AT) and PPAR-α agonist LY518674 (LY). Participants were initially divided into three groups: placebo, 10 mg atorvastatin, and 40 mg atorvastatin for four weeks. Study participants were then given either 10 µg or 50 µg LY518674 alone or placebo for 12 weeks. Serum was drawn at baseline (pre-study), four weeks, and sixteen weeks from start date. Samples were analyzed for HDL, LDL, triglycerides, and total cholesterol previously. Human apoM was measured using this MS-based assay.

146 Previously Reported Drug-Induced Effects on Lipid and Lipoprotein Levels

Triglycerides (TG), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and total cholesterol (TC), apoB100, apoAI, and apoAII were measured in mg/dL previously

[23] and reported here. Changes were measured from baseline to end of treatment and from four weeks to end of treatment. Statistical significance was set at p<0.05 compared with placebo group. No statistically significant changes (NS) were observed in the placebo group for all lipid measurements.

Measured from baseline to end of treatment, LY518674, a PPARα-agonist given at 10 µg and 50 µg doses, decreased triglycerides by 36.9% and 37.5% (p<0.001). LDL-

C levels decreased by 13.2% (p<0.05) and 15.8% (p<0.01), apoB100 by 13.9% and

14.9% (p<0.001), respectively. Total cholesterol levels decreased 11.6% (p<0.01) with

10 µg LY and 14.6% (p<0.001) with 50 µg LY. HDL-C levels increased by 15.0% (NS) and 12.5% (NS), apoAI by 6.7% (NS) and 12.4% (p<0.05), and apoAII by 12.7%

(p<0.01) and 33.7% (p<0.001), respectively.

10 mg atorvastatin treatment did not induce a statistically significant change in triglycerides (-18.5%), HDL-C (2.0%), apoAI (0.3%), and apoAII (-6.3%). Statistically significant decreases were measured in TC by 29.3%, LDL-C by 40.7%, and apoB100 by

33.7% (p<0.001). 40 mg atorvastatin treatment did not influence HDL-C levels (3.4%), apoAI (-0.2%), and apoAII (-4.2%), but significantly decreased triglycerides by 23.4%

(p<0.05), total cholesterol by 33.5% (p<0.001), LDL-C by 45.5% (p<0.001), and apoB100 by 37.6% (p<0.001).

When 10 µg or 50 µg LY518674 was given in combination with 10 mg atorvastatin (statin), triglycerides decreased significantly by 39.4% and 55.4% (p<0.001),

147 LDL-C by 36.6% and 41.1% (p<0.001), apoB100 by 31.3% and 38.1% (p<0.001), and total cholesterol by 27.8% and 32.3% (p<0.001). HDL-C increased by 12.6% (NS) and

17.6% (p<0.05), and apoAII by 11.9% (p<0.01) and 35.4% (p<0.001). ApoAI did not change significantly (6.5% and 8.9%) with LY518674 treatment.

Combined treatments of 40 mg atorvastatin and 10 µg or 50 µg LY518674 significantly decreased triglycerides 44.4% and 52.5% (p<0.001), LDL-C by 47.7% and

49.7% (p<0.001), apoB100 by 41.2% and 44.1% (p<0.001), and total cholesterol by

35.7% and 39.7% (p<0.001), respectively. HDL-C increased by 11.9% (NS) and 5.9%

(NS) and apoAII increased by 14.1% and 31.9% (p<0.001). ApoAI was not changed with 40 mg atorvastatin plus 10 µg LY 4.2% (NS), but significantly decreased with 40 mg atorvastatin plus 50 µg LY (2.4%, p<0.05). Percent changes and statistical analyses indicated here were calculated and reported previously by Nissen, et al [23].

Percent Changes in ApoM Concentration after Treatment versus Baseline

Human apoM concentration after placebo or drug treatment was compared against baseline apoM levels using one-way ANOVA to determine statistical difference in group means. Statistical significance was determined at p<0.05. Placebo and all drug treatments (except 40 mg atorvastatin) did not produce significant changes in apoM concentration compared to baseline. 40 mg dose of atorvastatin for four weeks significantly decreased apoM concentration from baseline levels (10.8%, p<0.001) at the end of the four weeks (Table 12). ApoM remained significantly decreased (13.4%, p<0.01) from baseline after 12 more weeks of placebo treatment in this group (Table 13).

148 A significant decrease in apoM was measured with 10 mg atorvastatin after four weeks of treatment (7.9%, p<0.05), but was not maintained through 16 weeks.

Interestingly, combination treatment of 40 mg atorvastatin with 10 µg or 50 µg

LY518674 did not result in a significant decrease in apoM concentration compared to baseline levels, as measured with 40 mg atorvastatin treatment alone (Figure 33). ApoM concentration in these combination treatments remained at baseline levels. However,

LY518674 treatment alone did not result in significant increase in apoM concentration.

Thus, LY518674 in combination with atorvastatin treatment helped to maintain baseline levels of apoM concentration. In combination treatments, although apoM was not increased, maintenance of normal plasma levels in addition to the dramatic decrease in

LDL and slight increase in HDL may improve the overall effectiveness of statin treatment on anti-atherogenesis and CHD prevention. Further studies should be performed to understand the lipid and drug interactions involved, to better design treatments and gain optimal response in different lipid responses after treatment.

