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).