JPROT-01808; No of Pages 10 JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

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Anne von Zychlinski a,⁎, Michael Williams c , Sally McCormick a , Torsten Kleffmann a,b

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Department of Biochemistry, University of Otago, Dunedin, New Zealand Centre for Protein Research, University of Otago, Dunedin, New Zealand c Department of Medicine, University of Otago, Dunedin, New Zealand

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Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins

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AR TIC LE I NFO

ABSTR ACT

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Article history:

Lipoprotein-associated proteins form an intrinsic part of the major plasma lipoprotein

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Received 20 January 2014

classes. There is increasing evidence that the quantity of these proteins per lipoprotein

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Accepted 18 April 2014

particle determines lipoprotein function including redox, inflammatory and thrombotic

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properties and may impact on lipoprotein-related risks for developing heart disease.

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However, only limited information on the relative quantity of these proteins has been published and no comprehensive absolute quantitative data providing the stoichiometry of

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Absolute protein quantification

proteins associated with lipoproteins is available to date. To address this, we performed

Lipoproteins

extensive absolute quantification by mass spectrometry of 17 lipoprotein-associated

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Cardiovascular disease

proteins on VLDL, LDL, Lp(a) and HDL from healthy subjects. For the first time we show

Stoichiometry of apolipoproteins

the exact stoichiometry of apolipoproteins on different lipoprotein classes. The most

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Keywords:

distinct differences were seen in the abundance of all apoCs, apoE and apoF. We further

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revealed strong variations between individual samples, which indicates the complexity of

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the protein complement of lipoproteins and can provide additional insights into

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lipoprotein-related risk factors. This approach has the potential to determine alterations

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in the protein profiles of lipoproteins in disease states such as CVD or diabetes and, if

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performed on large cohorts, to translate into a tool for identifying new candidate

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biomarkers for risk of disease.

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Biological significance A more comprehensive picture about the protein complement on individual lipoprotein classes is the goal of lipoprotein proteomics analyses. Despite many such studies, there is a lack of absolute quantitative data on lipoproteins isolated from individual subjects. The stoichiometry of lipoprotein-associated proteins rather than their presence or absence could provide insights into an individual's predisposition for disease such as heart disease or diabetes. Our study provides a comprehensive overview of the absolute quantity of proteins on the major apolipoprotein classes VLDL, LDL, Lp(a) and HDL. © 2014 Published by Elsevier B.V.

⁎ Corresponding author at: Department of Biochemistry, University of Otago, 710 Cumberland Street, PO Box 56, Dunedin 9054, New Zealand. Tel.: +64 3 479 5190; fax: +64 3 479 7866. E-mail address: [email protected] (A. von Zychlinski).

http://dx.doi.org/10.1016/j.jprot.2014.04.030 1874-3919/© 2014 Published by Elsevier B.V.

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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2.1. Blood sampling

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Human plasma was isolated from 16 healthy participants after overnight fasting who had no self-reported personal or family history of cardiovascular disease. This study was approved by the local Ethics Committee and participants gave written informed consent. For the isolation of VLDL, LDL and HDL, we chose donors with low to non-detectable Lp(a) levels (0–3.8 mg/dl, samples 44, 57, 62, 64, 65, 75, 92 and 95) whereas for isolating Lp(a) we chose donors with elevated Lp(a) levels (>30 mg/dl, samples 53, 54, 56, 58, 112, 136, 139 and 141). Table 1 shows the lipid profile for each donor.

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2.2. Isolation of lipoproteins

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2.2.1. Isolation and purification of VLDL, LDL and HDL