149 Table 12

Placebo Atorvastatin

4 Weeks 10mg 40mg

ApoM 5.0 -7.9* -10.8*** n 26 19 29

Table 12: ApoM decreased significantly after four weeks of atorvastatin treatment compared to placebo

Human apoM concentrations were compared to baseline levels after four weeks of atorvastatin treatment. Changes in apoM concentration with four weeks placebo, or 10 mg or 40 mg atorvastatin treatment were expressed as percent of baseline apoM concentrations. Statistical significance was determined at p<0.05 compared to baseline levels. ApoM concentration after placebo treatment was not different than baseline. 10 mg and 40 mg atorvastatin significantly decreased apoM concentration after four weeks treatment by 7.9% (*p<0.05) and 10.8% (***p<0.001), respectively.

150 Table 13

Atorvastatin

Placebo 10mg 40mg

LY518674 LY518674 LY518674 16 Weeks Placebo 10µg 50µg Placebo 10µg 50µg Placebo 10µg 50µg

ApoM -0.6 11.0 7.6 -8.5 --- 4.2 -13.4** -7.5 -8.9 n 10 11 6 6 2 4 11 10 9

Table 13: Human apoM was significantly decreased after 16 weeks of 40 mg atorvastatin (4 weeks) and placebo (12 weeks) from baseline

Human apoM Concentrations were compared to baseline levels after 16 weeks of treatment. Mean apoM concentrations were expressed as a percentage of the baseline measurement (100%) to view changes with each treatment after 16 weeks (full study) of treatment compared to the pre-study measurement (baseline). ApoM data is shown here as difference (+/-) from baseline. Statistical differences are shown by ** p<0.01 compared to baseline. ApoM concentration was significantly lower than baseline measurement with 40 mg atorvastatin treatment. No significant change in apoM concentration compared to baseline was observed when LY518674 was added to 40 mg atorvastatin treatments. ApoM concentrations remained at baseline levels in 10 µg and

50 µg LY518674 alone, 10 mg atorvastatin alone and in combinations, and placebo treatment groups.

151 Figure 33

ApoM Baseline Levels Maintained with Combined Atorvastatin and LY518674 **

100

50 ApoM, % of % Baseline ApoM, 0

0LY 40AT 5 Baseline 40AT/10LY 40AT/

Figure 33: ApoM was maintained at baseline levels with combination treatments compared

From baseline measurement to end-of-study measurement (16 weeks), apoM decreased significantly (**p<0.01) compared to baseline apoM concentration after 40 mg atorvastatin treatment. ApoM was expressed here as a percent of baseline measurement

(+/-SEM). When 10 µg or 50 µg LY518674 was added in combination with 40 mg atorvastatin, baseline concentration of apoM was maintained.

152 Percent Changes in ApoM Concentration after Treatment versus Placebo Group

ApoM concentrations were measured in all treatment groups and compared to the placebo group using ANOVA. Treatment with 10 mg and 40 mg atorvastatin after the initial four-week period significantly decreased human apoM concentration by 7.9%

(p<0.05) and 10.8% (p<0.01) with 10 mg and 40 mg atorvastatin, respectively, compared to apoM concentration in the placebo group (Figure 34). After 16 weeks, however, apoM levels were not statistically different from the placebo group.

Mean apoM concentrations measured at 16 weeks were compared to previously measured lipid data. Placebo treatment did not significantly change apoM, lipid particles, or other apolipoprotein concentrations. ApoM concentrations were not significantly different from placebo after 16 weeks with any treatment compared to placebo (Table

14). A decrease in apoM concentration was measured with 40 mg atorvastatin (-13.4%) but was not significant. Statistical analysis was not performed on 10 mg atorvastatin/ 10

µg and 50 µg LY518674 due to small sample size. HDL cholesterol and HDL-associated apolipoproteins A-I and A-II were not changed with atorvastatin treatment.

ApoM did not replicate HDL behavior with atorvastatin treatment due to its slight decrease, but was more similar to LDL and LDL-associated apoB100, all of which decreased significantly with atorvastatin compared to placebo. LDL decreased significantly in all treatment groups from baseline to 16 weeks compared to placebo.

ApoB100, the main structural apolipoprotein of LDL, followed atorvastatin-induced decreases in LDL close to the same percentages. ApoM is associated with LDL particles to a smaller extent than HDL, but it may be possible that a large decrease (45.5%) in

LDL with 40 mg atorvastatin is associated with a small decrease in plasma apoM levels.

153 It may also be possible this mechanism of apoM decrease was unrelated to LDL changes.

Further experiments were not done to test these hypotheses.

HDL concentration increased slightly (12-15%) with LY518674 treatment, but was not statistically different from placebo. However, significant increases in apoAI (50

µg only) and AII were measured with LY518674 treatment. An increase in apoM (11%) was measured with 10 µg LY518674 treatment after 16 weeks, but was not significantly different from placebo. In this treatment, HDL increased 15%, but was also not statistically significant.

In this treatment, apoM concentration changes followed increases in HDL concentrations as hypothesized. ApoAI and AII, the two main HDL-associated apolipoproteins, represent a larger population of HDL and are more strongly correlated with HDL levels [38] as observed in this study. Since apoM is only associated with a small sub-population of HDL, a strong correlation may not exist. The resulting data indicate that small increases observed in plasma HDL levels were not a result of larger increases in apoM-containing HDL concentration.

154 Figure 34

Changes in ApoM Concentration With 4-Week Treatment vs. Placebo ** *

100

50

ApoM conc, baseline % of conc, ApoM 0 Placebo 10mg AT 40mg AT

Figure 34: ApoM concentrations decreased significantly after four weeks of initial atorvastatin treatment versus placebo

ApoM decreased 8% with 10 mg atorvastatin (*p<0.05) and 11% (**p<0.01) with 40 mg atorvastatin versus placebo. The decrease was not maintained through the next 12-week treatment period compared to placebo. ApoM concentration is shown as a percent of baseline measurement (+/-SEM).

