MRM Assay for Quantitation of Complement Components in Human Blood Plasma — a Feasibility Study on Multiple Sclerosis

JOURNAL OF PROTEOMICS 75 (2011) 211– 220

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MRM assay for quantitation of complement components in human blood plasma — a feasibility study on multiple sclerosis

Melinda Rezeli a,⁎, Ákos Végvári a, Jan Ottervald b, c, Tomas Olsson b, Thomas Laurell a, d, György Marko-Varga a a Division of Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Lund, Sweden b Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Hospital, Stockholm, Sweden c AstraZeneca, R&D Södertälje, Sweden d Department of Biomedical Engineering, Dongguk University, Seoul, Republic of Korea

ARTICLE INFO ABSTRACT

Article history: As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for Received 1 March 2011 quantitation of proteotypic peptides, representing seven plasma proteins associated with Accepted 17 May 2011 inflammation (complement components and C-reactive protein). The assay development Available online 15 June 2011 and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable ≤ Keywords: reproducibility over a 2 orders of magnitude linear range (RSD 25%). The assay panel was Selected reaction monitoring utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and Linear ion trap 8 control patients. Multiple sclerosis © 2011 Elsevier B.V. All rights reserved.

1. Introduction biomarkers are available to diagnose disease, to predict disease progression, or to monitor treatment effects. However, several Multiple sclerosis (MS) is a common inflammatory disease of the promising approaches have been proposed [2–6]. central nervous system in young adults. Multiple sclerosis can Quantitation of peptide and protein biomarkers in complex appear in three major forms, as i) relapsing-remitting (RRMS), ii) biological matrices, such as human plasma, is a challenging task primary progressive (PPMS) and iii) secondary progressive [7,8]. In biomarker discovery at the verification and the multiple sclerosis (SPMS). Clinically isolated syndrome (CIS), a validation phase, the determination of the levels of high number single neurological episode caused by demyelization, may be of candidate biomarkers in different body fluids in large sample the first indication of a later evolving multiple sclerosis [1]. sets is crucial in future health care developments. The re- Although, its etiology is still unclear, most likely various genetic quirements are rigorous: as the assay must be sensitive, and environmental factors have impact on the disease devel- accurate, and highly reproducible while the high throughput is opment and progression in individuals. Currently, no validated also an important aspect. Currently, the highly sensitive and

Abbreviations: RRMS, relapsing-remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; C3, Complement C3; C4, Complement C4; C5, Complement C5; C9, Complement C9; fB, Complement factor B; fH, Complement factor H; CRP, C-reactive protein. ⁎ Corresponding author at: Div. Clinical Protein Science & Imaging, Dept. of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, SE-221 84 Lund, Sweden. Tel.: +46 46 222 3721; fax: +46 46 222 4527. E-mail address: [email protected] (M. Rezeli).