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2. Material and methods

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Lipoproteins are the main lipid transport particles in circulation. Very low density lipoprotein (VLDL) is synthesized and secreted by the liver and transformed into the smaller and more cholesterol-rich, low density lipoprotein (LDL) via lipolysis by lipoprotein and hepatic lipases [1–3]. VLDL and LDL deliver triglycerides and cholesterol to the peripheral tissues for lipid and energy metabolism. They both contain one molecule of the major structural protein apolipoproteinB-100 (apoB-100), a 550 kDa glycoprotein, and are therefore referred to as apoB-100-containing lipoproteins. Human lipoprotein(a) [Lp(a)] is similar to LDL but contains an additional large glycoprotein, apolipoprotein(a) [apo(a)], which is attached to apoB-100 by one disulfide bridge. High density lipoprotein (HDL) is the only non-apoB-containing lipoprotein in circulation and has apoA1 as its major structural protein. HDL is mainly involved in transporting lipids from the periphery back to the liver, in a process known as reverse cholesterol transport. High levels of LDL are associated with an increased risk for CVD, whereas high HDL levels are favorable and implicate a reduced risk [4]. Increased levels of Lp(a) are an established independent and important risk factor for developing premature CVD [5]. Traditionally LDL-total and HDL-total cholesterol levels are used for CVD-risk predictions in patients. However, various studies have shown that particle number rather than cholesterol levels is a much better predictor for CVD [6] and that lipoprotein independent factors can improve the predictive power for CVD. For example, the addition of C-reactive protein measurements in conjunction with cholesterol levels is seen as a simple and inexpensive tool to improve CVD risk prediction [7]. The ratio between apoA1 and apoB-100 has been reported as a better predictor for CVD risk than LDL and HDL cholesterol levels alone [8]. It is likely that other apolipoproteins may serve as additional targets to refine the accuracy of predicting CVD risk in individual patients. The relative abundance of apoCs and apoE declines in the maturation of VLDL to LDL and other apolipoproteins may increase in abundance [9]. As well as structural components, apolipoproteins act as activators or inhibitors of lipases involved in lipolysis i.e. the apoCs [8] or as ligands for receptor-mediated triglyceride clearance in the liver and peripheral tissues i.e. apoE [10]. It is possible that the imbalance of apolipoproteins on specific lipoprotein particles is related to different disease states such as CVD and diabetes. Proteomic studies of different lipoproteins show that their protein load is much more diverse than originally anticipated with many unexpected or novel proteins (summarized in [11,12]) such as protease inhibitors, acute phase response proteins and proteins involved in the complement pathway and immune functions. This diversity coincides with the multiple functions attributed to lipoproteins beyond lipid transport, e.g. redox regulation, hemostasis, inflammation and immunity in HDL (summarized in [13]). For example, response to wounding is the main biological process attributed to Lp(a)-associated proteins, a link that could explain Lp(a)'s atherogenic and prothrombotic properties within sites of vascular injury through interference with coagulation and certain immune responses [14]. The quantity of lipoprotein-associated proteins may have a strong impact on the function of the respective lipoprotein population.

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Despite the great number of lipoprotein proteomic studies published to date, there is a lack of accurate quantitative information about the apolipoproteins and associated proteins on individual lipoprotein particles. The available information is very limited or spans only a small protein dataset [14]. For example, analyses of HDL-associated proteins are mainly of semi-quantitative nature [15,16] or have targeted reconstituted particles [17]. Larger scale quantitative analyses of apolipoproteins and associated proteins have only targeted whole plasma so far [18,19]. A global picture of their absolute amount on the most predominant classes of lipoproteins is still missing. Here, for the first time we show the exact stoichiometry of apolipoproteins on individual lipoprotein classes. We targeted 17 common apolipoproteins and associated proteins that have been identified by most proteomic studies to date or been attributed with interesting properties in conjunction with lipoprotein function and metabolism (summarized in [11]). We determined their exact stoichiometry per particle for VLDL, LDL, and Lp(a) by high resolution/accurate mass (HR/ AM) mass spectrometry using stable isotope labeled standard (SIS) peptides for absolute quantification (AQUA) of proteins. The exact molar ratio of one apoB-100 per VLDL, LDL and Lp(a) particle allows for the absolute quantification of other lipoprotein-associated proteins against it and determination of their stoichiometry per particle. There is no protein with a constant number of molecules per HDL particle. Intensive work has established the number of apoA1 molecules per particle to range between 2 and 5 [20,21]. To avoid any bias we normalized the measured protein quantities to one apoA1 molecule which does not reflect the number of HDL particles. AQUA of proteins using SIS peptides has been successfully applied to many different types of biological samples such as yeast, mice and human tissues, in vitro systems, human cell lines [22] and Lp(a) particles [14]. Our approach allows for simultaneous quantification of many target proteins in individual samples with high precision [23]. This will be critical for determining alterations in lipoprotein proteomes in larger sample numbers and the comparison between healthy and disease state samples.