155 Table 14

Atorvastatin

Placebo 10mg 40mg

LY518674 LY518674 LY518674

16 Weeks Placebo 10µg 50µg Placebo 10µg 50µg Placebo 10µg 50µg

ApoM -0.6 11.0 7.6 -8.5 --- 4.2 -13.4 -7.5 -8.9 HDL 6.9 15.0 12.5 2.0 12.6 17.6* 3.4 11..9 5.9 ApoA-I 4.9 6.7 12.4* 0.3 6.5 8.9 -0.2 4.2 -2.4* ApoA-II 0.3 12.7** 33.7† -6.3 11.9** 35.4† -4.2 14.1† 31.9† LDL -1.0 -13.2* -15.8** -40.7† -36.6† -41.1† -45.5† -47.7† -49.7† ApoB-100 1.8 -13.9† -14.9† -33.7† -31.3† -38.1† -37.6† -41.2† -44.1† TG -5.3 -36.9† -37.5† -18.5 -39.4† -55.4† -23.4* -44.4† -52.5† TC 0.12 -11.6** -14.6† -29.3† -27.8† -32.3† -33.5† -35.7† -39.7† n 10 11 6 6 2 4 11 10 9

Table 14: Changes in ApoM and other lipoprotein concentrations after 16 weeks of treatment compared to placebo group

Changes in apoM concentration were compared to placebo group after 16 weeks of treatment. Mean apoM concentrations were expressed as a percentage of the baseline measurement (100%) to view changes with each treatment after 16 weeks (full study) of treatment compared to placebo group. Statistical differences are shown by * p<0.05, ** p<0.01 and † p<0.001 compared to placebo group means. The sample size of each treatment group (based on the number of samples measured for apoM) is represented by

‘n’. Statistical analysis was not performed on 10 mg atorvastatin/ 10 µg and 50 µg

LY518674 due to small sample size. All lipid data except apoM measurements were done previously as part of the clinical study. ApoM was measured using this MS-based assay. Placebo treatment did not change apoM, lipid particles, or other apolipoprotein levels. ApoM concentration decreased with 10 mg and 40 mg atorvastatin treatments, but was not statistically significant compared to placebo group. HDL and associated apoAI

156 and AII did not change with this treatment. Atorvastatin caused a significant decrease in

LDL and associated apoB100, TC, and TG. ApoM remained unchanged in all treatment groups and did not follow significant increases measured in HDL and apoAI concentrations. Therefore, increases observed in HDL were not a result of larger increases in apoM concentration.

157 ApoM Correlation with HDL and LDL Particles in this Clinical Study

A very weak positive correlation (R 2=0.058) existed between apoM concentration and HDL cholesterol levels in this clinical study (Figure 35). As discussed above, apoM did not always mimic HDL behavior in response to various drug treatments and therefore a strong correlation could not be drawn. Since apoM is only present on a small subset of

HDL particles, it may be difficult to observe a strong correlation between the two concentrations, but the measurement of apoM included with apoAI, AII, and HDL concentrations results in a deeper understanding of the HDL particles present and what is happening at subpopulation levels.

LDL cholesterol and apoM concentrations were also very weakly correlated

(R 2=0.0776) in this study (Figure 36). ApoM has been found to associate to a smaller extent with LDL particles [10] and decreases in apoM were observed that followed decreases in LDL levels in this study. ApoM has been mainly implicated as an HDL- associated apolipoprotein due to its potent cholesterol efflux stimulation and initial acceptance [2], so a strong correlation between apoM and total plasma LDL levels was not likely or predicted. Thus, any decreases in apoM can be detrimental to cholesterol efflux, reverse cholesterol transport capacity and anti-atherogeneic properties of pre β-

HDL particles.

158 Figure 35

Relationship Between ApoM Concentration and HDL in Human Serum

0.8 0.7 0.6 0.5 0.4 0.3

ApoM(umol/L) 0.2 0.1 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 HDL Cholesterol (mol/L)

Figure 35: ApoM concentration was not correlated with HDL concentration

Little correlation (R²=0.0585, n=260) existed between apoM concentration (µg/mL) and

HDL-C concentration (mg/dL) in this study. Increases measured in HDL-C were not a result of larger increases in apoM. ApoM was not affected by atorvastatin treatments in the same manner as HDL-C.

159 Figure 36

Relationship Between LDL Cholesterol and ApoM

1.0 Concentration

0.8

0.6

0.4 ApoM ApoM (umol/L) 0.2

0.0 0 1 2 3 4 5 6 7 8 LDL Cholesterol (mol/L)

Figure 36: ApoM concentration was not correlated with LDL concentration

LDL-C had little correlation with apoM concentration in this human clinical study

(R 2=0.0776, n=260). ApoM has been found to be associated to a small extent with LDL particles and did mimic LDL responses to atorvastatin treatments. However, LDL was also decreased by LY518674 treatments, whereas apoM was slightly, but not significantly increased. Overall, apoM was not correlated with plasma LDL cholesterol levels in this study.

160 DISCUSSION

The development of a high-throughput, non-antibody, MS-based assay to quantify apoM in human, mouse, and rat serum was developed to provide an important tool in the advancement of pre-clinical and clinical research to increase the understanding of the biology of apoM in relation to cardiovascular disease, diabetes, and therapeutic treatments, among many others.