1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.05.042 212 JOURNAL OF PROTEOMICS 75 (2011) 211– 220 specific immunoassays (mostly sandwich assays) are common- ly used for verification and validation of new biomarkers [9].The success of the immunoassays based on the highly specific antibodies, which are usually not available for the novel Patient Database candidate proteins, and their development is a very expensive and time-consuming process. A further limitation is the Clinical MS restricted potential to develop multiplex immunoassays that data data in all cases will be quality determined by the antibodies utilized. Mass spectrometry-based targeted approaches, such as Primary Secondary selected reaction monitoring (SRM; or MRM in plural) with the Blood CSF Biobank Biobank use of stable isotope-labeled peptide standards, allow accurate peptide and protein quantitation and provide an alternative Samples for Karolinska Lund Primary Biobank Institute University method to antibody-based approaches [10]. SRM is a targeted MS-based technique, generally performed on triple-quadrupole – (QqQ) instruments. In SRM, the parent ion for the targeted Fig. 1 Overview of the current multiple sclerosis study. analyte is selected in the first quadrupole (Q1), followed by frag- mentation in the collision cell (Q2) whereas the third quadrupole In this paper, we report on a feasibility study conducted on a (Q3) is used to monitor selected specific fragment ions. Coherent multiple sclerosis related sample cohort. The study procedure is precursor and fragment ion pairs are called SRM transitions, and outlined in Fig. 1. We developed an MRM assay for quantitation several such transitions are monitored during a single chro- of plasma proteins associated with inflammation that was matographic run. The two mass filters result in highly specific utilized for the analysis of plasma samples from multiple and sensitive responses for the targeted peptides. MRM assays sclerosis patients with i) relapsing-remitting and ii) secondary provide good sensitivity and quantitation of proteins/peptides in progressive status and iii) from control patients. Seven medium a broad dynamic range (exceeding four orders of magnitude abundant proteins were selected within the concentration [11,12]). Clinical Proteomics Technology Assessment for Cancer range from 1 μg/mL to 1.5 mg/mL in human plasma. network of the National Cancer Institute (NCI-CPTAC) conducted a multilaboratory study to demonstrate that the MRM assays based on standardized protocols can be highly reproducible 2. Materials and methods across 8 laboratories and various instrument platforms [13]. The limit of quantitation (LOQ) in human blood plasma 2.1. Materials without any fractionation is ~1 μg/mL [10], which is insufficient to achieve the requirements for plasma biomarker measure- Water (Chromasolv® Plus for HPLC), formic acid (Reagent ments at the medium- and the low abundant proteins. Different grade≥95%), dithiothreitol and iodoacetamide were purchased strategies have been developed to decrease sample complexity from Sigma-Aldrich (Steinheim, Germany); acetonitrile (Hyper- and simultaneously improve sensitivity of MRM assays. With grade for LC-MS) was from Merck (Darmstadt, Germany). the combination of depletion and strong-cation exchange Sequence grade trypsin was purchased from Promega (Madison, chromatographic (SCX) separation, the LOQ was improved to WI). Light and heavy sequences of the target peptides with 1–10 ng/mL by Keshishian et al. [14]. Whiteaker et al. developed a purity higher than 97% were purchased from Thermo Fischer method, called Stable Isotope Standards with Capture by Anti- Scientific (Ulm, Germany). The C-terminal Arginine or Lysine Peptide Antibodies (SISCAPA), by which they could reach the was labeled with 13Cand15N in the heavy forms, providing an detection limit as low as 1.6 ng/mL and with increased sample increased nominal mass of 10 and 8 Da, respectively. volume they could improve it to tens of pg/mL in multiplex assay [15]. Anti-peptide antibodies were used to capture the target 2.2. Clinical materials peptides and their corresponding stable isotope labeled standards, and the sample processing were automatized, therefore, Human plasma samples were provided by Karolinska University the approach has the ability to achieve high throughput. Hospital, from the Neuro Biobank (Prof. Tomas Olsson). A Most of the proteomic MRM assays are performed on triple secondary biobank was established at Lund University, where quadrupole or hybrid triple quadrupole ion trap instruments all samples were logged into the archive with clinical data [11,16–19]. However, linear ion trap mass spectrometers can associated (Fig. 1). All study enrolment followed the recom- also offer an assay platform for protein quantification, as mendations of the Declaration of Helsinki and the study was reported previously [20–22]. approved by the Ethics Committee of the Karolinska Institute The complement system is part of the innate immune #02-365. Oral and written information was given to the patients system, consisting of a number of circulating and membrane- and confirmed consent in writing was received before inclusion. associated proteins. The complement system provides a defense against pathogens in the human body, as the 2.3. Sample preparation activation of the complement system results in the formation of membrane attack complex (MAC) and the release of The seven most abundant proteins were depleted in the plasma anaphylotoxins. It is also implicated in the pathology of samples by using Plasma 7 Multiple Affinity Removal Spin neurodegenerative disorders [23–25], such as Alzheimer's Cartridge (Agilent Technologies, Santa Clara, CA). The first flow- disease, Parkinson's disease and multiple sclerosis. through fractions were denatured by the addition of 8 M urea; JOURNAL OF PROTEOMICS 75 (2011) 211– 220 213 and reduced with 10 mM dithiothreitol (60 min, 37 °C) and plasma equivalent) were injected onto a 0.5×2 mm CapTrap C8 alkylated with 50 mM iodoacetamide (30 min, at room temper- column (Michrom BioResources, Auburn, CA), and following on- ature in dark). The excess of the reagents was removed by buffer line desalting and concentration the tryptic peptides were exchange with 50 mM ammonium bicarbonate buffer using a separated on a 75 μm×150 mm fused silica column packed 10 kDa cut-off spin filter (Millipore, Billerica, MA). The samples with ReproSil C18 (3 μm, 120 Å from Dr. Maisch GmbH, were digested with sequence grade trypsin overnight at 37 °C. Germany). Separations were performed in a 30-min linear The digested samples were then acidified with 1% formic acid, gradient from 5 to 40% acetonitrile containing 0.1% formic acid; dried, re-dissolved in 0.1% formic acid and spiked with a mixture at the flow rate of 250 nL/min. During the assay development of heavy isotope-labeled peptide standards and finally analyzed three transitions per target peptide were monitored by using two by nanoLC-ESI-MS/MS. time segments, and in the final assay a single transition was selected for each peptide (see Table 1). The parent ion was 2.4. LC-MS/MS analysis isolated with a mass window of 2.0 m/z units, fragmented (collision energy=35%, activation time=30 ms at Q=0.25), while An MRM assay was developed on an LTQ XL mass spectrometer the resulting daughter ion was scanned in profile mode with a (Thermo Scientific, Waltham, MA) equipped with an Eksigent mass window of 2.0 m/z unit. The maximum ion accumulation nanoLC-1D plus pump. Two microliters of samples (0.04 μl time was 100 ms, and the number of microscans was set to 1.