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1. Introduction

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VLDL, LDL and HDL were isolated from individual 164 plasma samples by a combination of sequential density 165

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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LDL cholesterol mmol/L

HDL cholesterol mmol/L

Trigs mmol/L

Lp(a) mg/dl

3.3 6 4.1 6.6 4.7 3.2 4 4 5.8 5.6 6.1 4.7 5.5 5.5 6.4 6.4

1.5 4.4 2.2 4.7 2.8 1.8 2.6 2.4 4.1 3.8 4.1 2.4 4 3.6 4 3.6

1.37 1.24 1.55 1.23 1.25 1.28 1.09 1.09 1.28 1.35 1.36 2 0.93 1.38 1.77 2.46

1 0.7 0.8 1.5 1.4 0.3 0.7 1.2 0.9 1.1 1.3 0.6 1.2 1.1 1.3 0.7

1 3.8 1 3.4 1 1 1 1 55.8 32.5 62.2 35.9 62.6 37.9 150.1 90.0

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2.2.2. Isolation and purification of Lp(a)

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Lp(a) was isolated and purified by sequential density ultracentrifugation and SE-FPLC according to the procedure described in [14].

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2.3. Preparation of tryptic peptides

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Purified lipoproteins were digested with trypsin (proteomics grade trypsin, Promega Corporation, Madison, WI) in an aqueous

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solution of 25% acetonitrile and 50 mM ammonium bicarbonate using a trypsin/protein ratio of 1/10 (w/w). Digests were incubated overnight at 37 °C. To enhance digestion efficiency, more trypsin [1/20 (w/w)] was added in the morning and digested for an additional 3 h. Digestion was stopped by adding formic acid (FA) to a final concentration of 0.2%.

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ultracentrifugation [24] and size exclusion-fast performance liquid chromatography (SE-FPLC) [15]. Briefly, 2 ml of plasma was over layered with 1 ml of KBr solution of 1.006 density. The sample was centrifuged at 425,000 ×g for 2 h at 10 °C in a Beckman Coulter Optima TL ultracentrifuge using a fixed angle TLA-100.3 rotor. The top layer (d ≤ 1.006) containing VLDL was removed and set aside. The density of the remaining plasma was re-adjusted with KBr to 1.063 and subjected to a second density ultracentrifugation step. The resulting top layer (d = 1.006–1.063) containing LDL was removed and set aside. The density of the remaining plasma was re-adjusted to 1.21 with KBr and subjected to a third ultracentrifugation step. The lop layer containing HDL was removed and set aside. The isolated VLDL, LDL and HDL fractions were subjected to SE-FPLC under isocratic conditions on a Superose™6 10/300GL column (GE Healthcare, Uppsala, Sweden) using a Biorad BioLogic DuoFlow Workstation (Biorad, Hercules, CA) at a flow rate of 0.5 ml PBS/min. Lipoproteins were collected in 0.5 ml fractions in a collection window from 3 to 29 ml. Centered peak fractions [at full width at half maximum (FWHM) of peaks] from SE-FPLC (fractions 15 and 16 for VLDL, fractions 21–24 for LDL and fractions 30–32 for HDL) were pooled, concentrated through Vivaspin500 concentrators (GE Healthcare, Upsala, Sweden) and analyzed via agarose gel electrophoresis using the Helena TITAN™ Lipoprotein gel electrophoresis system according to manufacturer's instructions (Helena Laboratories, Beaumont, TX). Lipoproteins were visualized by Fat Red 7B staining. The FWHM peak fractions for each lipoprotein subclass were used for absolute protein quantification by mass spectrometry. One third of the FWHM peak fractions were subjected to a second FPLC analysis to monitor the purity of the isolated fraction.

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t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20

Total cholesterol mmol/L

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Table 1 – Lipid profile and age of plasma donors. The top 8 were used for VLDL, LDL and HDL profiling, the bottom 8 were used for Lp(a) profiling.

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2.4. Absolute quantification of targeted proteins

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Custom made stable isotope labeled synthetic peptides (SigmaAldrich custom AQUA peptide synthesis, MO, USA) corresponding to endogenous peptides of tryptically digested lipoproteins were used as internal standards for mass spectrometry-based absolute quantification [25]. The following apolipoproteins and associated proteins were quantified: apoA1, apoA2, apoB-100, apoE, apoC1, apoC2, apoC3, apoC4, apoM, apoF, apoD, clusterin, PON1, PAF-AH, complement C3, complement C4-A and apo(a). All peptide sequences (see Table 2) were unique for the targeted proteins as assessed by BLAST searches against the NCBI nr database and are from non-repeat sequences within the protein primary structure.