This non-antibody, MS-based assay for apoM was developed over other types of proteomic assays for a few reasons. Previous attempts to develop an antibody-based assay were unsuccessful. The use of a primary anti-apoM antibody to capture human recombinant apoM in solution and identify apoM using Western blot was successful, however, the same antibody did not capture apoM in human serum. Identification of apoM in human serum could only be achieved by Western blot, not from a solution

(unpublished data) and an ELISA that was developed for apoM [14] was not widely available. A drawback to an antibody-based technique is the likelihood that primary antibodies will cross-react with apoM from different species. Typically, cross-reactivity may not present a problem if apoM is measured in a single species. However, human apoM-transgenic mice have been described and used in apoM studies [25]. In human- transgenic animal models, human proteins are expressed, but endogenous proteins can still be present. The specific measurement of human apoM in the mouse serum without cross-reacting with the typically higher level of endogenous mouse apoM is impossible using an antibody due to the high percentage of apoM AA sequence homology between the two species. If an antibody-based assay, such as ELISA, is used to quantify human

161 apoM, a small amount of cross-reactivity of the antibody with mouse apoM will greatly distort the measurement of the low concentration of human apoM. The MS-based assay can measure human apoM using a unique tryptic peptide (AFLLTPR), the AA sequence of which has only one residue difference compared to the mouse sequence of AFLVTPR.

However, these two peptides are easily distinguishable by MS due to the difference in mass between leucine and valine. A common peptide between the two species,

FLLYNR, was also measured to support the two species-specific peptide measurements and measure total apoM. The quantification of apoM in these experiments is not possible using a conventional antibody-based method. Thus, a non antibody-based, MS assay for apoM is essential to maximizing the value of the results and interpretations obtained from studies involving increasingly-popular human transgenic animal models.

Antibodies are commonly used, not only for quantification of a target protein, but also for purification of a target protein from a more complex matrix (i.e. serum, cell lysate), especially if the concentration of the target analyte is low to maximize the sensitivity of the assay. ApoM circulates in relatively low abundance (~370-400 nM) in serum and was selectively purified from serum prior to MS analysis without the use of an antibody. In this assay, another purification approach was taken to purify apoM from serum. PHM-Liposorb was used to selectively and quantitatively purify apolipoproteins, including apoM, from serum in one simple enrichment step.

The result was a versatile MS assay that can measure apoM concentration in many species, including human, rat, mouse, horse, dog, monkey, sheep, goat, pig, and bovine. At least one of the three tryptic peptides included in the MRM method can be found in these species and used for the quantification of apoM. The sample preparation

162 and instrument methods do not need to be modified with the selection of a new species as long as apoM in that species contains one of the three selected apoM-derived tryptic peptides. A set of calibration samples of serum from the new target species diluted into a selected background matrix serum that does not contain the selected tryptic peptide can be made and the experimental samples should be diluted into the same background matrix as the calibration samples at a constant ratio. For example, any species containing

FLLYNR can be diluted into rabbit serum and analyzed using the MS assay without making any changes. The transition from one species to another to measure apoM using this assay can be achieved in a few hours.

Calibration standards diluted into a serum matrix mimicked the complex environment of the experimental serum samples and were used instead of recombinant or purified apoM. Serum from a non-target species was used as a dilution matrix to prepare serial dilutions of serum from the target species to use as calibration samples. PBS was not used to prepare serial dilutions of the serum in the calibration samples. Otherwise, the volume of serum will be different at each calibration sample and may have different recoveries from Liposorb binding and digestion with trypsin. The use of serum as a dilution matrix maintained the same volume of total serum per calibration sample. The samples were exposed to the same sample preparation procedure (Liposorb binding, digestion with trypsin) as the experimental samples so apoM recovery is maintained between calibration and experimental samples. The apoM recovery from the serum calibration samples can provide a direct comparison of apoM recovery from the experimental samples and quantification should be more accurate than using a calibration

163 standard prepared from a different source as the experimental samples (i.e. recombinant protein).

This assay was validated for reproducibility and accuracy of the measurement of apoM in human serum by repeated spike recovery analysis. The concentration of apoM in Cal-G samples was accurately predicted using Val-H as a calibration standard and vice versa. The validated working ranges of the apoM assay were 0.29-13.0 µg/mL (11.2-500 nM) in the urea protocol and 0.23-13.0 µg/mL (8.8-500 nM) in the R/A protocol. The average apoM concentration in human serum was measured at an average 9.6 µg/mL

(370 nM). The experimental samples were typically diluted 1:2 in order to bring the concentration of apoM in the experimental samples within the middle range of the concentration of apoM in the calibration samples. Dilution of the experimental samples at this ratio brought the average concentration to ~150 nM, which falls near the middle of the linear range of the calibration standards (8.8-500 nM). Therefore, the validated working range of this assay is wide enough to provide a measurement of apoM in experimental samples with the dilution of the experimental samples to bring the concentration of apoM within the middle of the linear range of the calibration standards.