Table 1 – Proteotypic peptide sequences and selected SRM transitions for the seven analyzed plasma proteins. Accession no Protein Position Peptide sequence Q1 Q3

1+ P01024 Complement C3 905–913 TGLQEVEVK 501.8 (2+) 731.4 (y6 ) 603.3 (y51+) 422.75 (y72+) 1+ TGLQEVEVK 505.8 (2+) 739.4 (y6 ) 611.3 (y51+) 426.75 (y72+) 1+ P0C0L4 P0C0L5 Complement C4-A and C4-B 485-494 VGDTLNLNLR 557.8 (2+) 629.4 (y5 ) 402.2 (y31+) 742.5 (y61+) 1+ VGDTLNLNLR 562.8 (2+) 639.4 (y5 ) 412.2 (y31+) 752.5 (y61+) 1+ P00751 Complement factor B 620–629 EELLPAQDIK 578.3 (2+) 671.4 (y6 ) 784.5 (y71+) 897.5 (y81+) 1+ EELLPAQDIK 582.3 (2+) 679.4 (y6 ) 792.5 (y71+) 905.6 (y81+) 1+ P08603 Complement factor H 212–224 SPDVINGSPISQK 671.7 (2+) 830.7 (y8 ) 943.5 (y91+) 716.4 (y71+) 1+ SPDVINGSPISQK 675.7 (2+) 838.8 (y8 ) 951.5 (y91+) 724.4 (y71+) 2+ P01031 Complement C5 1381–1392 IDTQDIEASHYR 483.3 (3+) 610.3 (y10 ) 667.8 (y112+) 495.7 (y82+) 2+ IDTQDIEASHYR 486.7 (3+) 615.5 (y10 ) 673.0 (y112+) 500.8 (y82+) 1+ P02748 Complement C9 214-225 TEHYEEQIEAFK 508.6 (3+) 607.3 (y5 ) 365.2 (y31+) 735.4 (y61+) 1+ TEHYEEQIEAFK 511.3 (3+) 615.4 (y5 ) 373.2 (y31+) 743.4 (y61+) 1+ P02741 C-reactive protein 32–41 ESDTSYVSLK 564.8 (2+) 347.3 (y3 ) 609.4 (y51+) 696.4 (y61+) 1+ ESDTSYVSLK 568.8 (2+) 355.3 (y3 ) 617.4 (y51+) 704.4 (y61+)

Transitions, containing the Q3 y-ions as highlighted in bold, were selected in the final assay as best transitions. 214 JOURNAL OF PROTEOMICS 75 (2011) 211– 220