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Table 2 – Targeted proteins and sequences of SIS peptides.

t2:1 t2:2

Target protein Apo(a) ApoA1 ApoA2 ApoB-100 ApoC1 ApoC2 ApoC3 ApoC4 ApoD ApoE ApoF ApoM C3 C4-A Clusterin PAF-AH PON 1

Peptide sequence LFLEPTQADIALLK LLDNWDSVTSTFSK EQLTPLIK ALVEQGFTVPEIK EWFSETFQK ESLSSYWESAK GWVTDGFSSLK DGWQWFWSPSTFR NILTSNNIDVK AATVGSLAGQPLQER SGVQQLIQYYQDQK AFLLTPR TELRPGETLNVNFLLR DSSTWLTAFVLK ELDESLQVAER GSVHQNFADFTFATGK IFFYDSENPPASEVLR

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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2.5. Hierarchical clustering and statistics

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Hierarchical clustering and statistics were performed using the R software package (version 3.0.1, http://www.r-project.org/). Input data for hierarchical clustering was the square root of the individual protein frequencies. VLDL, LDL and HDL proteins are depicted with an “V”, “L” and “H” in front of the protein name respectively. Fold change differences of protein frequencies per particle between apoB-100-containing lipoproteins were given as the log2 of the frequency ratios. The significance of the fold change differences was tested using the Mann–Whitney U test.

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3. Results

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3.1. Isolation and purification of lipoprotein particles

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We previously described an isolation protocol that yields highly purified Lp(a) [14]. The purity was confirmed by an

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3.2. Protein frequencies of apolipoproteins and associated 291 proteins on VLDL, LDL, Lp(a), and HDL 292 We performed an AQUA approach on 17 targeted apolipoproteins and associated proteins using SIS peptides (Table 2). Previously we demonstrated that our quantification of apolipoproteins by HR/AM measurements using apoB-100, apo(a) and apoA1 in a Lp(a) sample with a ratio of 1:1:1 of all three apolipoproteins shows an excellent linearity (R2 > 0.998 for all linearity of calibration curves) and reproducibility in a range of 2–200 fmol on column loading of endogenous protein [14]. We further confirmed for a set of four proteins ranging between 6 and 500 fmol on column loading that the quantification by HR/AM using either the average peak intensity along the peak at FWHM or the area under the curve at FWHM gives the same results as quantification by SRM using the linear ion trap mass analyzer (Supplemental Fig. 1).

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extensive quality control combining agarose gel electrophoresis, western blot analyses and mass spectrometry [11]. The Lp(a) particles used in the present study were subjected to the same isolation protocol and quality controls. The isolation protocol was then adapted for the purification of VLDL, LDL and HDL. To avoid co-purification of Lp(a) within VLDL, LDL and HDL we chose participants with very low Lp(a) levels. We monitored lipoprotein purity at each step and found that although the lipoproteins isolated by the density centrifugation step appeared as single bands on agarose gels (Fig. 1A), contaminating protein peaks were evident in the FLPC chromatograms (Fig. 1B). Following the SE-FPLC step, however, the final lipoprotein fractions showed only single peaks indicating the removal of contaminating proteins (Fig. 1C).

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Furthermore, they did not contain cysteines, methionines or internal tryptic cleavage sites. Spiking of lipoprotein digests with internal standard peptides was performed as previously described [14]. Briefly 500– 1000 fmol of each digested lipoprotein sample was spiked with a mixture of heavy peptide standards tailored to the different lipoprotein classes. The VLDL standard contained 500 fmol apoE, apoC1/C2/C3/C4, 250 fmol apoB-100, apoA1, 100 fmol apoA2, apoM and 25 fmol of the remaining peptides. The Lp(a) standard contained 500 fmol apoB, apo(a), apoA1, apoC1/C3, 200 fmol apoF, 100 fmol apoC2/C4, 50 fmol apoE, complement C3, apoA2 and 25 fmol of the remaining peptides. The LDL standard contained 500 fmol apoB-100, 100 fmol apoA1, apoC2/C3, apoE, apoF and 25 fmol of the remaining peptides. The HDL standard contained 500 fmol apoA1, apoC1, 250 fmol apoA2, apoC2/C3, apoM, 50 fmol apoF, apoM and 25 fmol of the remaining peptides. Sample aliquots containing 500 fmol of the heavy peptide standard apoE (for VLDL), apoB-100 [for LDL and Lp(a)] and apoA1 (for HDL)/5 μl were loaded onto an in-house packed emitter tip analytical column (75 μm ID Pickotip fused silica packed with C18 beads (5 μm 100 Å) on a length of 8 cm) using an Ultimate 3000 uHPLC system (Dionex Corporation, Sunnyvale, CA). Peptides were eluted through an acetonitrile gradient developed from 100% solvent A (5% acetonitrile, 0.2% FA in water) to 45% solvent B (90% acetonitrile, 0.2% FA in water) in solvent A over either 40 min (for VLDL, LDL and HDL) or 25 min [for Lp(a)] at a flow rate of 800 nl/min. The Orbitrap analyzer was operated in MS mode with a resolution of 100,000 FWHM at m/z 400. The lock mass option of the Orbitrap analyzer was enabled using the signal of [Si(CH3)2O]6 at m/z 445.12003 [26]. Note, for VLDL a second MS run using a 10th of the sample was performed for the quantification of apoC2 and apoC3. The signals of the target peptides were identified by accurate mass retention time pairs based on the procedure described by Silva et al. [27]. The internal heavy peptide standards were used as retention time markers. The intensity ratios between heavy peptide standard and endogenous peptide were calculated based on the MS peak area at FWHM of the chromatographic elution profile of each peptide pair using the Xcalibur software (Thermo Scientific, San Jose, CA).