The dynamic range of the MS is three-to-four orders of magnitude whereas the range of antibody-based assays is much smaller. To maximize the advantages of using a linear ion trap MS for the development of a MRM method, the isotopic distribution of the fragment ions was captured during data collection. The isotopic distribution of each tryptic peptide was used as its unique fingerprint to identify and confirm the presence of the tryptic peptide and visualize any contaminating ions. The average mass of the tryptic peptide was added to the MS-measured charge state of the tryptic peptide and then the total was

164 divided by the same charge state to obtain the mass-to-charge ratio [Avg. MW + charge)/charge] used in the targeted full MS and MRM scans. The use of average mass of the precursor ion instead of the monoisotopic mass as the center of the isolation window in the MRM method allowed the acquisition of the natural isotopes of the precursor ion. The collection of these isotopes gives additional information as to the cleanliness of the precursor ion based on interferences in the isotopic distributions of the product ions in the MRM ion spectra. Interferences that continue to be present after targeted selection using the MRM method can be visualized by an alteration in the isotopic distribution pattern of the product ions

In a pre-clinical application of this assay, apoM concentration was measured in serum from mice treated with different doses of an MTTP-inhibitor. ApoB, apoE and

VLDL particles showed the largest decrease in serum concentration in an inverse dose- dependent manner. The function of the MTTP-inhibitor was to prevent the intracellular assembly of apoB-containing VLDL particles in the liver, decreasing VLDL particle secretion and therefore lowering apoB and VLDL levels in circulation. ApoB is the main apolipoprotein component of LDL and VLDL and its circulation levels were closely associated with the levels of these particles. Triglycerides can accumulate in the liver due to MTTP-inhibitor suppression of VLDL secretion and assembly and the decreases observed in the circulation levels of HDL, apoAI and apoM with MTTP-inhibitor treatments as compared to control are possibly due to the suppression of reverse cholesterol transport due to the elevated hepatic triglyceride levels. Decreases in HDL, apoAI and apoM are detrimental to the protective anti-atherogenic effects that they can offer. ApoAI was more closely correlated with HDL levels than apoM, likely due to the

165 presence of at least two apoAI particles per HDL molecule and the presence of apoM on

HDL to a much lesser extent. The decrease in VLDL and LDL by MTTP-inhibition may not be as beneficial of a treatment due to the ensuing decreases in HDL, apoAI, and apoM. The results of this analysis led our collaborators to change the study design and dose selection in subsequent experiments, to obtain an optimal dose of MTTP inhibitor that did not decrease HDL-associated apolipoproteins.

Clinical application of this assay measured human apoM in serum collected from participants in a clinical study of statin and PPAR-α agonists drug therapies to improve lipid profiles and decrease the risk of cardiovascular events. Statin treatment is known to decrease LDL and total cholesterol significantly [31] and was observed in this study.

Statin-induced decrease in plasma LDL concentration has anti-oxidative and anti- atherogenic properties. ApoB-containing LDL in plasma is a substrate for oxidation and foam cell formation [9]. Decrease in plasma LDL concentrations can therefore reduce the formation of atherosclerotic plaque, which is also a property of high apoM levels.

Increasing apoM in conjunction with decreasing LDL could potentially give additional value to the anti-atherogenic properties of this treatment. However, high-dose statin treatment also decreased apoM concentration significantly from baseline, resulting in a very weak correlation between LDL cholesterol and apoM concentration (R²=0.0776, n=260). Activation of PPAR-α with agonists has been shown to clearly reduce hepatic triglyceride synthesis and promote triglyceride clearance from circulation. PPAR-α agonists have also been shown to increase plasma HDL levels 10-11% in humans [36].

The initial question as to the nature of the increased HDL particles was explored in this experiment utilizing this MS-based assay to quantify apoM. Concentrations of human

166 apoM in circulation were hypothesized to increase with an increase in HDL levels, attributed to the action of PPAR-α agonists. ApoM-containing HDL particles are a small subset of total HDL, so a dramatic increase in apoM is necessary to influence a small increase in total HDL. However, apoM concentration did not change significantly within these treatment groups. Two dose concentrations of the PPAR-α agonist resulted in significant ~20% increases in HDL levels, but these changes can not be attributed to a larger increase in apoM levels. However, this potent PPAR-α agonist alone was not enough to induce a dramatic increase in apoM.

Forty milligrams of Atorvastatin significantly decreased apoM concentrations after four weeks of treatment and was sustained after 12 more weeks of placebo. A decrease in apoM may be a negative affect of this stain treatment; however statins are widely used and successful in maintaining lower cholesterol levels. The measurement of apoM provides a deeper understanding of other statin effects and may be used to find optimal doses. Statistical significance was used here to indicate that apoM in the treatment group was truly lower than the baseline within 5% error. Biological significance can not be calculated in this way and involves further study into the biological mechanisms involved in statin treatment and resulting biological effects of this percentage decrease in apoM. The complete significance of this decrease is unknown; however, it has been shown that decrease in apoM is indicative of less lipid-poor pre β-

HDL formation [2], lowering peripheral cholesterol removal capacity.

The decrease observed in serum apoM levels is not considered a positive attribute of atorvastatin treatment due to apoM’s anti-atherogenic effects and coronary heart disease (CHD) prevention as well. Wolfrum et al demonstrated a decrease or absence of

167 apoM in mice lead to high levels of atherosclerotic lesion formation, associated with increased CHD [2]. A decrease in apoM concentration is detrimental to the prevention of atherosclerotic lesion formation. Statin therapy is attributed to drastic decreases in serum

LDL levels, but may not be as beneficial if anti-atherogenic agent apoM is decreased as well. One scenario for decreased apoM concentrations may be in the relationship between apoM and LDL particles. ApoM is present in LDL density fractions and so a significantly lower LDL may result in a slight, and even significant, decrease in apoM levels in circulation. In these treatments, plasma LDL levels decreased between 35-50%.

The 13% apoM decrease observed with 40 mg atorvastatin treatment may be due to the larger decrease in LDL particles, although this hypothesis was not investigated further.