2.5. Standard curves of synthetic peptides spectra were used for validation of the peptides by performing database search using SEQUEST search engine (Proteome For characterization of the assay linearity, a dilution series of Discoverer v1.0, Thermo Scientific). The three highest intensity non-labeled synthetic peptide mixtures (in the range of 0.25– fragment ions (y-ions in most cases) were selected for SRM 125, and 0.5–50 fmol/μL in pure solution and in plasma digest, transitions for each peptide (Table 1). The selected transitions respectively) containing constant amount of isotope-labeled were verified by comparing the relative signal intensities of the synthetic peptides (12.5 fmol/μL) both in pure solution and daughter ions, generated from endogenous peptides with those spiked into plasma digest were analyzed in triplicates. from heavy peptide standards. The pattern of product ions from Calibration curves were generated by linear regression anal- the endogenous peptide was identical to that of the standard ysis on the peak area ratios (light/heavy) versus concentration peptide (Fig. 2). Only slight differences were observed between ratios for all target peptides. the endogenous and the synthetic peptides, which ensure that the selected transitions are free of matrix interference. In order 2.6. Data analysis to maximize the number of data points across the peaks, in the final assay a single proteotypic peptide and a single fragment The peak area responses recorded for each sample were ion (i.e., one SRM transition) was chosen for each target protein analyzed by using Qual Browser, part of the Xcalibur 2.0 (Table 1, highlighted in bold). software (Thermo Scientific). Peak areas were manually adjusted when signals were not intense and the software 3.2. Quantitation and analytical linearity could not reliably determine the peak. SRM signals in regards to multiple sclerosis patient groups For investigation of the assay linearity a dilution series of non- were compared with Kruskal-Wallis non-parametric tests labeled synthetic peptide mixtures containing constant using Matlab v 7.11 (Mathworks, Natick, MA). amount of isotope-labeled synthetic peptides were analyzed in triplicates in both pure solution and spiked into plasma digest. Calibration curves were generated by linear regression 3. Results analysis on the peak area ratios (light/heavy) versus concentration ratios for all target peptides (Fig. 3). Linear plots were As a proof-of-principle study, a multiple reaction monitoring observed (R2 ≥ 0.955) for each target peptide within the (MRM) assay was developed for quantitation of tryptic analyzed concentration range of 0.25–125 fmol/μL (while the peptides, representing seven plasma proteins associated concentration of heavy peptides was kept constant at with inflammation (complement components with C-reactive 12.5 fmol/μL) in pure solution. In plasma digest, linearity protein in addition) using a linear ion trap (LTQ XL) mass with a regression coefficient higher than 0.990 was observed; spectrometer. Previous studies have established the role of except for the complement component 4 peptide. The complement system in the pathology of multiple sclerosis and standard deviations for the respective target peptide of each complement proteins have been investigated as potential biomarker are indicated as error bars on the plots, and found biomarkers of disease activity. We analyzed 18 multiple to be lower than 20% in both cases. sclerosis related blood plasma samples on a linear ion trap mass spectrometer by using our inflammatory MRM assay 3.3. MRM analysis of plasma samples panel. In order to have a high assay turn-around cycle, we optimized 3.1. Selection of transitions for SRM the separation conditions with respect to the physical and chemical properties of all biomarkers of the assay. As assay The selection of target peptides was based on the results of cycle time is critical in most laboratories, an optimization was previous non-targeted LC-MS/MS experiments of plasma di- performed with respect to the separation power of the nano- gests. In addition publicly accessible databases, like Peptide LC separation, and the interfacing mass spectrometry. Atlas (http://www.peptideatlas.org/), MRM Atlas (http://www. The feasibility study comprised biobank samples from the mrmatlas.org/) and Global Proteome Machine Proteomics Karolinska Institute, where patient samples had been sam- Database (http://gpmdb.thegpm.org/), were utilized to choose pled, fractionated and stored at −80 °C prior to analysis. We the appropriate peptides representing our seven target proteins. analyzed plasma samples from patients with relapsing- The selected tryptic peptides were synthesized in both heavy- remitting multiple sclerosis (RRMS), secondary progressive labeled and unlabeled forms with purity higher than 97%; in multiple sclerosis (SPMS) and samples from controls. Equal order to determine the best SRM transitions. A mixture of volumes of patient samples were used for depletion and synthetic peptides was analyzed by nanoLC-MS/MS on LTQ XL tryptic digestion, with the net dilution factor of 10 during in data dependent full MS/MS scan mode. The acquired MS/MS sample processing. The protein digests were further diluted