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Fig. 1 – Purification of VLDL, LDL and HDL. (A) Native agarose gel electrophoresis of lipoproteins after first (ultracentrifugation — even numbers) and second (FPLC — uneven numbers) purification step. Lane 1: plasma control, lanes 2/3: VLDL, lanes 4/5: LDL, lanes 6/7: HDL. Lipoproteins were stained with lipid specific Fat Red 7B stain. (B) Elution profile of lipoproteins after ultracentrifugation and (C) after size exclusion chromatography. All lipoprotein fractions used for proteomic analysis elute as a single peak for each class at the expected position.

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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PON 1 PAF-AH C4a C apoC4 apoM Clusterin C apoD apo E C3 apoA2 apoF

apoC3 apoC2 apoA1 apoC1 apo(a) apoB

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apoC1

apoC4

apoF

apoM

apo E

apoC3

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between 5 and 124 molecules per particle. In contrast, LDL showed no proteins with an average frequency > 1 indicating a massive loss of protein content compared to VLDL. ApoB-100 was the most abundant protein on LDL although in two samples apoA1 was measured at a slightly higher frequency per particle. Lp(a) showed the expected average frequency of one apo(a) per apoB-100. Both apoC1 and apoA1 had an average frequency of >1 and apoC2 and apoC3 were also more abundant than apoB-100 in one and two individual Lp(a) samples respectively. Interestingly LDL and Lp(a) had a very similar apoE content (frequencies of 0.17 and 0.15 respectively). Other analyzed apolipoproteins (apoD, apoM and apoF) were measured at lower average frequencies per apoB-100 containing particle ranging between 0.02 and 0.44 with the highest values of 0.29 for apoM on VLDL and 0.44 for apoF on Lp(a). Other associated proteins (clusterin, PON1, C3, C4-A, PAF-AH) were significantly lower in abundance on all of the apoB-100 containing particles and only two proteins had an average frequency of > 0.1 i.e. complement component C3 with an average frequency of 0.22 per Lp(a) particle and clusterin with an average frequency of 0.12 on VLDL. Considering positive protein identifications in at least six out of eight samples as a minimum count for quantification we were able to measure average frequencies per particle of clusterin and PAF-AH on all apoB-100 containing particles. PON1 was not quantified on LDL (only 2 positive samples), C3 not on VLDL and LDL (only 2 and 1 positive sample respectively) and C4-A not on VLDL (no positive sample). On HDL the frequency of apolipoproteins and associated proteins was normalized to the number of apoA1 molecules. However, apoA1 occurs at a frequency of 2 to 5 molecules per HDL particle which does not allow for an accurate measurement of frequency per particle using the here described method. It still allows quantitative comparison of the composition of targeted proteins relative to apoA1 on HDL particles isolated from different plasma samples. ApoA1 and apoA2 were the most abundant proteins on HDL with an average frequency of apoA2 of 0.85 per apoA1 molecule, followed by apoC2, apoC1 and apoC3 with average frequencies per apoA1 molecule of 0.54, 0.38 and 0.31 respectively. All other apolipoproteins and associated proteins occur at frequencies below 0.2. We did not identify PAF-AH and C3 on HDL and C4-A and clusterin were only detected in 3 and 5 samples respectively. The protein profiles of lipoproteins were visualized by SDS-PAGE which resolved very distinct banding patterns for each of the respective lipoprotein classes (see Supplemental Fig. 2). The banding pattern of two different VLDL samples with variations in the observed frequencies supported the results from the AQUA approach for the larger proteins >15 kDa (for details see Supplemental Fig. 2 and supplemental results and discussion). However, smaller proteins were not sufficiently separated to gain quantitative information based on in gel staining intensities. To illustrate the impact of the numbers of individual apolipoproteins and associated proteins on the total mass of the different particle classes we calculated their molecular weight contribution based on their average frequencies per lipoprotein particle. Fig. 3A shows the average frequency of targeted proteins while Fig. 3B shows the mass contribution of targeted proteins per particle for each lipoprotein class.