The HDL concentrations remained steady, and apoM unexpectedly did not mimic this behavior.

The addition of LY518674 to atorvastatin treatment did not result in significantly decreased apoM. Baseline levels of apoM were maintained in these treatments, although a slight decrease was measured. It is unclear as to the level of decrease in apoM that can be detrimental to cholesterol efflux and pre β-HDL formation, but a significant decrease in apoM from atorvastatin treatment may be buffered by combination with PPAR-α agonists.

ApoM was not significantly different from placebo after 16 weeks of treatment.

ApoM was significantly decreased by four weeks of 10 mg and 40 mg atorvastatin treatment compared to placebo, but this was not maintained with 12 more weeks of placebo. Overall, statin therapy and PPAR-α agonists combinations did not affect apoM concentration compared to placebo treatments.

168 Due to the development of an apoM assay, a deeper understanding of a drug’s effects on lipid levels can be explored. The nature of the influence of PPAR-α agonists on HDL levels was not a result of a dramatic positive change in apoM concentrations.

High doses of atorvastatin treatment lowered LDL, total cholesterol, and triglyceride levels, but an underlying negative affect on apoM levels has been measured. A more thorough understanding of the mechanisms and overall affects of drug treatments can benefit the development of new therapies.

Average apoM concentration in human sample population (n=105) was 9.6

µg/mL +/- 2.4 (370 nM). HDL levels have been previously correlated with apoM concentration [14], but were weakly correlated with apoM concentration (R²=0.0585, n=260) in this study. A stronger correlation to HDL levels is calculated using plasma levels of apoAI and AII, the two major HDL-associated apolipoproteins [14, 38].

Approximate apoM association with HDL was calculated using apoAI plasma concentration. Two to four apoAI particles are present per HDL particle, and apoAI

(28.3 kDa) typically circulates in plasma ~1 mg/mL, or 35 µM [38]. Comparison of molar concentration indicated that apoM levels are approximately 1% of the level of apoAI in circulation and therefore may be present in approximately 2-4% of HDL particles. This measurement is in close agreement with previous calculations made by

Axler, et al that ~5% of HDL contains apoM [14]. The weak correlation between apoM and HDL may be due to the small population size of apoM-containing HDL, represented by apoM concentration. A small change in apoM, even statistically significant, may not be enough to influence a change in total HDL concentration and was not well-correlated to HDL levels in this study.

169 The apoM data from the human clinical study demonstrate the importance of an assay for quantification of apoM. In this study, quantification of apoM uncovers more information about the affects of these drugs on lipid metabolism. Measuring changes in lipoprotein particles or direct apolipoprotein components, such as apoAI or apoB100, allows a glimpse of only part of the biological picture. By measuring apolipoproteins that are not completely associated with one specific particle, such as apoM, different aspects of lipid metabolism can be seen. For example, apoM has additional anti- atherogenic effects, so an increase in apoM due to drug treatment would be a greater beneficial effect of that treatment compared to increasing HDL alone. Increases in HDL indicate that more cholesterol is being returned to the liver, but apoM can give further information as to the lipid status of these particles. An increase in apoM indicates an increase in lipid-poor pre β-HDL formation, whereas the measurement of HDL or apoAI does not disclose these details. A decrease in apoM is detrimental to the desired effects of treatment, but may not be detected by total HDL measurement alone. Without apoM measurement, it is shown that HDL does not change and LDL, TC, and TG decrease with these drug treatments. Without the measurement of apoM, the results of the statin treatment are positive to lower LDL, TC, and TG. The addition of the measurement of apoM uncovers another layer in drug action to gain a better understanding of how humans are affected by drug therapies, at the target site and off-target sites in the body.

Other treatments may affect apoM to different extents and that information should be considered to maintain or increase cholesterol clearance capacity with normal or elevated apoM levels.

170 This assay can be applied to many different situations like the one demonstrated here. Cholesterol and lipid-altering drug evaluations, cardiovascular disease research, and other applications in which lipid metabolism is being studied additional uses for this

MS-based assay. The versatility of the assay to measure apoM concentration in rat, mouse, and human serum can create a smooth transition between the measurement of apoM concentration in pre-clinical and clinical studies.

171 CONCLUSION

Apolipoprotein M is an important component in lipid metabolism and is believed to have anti-atherogenic properties. ApoM is necessary for the formation of lipid-poor pre β-HDL particles, the initial promoters and acceptors of cholesterol efflux from peripheral cells. Research has been hindered thus far due to lack of a widely-available assay for apoM. Thus, a non-antibody, high throughput MS-based assay for quantification of human and mouse apoM in serum or plasma was developed. This assay measures apoM using its unique peptides for identification and quantification. FLLYNR peptide is common between many species, whereas AFLLTPR is human-specific and

ALFVTPR was specific to mouse and rat apoM. A lipoprotein-binding agent, Liposorb, was used to selectively removed apolipoproteins from serum. The proteins were digested with trypsin in the presence of detergent and apoM tryptic peptides were measured by

LC-MS via mass-to-charge ratio ( m/z ) of the intact and fragmented peptides. The AUC was integrated and absolute quantification was achieved using an external standard curve.

The assay was validated with repeated spike-recovery measurement of apoM in calibration standards. Human apoM in serum was measured using the MS assay and

Western blot to produce comparable results with the MS assay and a more conventional method.