Fig. 2 – (A) Extracted ion chromatograms show the 3 monitored transitions for the endogenous (red) and the heavy labeled peptides (blue) in spiked plasma digest. (B) MRM mass spectra of the 5 analyzed endogenous peptides (red) and the corresponding heavy labeled peptides (blue) in plasma digest. JOURNAL OF PROTEOMICS 75 (2011) 211– 220 215

A B IDTQDIEASHYR 610.3 100 615.5 100 100 80 80 80

60 60 60 667.8 673.0 40 40 40

20 20 495.7 Relative Abundance 20 Relative Abundance 500.8 0 0 16.0 17.0 18.0 19.0 16.0 17.0 18.0 19.0 0 500 550 600 650 700 Time (min) Time (min) m/z

TGLQEVEVK 731.4 739.4 100 100 100 80 80 80

60 60 60 603.3 611.3 422.75 40 40 40 426.75 20 20 Relative Abundance 20 Relative Abundance 0 0 16.0 17.0 18.0 19.0 16.0 17.0 18.0 19.0 0 400 450 500 550 600 650 700 750 Time (min) Time (min) m/z

TEHYEEQIEAFK 607.3 615.4 100 100 100 80 80 80

60 60 60

40 40 40 365.2 743.4 20 20 735.4 20 373.2 Relative Abundance Relative Abundance 0 0 19.5 20.5 21.5 22.5 19.5 20.5 21.5 22.5 0 400 500 600 700 Time (min) Time (min) m/z

EELLPAQDIK 671.4 100 679.4 100 100 80 80 80

60 60 60

40 40 40

20 20 Abundance Relative Relative Abundance 20 784.5 905.6 0 0 792.5 897.5 21.0 22.0 23.0 24.0 21.0 22.0 23.0 24.0 0 650 700 750 800 850 900 Time (min) Time (min) m/z

SPDVINGSPISQK 830.7 100 838.8 951.5 100 100 943.5 80 80 80

60 60 60 716.4 724.4 40 40 40

20 20

Relative Abundance 20 Relative Abundance 0 0 16 17 18 19 20 21 0 16 17 18 19 20 21 700 750 800 850 900 950 Time (min) Time (min) m/z 216 JOURNAL OF PROTEOMICS 75 (2011) 211– 220

C5 C3 5 5

4 y = 1.081x - 0.146 4 y = 0.944x + 0.019 R² = 0.995 3 3 R² = 0.998 2 2 1 y = 0.713x - 0.116 1 y = 0.909x - 0.032 R² = 0.987 R² = 0.999

peak area ratio (L/H) 0 peak area ratio (L/H) 0 01234 01234 conc. ratio (L/H) conc. ratio (L/H) C9 C4

6 10 y = 8.057x - 1.485 5 y = 1.427x - 0.246 8 R² = 0.878 R² = 0.989 4 6 3 4 y = 0.839x - 0.221 2 R² = 0.955 1 y = 0.574x - 0.087 2 R² = 0.991 0

peak area ratio (L/H) 0 peak area ratio (L/H) 01234 01234 conc. ratio (L/H) conc. ratio (L/H) CRP fB 5 6

4 y = 1.062x - 0.182 5 y = 1.452x - 0.181 R² = 0.993 4 R² = 0.993 3 3 2 2 y = 0.883x - 0.072 1 y = 0.839x - 0.109 R² = 0.997 1 R² = 0.994

peak area ratio (L/H) 0 peak area ratio (L/H) 0 01234 01234 conc. ratio (L/H) conc. ratio (L/H)

fH 4

3 y = 0.879x - 0.041 R² = 0.999 2 in plasma digest in pure solution 1 y = 0.795x - 0.050 R² = 0.997 0 peak area ratio (L/H) 01234 conc. ratio (L/H)

Fig. 3 – Standard curves for the seven target proteins in pure solution (red) and in spiked plasma digest (blue). Dilution series of light peptides (in the range of 0.25–125 fmol/μL and 0.5–50 fmol/μL in pure solution and in spiked plasma digests, respectively) were analyzed where the concentrations of heavy peptides were kept at 12.5 fmol/μL. The analyses were performed on an LTQ XL instrument in triplicates.

and spiked with a mixture of heavy isotope labeled peptides proteins was elevated in the secondary progressive multiple prior to LC-MS/MS analysis; resulting in a final plasma dilution sclerosis pool compared to the other two samples; whereas factor of 50. The concentration of every synthetic peptide was the protein level was slightly higher in relapsing-remitting 12.5 fmol/μL in all samples. Besides the individual samples, multiple sclerosis pool than in the control in both cases. We three disease specific pools were created and analyzed observed similar tendency of the complement component 5 randomly in triplicates of each sample. A typical MRM assay (C5) levels that indicated only moderate differences (p=0.0794) readout of human plasma is illustrated in Fig. 4, with a 30- between the three groups (Fig. 5). On the contrary, comple- minute cycle period, quantifying and sequence confirming 5 ment component 3 (C3) level was lower in the disease related biomarkers in the assay. samples compared to control (p=0.0509) (Fig. 5). Unfortunately Figs. 5 and 6 show the results of the MRM assay performed we could not confirm these findings on the basis of the on pooled and individual plasma samples. The analysis of the analysis of individual samples (Fig. 6). The variation of the pooled samples indicated pronounced differences among the relative peak areas within groups was considerable, which three groups in the level of complement component 9 (C9) and prevented us from detecting any significant differences factor H (fH) (Fig. 5), with a p-value of 0.0608 and 0.0628, between the three patient groups (see Table 2). The control respectively (see Table 2). The plasma level of these two group showed the highest homogeneity among the three JOURNAL OF PROTEOMICS 75 (2011) 211– 220 217

4. Discussion

In this study we present an MRM assay for medium abundance plasma proteins using a linear ion trap mass spectrometer. The proteotypic peptide selection was based on empirical 508.6 607.3 C9 data publicly available and from our own experiments, taking 511.3 615.4 C9* trivial rules into account in choosing tryptic peptides [26,27]. 483.3 610.3 C5 To simplify the assay panel, we decided to choose a single representative peptide for each target protein, which gave 486.7 615.5 C5* good and reproducible signal responses in the previous fB 578.3 671.4 plasma MS/MS analysis. During the assay development, we 582.3 679.4 fB* followed more than one fragment ion per single parent ion, which provided a possibility to validate an SRM transition 671.7 830.7 fH beside the collection of full MS/MS spectra and peptide fH* 675.7 838.8 identification. Important criteria of the assay were that the 501.8 731.4 C3 parallel transitions originating from the same peptide should 505.8 739.4 C3* be co-eluted and the relative intensities of the product ions 5 10 15 20 25 30 originated from an endogenous peptide should be identical to Time (min) those from an isotope labeled peptide standard. In order to get sufficient number of data points across the peaks in the final Fig. 4 – SRM signals of 10 transitions in spiked human plasma assay we monitored only one transition for each target digest (SPMS pool). Signals originated from the corresponding peptide, which gives the best signal. Due to the relatively heavy-labeled and endogenous peptide pairs are indicated low scan speed of the linear ion trap instrument, compared to with the same tone. Heavy labeled peptides are indicated with the triple quadrupole instruments, the number of monitored asterisk (*). transitions during a chromatographic run is limited. The total cycle time required for each transition was 200 ms, which resulted in 15 measured points across a 30 s peak width, by monitoring 10 transitions. By increasing the number of monitored daughter ions the number of data points is sample groups. C-reactive protein (CRP) level in the majority of decreasing. Even with the application of two time segments samples was below quantification limit and in most cases CRP resulted in a loss of one third of the data points, which peaks could only be integrated by manually jeopardizing their ultimately insufficient for peak detection and integration. reliable quantification due to high standard deviation. However, the number of transitions may be increased with Two thirds of the RSD values of the triplicate measure- respect of short analysis time by using time segments, where ments were below 20% and the average RSD % of the plasma transitions of only a limited number of peptides are acquired. experiments was 17.6%. Fig. 7 shows two representative This functionality is supported by the new software and is extracted ion chromatograms of the corresponding peptide called scheduled SRM. pairs, where signals from three repeated measurements on We spiked the plasma samples with heavy isotope-labeled the same sample were overlaid. peptides that are identical sequences as the target peptides, prior to analysis. These peptides precisely co-elute with the endogenous peptides, consequently are not only involved in quantification of the peptides but also in validation of the 0.6 selected transitions, as it is represented in Figs. 2 and 7. The