Pre-clinical study application of this assay measured apoM concentration in mouse serum after treatment with different doses of an MTTP-inhibitor or control vehicle solution. Decreases in apoB and apoE were closely associated with decreases in LDL and VLDL due to MTTP-inhibition of VLDL assembly and secretion and apoB is the

172 main protein component of VLDL and LDL. ApoE is necessary for the assembly of these particles and so was decreased as well. ApoAI, apoM and HDL were also decreased with these treatments possibly due an increase in hepatic triglyceride levels that suppressed the reverse cholesterol transport actions of these proteins to transport cholesterol from the peripheral tissues to the liver for degradation. The decreases in apoAI, apoM, and HDL were reflective of a decrease in lipid-poor and mature HDL particles, thus a decrease in the capacity for cholesterol removal. This application demonstrates the importance of the measurement of apoM concentration to evaluate the lipid-poor status of HDL and thus the capacity of HDL particles for cholesterol removal from the peripheral tissues. A decrease in this capacity, shown by a decrease in apoM, is detrimental to the anti-atherogenic effects of apoM.

A clinical study application of this assay measured apoM concentration in human serum before and after administration of two drugs, statin drug atorvastatin and PPAR-α agonist LY518674, in a human clinical study. Serum levels of apoM have been shown to negatively correlate with cardiovascular (CV) risk, and these treatments have been shown to lower this risk as well. Both treatments lower LDL cholesterol and PPAR-α agonists additionally increase HDL. We hypothesized that a modest increase in HDL by the

PPAR-α agonist may be due to a large increase in the small subpopulation of apoM- containing HDL particles and thus an increase in lipid-poor pre β-HDL. However, apoM levels did not change with PPAR-α agonist treatment and did not have a strong correlation with HDL levels. However, apoM was significantly decreased with atorvastatin, but combination treatments did not change plasma apoM levels.

173 Measurement of apoM provides additional information in the lipid profile that was not measured before. Availability of a quantitative and high-throughput assay for apoM will be extremely valuable for development of anti-atherogenic drugs and understanding the mechanisms of such drugs. It is clear apoM is an important component of cholesterol metabolism that demands further evaluation and much of the information surrounding its biological mechanisms is left to be understood. Its implications in coronary heart disease have been demonstrated and the development of an additional tool to use in this research field is beneficial to the scientific and medical communities.

174 REFERENCES

1. Charlton-Menys, V. and P.N. Durrington, Human cholesterol metabolism and

therapeutic molecules. Experimental Physiology, 2007. 93 (1): p. 27-42.

2. Wolfrum, C., M.N. Poy, and M. Stoffel, Apolipoprotein M is required for pre β-

HDL formation and cholesterol efflux to HDL and protects against

atherosclerosis. Nature Medicine, 2005. 11 (4): p. 418-422.

3. Söderlund, S., et al., Hypertriglyceridemia is associated with pre β-HDL

concentrations in subjects with familial low HDL. Journal of Lipid Research,

2005. 46 : p. 1643-1651.

4. Xu, N. and B. Dahlbäck, A Novel Human Apolipoprotein (apoM). The Journal of

Biological Chemistry, 1999. 274 (44): p. 31286-31290.

5. Christoffersen, C., et al., Isolation and characterization of human apolipoprotein

M-containing lipoproteins. Journal of Lipid Research, 2006. 27 : p. 1833-1843.

6. Lusis, A., Atherosclerosis. Nature, 2000. 407 : p. 233-241.

7. Dahlbäck, B. and L.B. Nielsen, Apolipoprotein M- a novel player in high-density

lipoprotein metabolism and atherosclerosis. Current Opinion in Lipidology, 2006.

17 : p. 291-295.

8. Botham, K.M., et al., The induction of macrophage foam cell formation by

chylomicron remnants. Biochemical Soc. Trans., 2007. 35 (3): p. 454-458.

9. Hofnagel, O., et al., Statins and foam cell formation: Impact on LDL oxidation

and uptake of oxidized lipoproteins via scavenger receptors. Biochimica et

Biochysica Acta, 2007. 1771 : p. 1117-1124.

175 10. Karlsson, H., et al., Characterization of Apolipoprotein M Isoforms in Low-

Density Lipoprotein. Journal of Proteome Research, 2006. 5(10): p. 2685-2690.

11. Xu, N., P. Nilsson-Ehle, and B. Ahrén, Suppression of apolipoprotein M

expression and secretion in alloxan-diabetic mouse: Partial reversal by insulin.

Biochemical and Biophysical Research Communications, 2006. 342 : p. 1174-

1177.

12. Richter, S., et al., Regulation of Apolipoprotein M Gene Expression by MODY3

Gene Hepatocyte Nuclear Factor-1α. Diabetes, 2003. 52 : p. 2989-2995.

13. Zhang, X., et al., Hyperglycemia down-regulates apolipoprotein M expression in

vivo and in vitro. Biochimica et Biochysica Acta, 2007. 1771 : p. 879-882.

14. Axler, O., J. Ahnstrom, and B. Dahlbäck, An ELISA for apolipoprotein M reveals

a strong correlation to total cholesterol in human plasma. Journal of Lipid

Research, 2007. 48 : p. 1772-1780.

15. Bantscheff, M., et al., Quantitative mass spectrometry in proteomics: a critical

review. Analytical and Bioanalytical Chemistry, 2007. 389 : p. 1017-1031.

16. Ahmed, N. and G.E. Rice, Strategies for revealing lower abundance protein in

two-dimensional protein maps. Journal of Chromatography B, 2005. 815 (1-2): p.

39-50.

17. Higgs, R.E., et al., Comprehensive Label-Free Method for the Relative

Quantification of Proteins from Biological Samples. Journal of Proteome

Research, 2005. 4: p. 1442-1450.

18. Liebler, D.C., Introduction to Proteomics: Tools for the New Biology . 2002,

Totowa, NJ: Humana Press, Inc.

176 19. Han, B. and R.E. Higgs, Proteomics: from hypothesis to quantitative assay on a

single platform. Guidelines for developing MRM assays using ion trap mass

spectrometers. Briefings in Functional Genomics and Proteomics, 2008.

20. Cartwright, I.J. and J.A. Higgins, Intracellular degradation in the regulation of

secretion of apolipoprotein B-100 by rabbit hepatocytes. Biochem Journal, 1996.

314 : p. 977-984.

21. Altschul, S., et al., Basic local alignment search tool. Journal of Molecular

Biology, 1990. 215 : p. 403-410.

22. Finnigan XCalibur, Getting Productive: Processing Setup and the Analysis of

Quantitation Data. 2000. XCALI_97019 (Revision B).

23. Nissen, S.E., et al., Effects of a Potent and Selective PPAR-α Agonist in Patients

With Atherogenic Dyslipidemia or Hypercholesterolemia: Two Randomized

Controlled Trials. Journal of American Medical Association (JAMA), 2007.

297 (12): p. 1362-1373.

24. Schneider, M.R. and E. Wolf, Genotyping of transgenic mice: Old principles and

recent developments. Analytical Biochemistry, 2005. 344 (1): p. 1-7.

25. Christoffersen, C., et al., The Signal Peptide Anchors Apolipoprotein M in Plasma

Lipoproteins and Prevents Rapid Clearance of Apolipoprotein M from Plasma.

The Journal of Biological Chemistry, 2008. 283 (27): p. 18765-18772.

26. Hale, J.E., et al., A simplified procedure for the reduction and alkylation of

cysteine residues in proteins prior to proteolytic digestion and mass spectral

analysis. Analytical Biochemistry, 2004. 333 : p. 174-181.

177 27. Duan, J., B. Dahlbäck, and B.O. Villoutreix, Proposed lipocalin fold for

apolipoprotein M based on bioinformatics and site-directed mutagenesis.

Federation of European Biochemical Societies, 2001. 499 : p. 127-132.

28. Hussain, M.M., J. Shi, and P. Dreizen, Microsomal triglyceride transfer protein

and its role in apoB-lipoprotein assembly. Journal of Lipid Research, 2003. 44 : p.

22-32.

29. Olofsson, S.-O., P. Stillemark-Billton, and L. Asp, Intracellular Assembly of

VLDL. Trends in Cardiovascular Medicine, 2000. 10 (8): p. 338-345.

30. Olofsson, S.-O., L. Asp, and J. Boren, The assembly and secretion of

apolipoprotein B-containing lipoproteins. Current Opinion in Lipidology, 1999.

10 : p. 341-346.

31. Soedamah-Muthu, S.S., et al., The effect of atorvastatin on serum lipids,

lipoproteins and NMR spectroscopy defined lipoprotein subclasses in type 2

diabetic patients with ischaemic heart disease. Atherosclerosis, 2003. 167 : p. 243-

255.

32. Stancu, C. and A. Sima, Statins: mechanism of action and effects. Journal of

Cellular and Molecular Medicine, 2001. 5(4): p. 378-387.

33. Feig, J.E., R. Shamir, and E.A. Fisher, Atheroprotective effects of HDL: beyond

reverse cholesterol transport. Current Drug Targets, 2008. 9(3): p. 196-203.

34. Aviram, M., et al., Interactions of platelets, macrophages, and lipoproteins in

hypercholesterolemia: antiatherogenic effects of HMG-CoA reductase inhibitor

therapy. Journal of Caridovascular Pharmacology, 1998. 31 : p. 39-45.

178 35. Chawla, A., et al., Nuclear receptors and lipid physiology: opening the X-files.

Science, 2001. 294 (5548): p. 1866-1870.

36. Kersten, S., Peroxisome Proliferator Activated Receptors and Lipoprotein

Metabolism. PPAR Research, 2007. 2008 .

37. Gilde, A.J., J.-C. Fruchart, and B. Staels, Peroxisome Proliferator-Activated

Receptors at the Crossroads of Obesity, Diabetes, and Cardiovascular Disease.

Journal of the American College of Cardiology, 2006. 48 (9): p. A24-32.

38. Birjmohun, R.S., et al., Apolipoprotein A-II is Inversly Associated with Risk of

Future Coronary Artery Disease. Circulation, Journal of the American Heart

Association, 2007. 116 : p. 2029-2035.

39. Blanco-Vaca, F., et al., Role of ApoA-II in lipid metabolism and atherosclerosis:

advances in the study of an enigmatic protein. Journal of Lipid Research, 2001.

42 : p. 1727-1739.

40. Jones, P., et al., Comparative dose efficacy study of atorvastatin versus

simvastatin, pravastatin, lovastatin, and fluvastatin in patients with

hypercholesterolemia (the CURVES study). American Journal of Cardiology,

1998. 81 : p. 582-587.

41. Bakker-Arkema, R., M. Davidson, and R. Goldstein, Efficacy and safety of a new

HMG-CoA reductase inhibitor, atorvastatin, in patients with

hypertriglyceridemia. Journal of American Medical Association (JAMA), 1996.

275 : p. 128-133.

179 42. Careskey, H.E., et al., Atorvastatin increases human serum levels of proprotein

convertase subtilisin/kexin type 9. Journal of Lipid Research, 2008. 49 : p. 394-

398.

180