0.5 importance of the isotope standards is nicely illustrated in Fig. 7 where an additional peptide of the plasma sample gives 0.4 higher signal as the target peptide of complement component SP 9 by using two mass filters with 2 Da widths. Another example 0.3 RR is given by complement factor H in a patient plasma sample CTRL where high selectivity is achieved. L/H area ratio 0.2 By the addition of heavy isotopically labeled peptides, the quantification is based on relative intensity of analyte signal 0.1 compared with that of the internal standard. This approach allows more precise quantification than the label free 0.0 C5 C3 C9 CRP f B f H approaches, because the matrix effects are eliminated in most cases. However, the cost of the heavy peptide standards Fig. 5 – Relative peak areas of the complement components in makes the assay more expensive. In general, the internal pooled plasma samples. Samples were analyzed in triplicates standards should be introduced in the sample processing on an LTQ XL mass spectrometer. (SP: secondary progressive procedure as early as possible. In our case, the addition of MS, RR: remitting-relapsing MS, CTRL: control). heavy peptides into the sample before reduction and 218 JOURNAL OF PROTEOMICS 75 (2011) 211– 220

C5 C3 0.18 0.60 0.16 0.50 0.14 0.12 0.40 0.10 0.30 0.08 0.06 0.20 L/H area ratio L/H area ratio 0.04 0.10 0.02 0.00 0.00

C9 f B

0.40 0.70

0.35 0.60 0.30 0.50 0.25 0.40 0.20 0.30 0.15 L/H area ratio L/H area ratio 0.20 0.10 0.05 0.10 0.00 0.00

f H 0.60

0.50

0.40

0.30

0.20 L/H area ratio

0.10

0.00

Fig. 6 – Relative peak areas of the complement components in 18 patient samples. Samples were analyzed in triplicates on an LTQ XL mass spectrometer. (SP: secondary progressive MS samples, RR: remitting-relapsing MS samples, C: control samples).

alkylation is impossible, because of the necessary buffer alternative, recent reports have provided data on applications exchange before trypsination. Based on previous findings by where heavy isotope labeled proteins instead of tryptic Anderson's group, that the analysis variability is increased by peptides as standards allow for a reduction in technical the addition of heavy peptides prior to digestion [10]. variability as well as more accurate quantification [28,29]. Correspondingly, we added the isotope labeled standards to The ionization efficiency of the peptides will be dependent the plasma digests prior to the LC-MS analysis. As an on the background; where the effect of matrix complexity can lead to ion suppression, or, even ion enhancement effects. This matrix effect can be kept under control by the application of co- eluting isotope labeled standards, and by the use of relative – Table 2 Statistical analysis of the multiple sclerosis signal intensities instead of absolute for quantification [30].This patient samples. was confirmed by our experiments in most cases, as the Protein p-value of Kruskal–Wallis test standard curves in pure solution and in complex matrix were very similar (Fig. 3). However, the standard curves of C9 and fB Pooled samples Individual samples peptides were found to diverge in different matrixes (see Fig. 3). Complement C5 0.0794 0.6183 We also observed a variability that could result in RSD% values Complement C3 0.0509 0.4587 in the MRM assay, whereas the average RSD% values in triplicate Complement C9 0.0608 0.4000 measurements typically were found to be 13.84%, 10.42%, Complement factor B 0.1133 0.4114 Complement factor H 0.0628 0.3196 17.77%, 40.44% and 12.08% for C5, C3, C9, fB and fH peptides, respectively. Consequently, we verified that the quantitative JOURNAL OF PROTEOMICS 75 (2011) 211– 220 219

2000 Complement component 9 4000 Complement factor H 1800 3500 1600 3000 1400 2500 1200

1000 2000 Intensity Intensity 800 1500 600 1000 400 500 200

0 0 14 16 18 20 22 24 12 14 16 18 20 Time (min) Time (min)

Fig. 7 – Analytical reproducibility of the MRM-based assay performed on depleted plasma digests. Extracted ion chromatograms of co-eluting ion pairs for heavy labeled (blue) and endogenous (red) peptides. The figure represents three replicate runs on a pooled RRMS plasma digest.

precision will be dependent on the peptide sequence and this should also be taking into consideration in peptide selection Acknowledgement when MRM assay is being developed. Screening through the patient groups, interesting out- We thank Diemut König-Greger and Joel Luette for peptide comes were noticed. We could detect some differences synthesis, Egon Rosén and Martin Hornshaw for instrumen- between the three patient groups by the analysis of pooled tal support at Thermo Fisher Scientific. The authors are samples, in accordance with some earlier findings. We found grateful for funding support from the Swedish Research that the factor H level is increased in multiple sclerosis Council, Vinnova and the Foundation for Strategic Research – samples compared to controls, as it was stated before related The Programme: Biomedical Engineering for Better Health – to serum samples [24]. Similarly, the measured C3 level was Grant No.: 2006–7600, Swedish Research Council grant No: decreased in diseased samples compared to controls that are 2006–6020 and grant No: K2009-54X-20095-04-3, Swedish in correlation with earlier findings by Jongen et al. [31]. Cancer Society (08–0345), Knut and Alice Wallenberg Foun- Nevertheless, the results originated from different studies dation, Crafoord Foundation, Carl Trygger Foundation, Royal are often conflicting for reasons that are due to: i) the small Physiographic Society, Sten Lexner Foundation and Funda- patient number, ii) the patient group selection, iii) the lack of cion Federico. standards in sample handling, as well as iv) the differences in assay type selection. Due to the heterogeneity, and the low sample numbers it was not possible to statistically verify our previous findings by the analysis of individual samples. Hence, it is our conclusion that will be necessary to increase REFERENCES the sample size in further studies, and also to additionally extend the number of target proteins. By extending the MRM [1] Miller D, Barkhof F, Montalban X, Thompson A, Filippi M. assay, we would be able to include different isoforms as well Clinically isolated syndromes suggestive of multiple sclerosis, as other complement components. part 1: natural history, pathogenesis, diagnosis, and prognosis. Lancet Neurol 2005;4:281–8. [2] Harris VK, Sadiq SA. Disease biomarkers in multiple sclerosis potential for use in therapeutic decision making. Mol Diagn 5. Conclusions Ther 2009;13:225–44. [3] Tumani H, Hartung HP, Hemmer B, Teunissen C, Deisenhammer We have developed and applied an MRM assay for quantita- F, Giovannoni G, et al. Cerebrospinal fluid biomarkers in multiple tion of 5 high and medium abundant plasma proteins by sclerosis. Neurobiol Dis 2009;35:117–27. using isotope dilution mass spectrometry strategy. We have [4] Adam P, Sobek O, Taborsky L, Hildebrand T, Tutterova O, Zacek P. successively utilized an “unconventional” linear ion trap CSF and serum orosomucoid (alpha-1-acid glycoprotein) in mass spectrometer for this quantitative work. We have patients with multiple sclerosis: a analyzed a small sample cohort from multiple sclerosis comparison among particular subgroups of MS patients. Clin – patients by using an inflammatory assay panel. Although, we Chim Acta 2003;334:107 10. [5] Vladic A, Horvat G, Vukadin S, Sucic Z, Simaga S. Cerebrospinal could detect differences between the three patient groups in fluid and serum protein levels of tumour necrosis factor-alpha the plasma level of the target proteins, in the future, we need (TNF-alpha), interleukin-6 (IL-6) and soluble interleukin-6 to further increase the sample number in each patient group receptor (sIL-6R gp80) in multiple sclerosis patients. Cytokine to get more reliable results. 2002;20:86–9. 220 JOURNAL OF PROTEOMICS 75 (2011) 211– 220

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