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We were able to detect and quantify all targeted apolipoproteins [apoB-100, apo(a), apoA1, apoA2, apoC1, apoC2, apoC3, apoC4, apoD, apoE, apoF, apoM] on all lipoprotein classes with the expected negative detection of apoB-100 on HDL and apo(a) on VLDL, LDL and HDL. For VLDL, LDL and Lp(a), which contain one molecule of apoB-100, we were able to accurately measure the numbers of particles per sample volume based on the measured number of apoB-100 molecules and thus correlate the measured amount of every target protein to its average frequency per particle. We found apoC2 to be the most abundant protein on VLDL with an average of 48 molecules per particle followed by apoC3 with an average of 37 molecules per particle (Fig. 2, Supplemental Table 1). Several other apolipoproteins had an average frequency of > 1 on VLDL including apoC1, apoC4 and apoE. There was however, significant variation between the different samples and some of the targeted proteins showed a broad range of measured frequencies such as apoC3 on VLDL which ranged

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Fig. 2 – Box plots of numbers of target proteins on (A) VLDL, (B) LDL, (C) Lp(a) and (D) HDL. The absolute quantities of targeted proteins are normalized to apoB-100 for VLDL, LDL and Lp(a) and apoA1 for HDL. Asterisks indicate minimum and maximum outliers.

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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the particle structure. In all apoB-100 containing particles apoB-100 has a great mass contribution with 30% in VLDL, 93% in LDL and 42% in Lp(a). An average sized apo(a) isoform of 500 kDa contributes another 46% to the protein mass on Lp(a) resulting in 88% of the total protein mass occupied by apoB-100 and apo(a) (Fig. 3B). The average total molecular weight of proteins (excluding post translational modifications) on the different lipoprotein particles is 1694 kDa, 552 kDa and 1218 kDa for VLDL, LDL and Lp(a) respectively. However, this can vary significantly for those particles that carry larger lipoprotein-associated proteins such as the complement components C3 and C4-A which together add an additional 380 kDa to the total molecular weight of Lp(a) particles. It is however, unlikely that a single particle carries both of those large associated proteins. The frequency data in combination with the calculated mass contribution of different proteins to the total protein mass per particle indicates that there might be various lipoprotein populations that differ significantly not only by their protein composition but also by their total protein mass.

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Fig. 3 – Composition of lipoprotein particles. (A) Average frequencies of targeted proteins per particle for VLDL, LDL and Lp(a) or per apoA1 molecule for HDL. The size of the apoB-100 slice corresponds to a protein frequency of one for VLDL, LDL and Lp(a). For HDL the size of the apoA1 slice represents a frequency of one. (B) Mass contribution of targeted proteins to the total mass of lipoprotein particles. Here the average frequencies were multiplied with the molecular weight of the targeted protein. Note: All targeted proteins with a frequency of less than 0.05 are combined into “others”. ApoA4, apoA5 and apoL-1 were not considered in this study (see text for details).

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Next we compared the average frequencies of apolipoproteins and associated proteins between the different lipoprotein classes. The most abundant proteins on VLDL i.e. apoC1, apoC2, apoC3, apoC4 and apoE are all significantly decreased on LDL (Mann–Whitney U test; p < 0.05, Supplemental Table 2) by fold changes (log2 of the ratios of frequencies on LDL versus VLDL) between − 4 and −7 (Fig. 4A). Although the average frequencies of these proteins are all reduced on Lp(a) compared to VLDL (Fig. 4B), the frequencies of apoC1 (3.4), and apoC3 (0.7) are significantly higher on Lp(a) [p < 0.05] compared to LDL with fold changes between 1 and 5 (Fig. 4C). With the exception of apoM which also was strongest on VLDL all other apolipoproteins (apoA1, apoA2, apoD and apoF) showed higher frequencies on Lp(a) compared to VLDL and LDL. ApoA1 and apoA2 are the major protein components on HDL in numbers and molecular weight. The average ratio of apoA2 molecules versus apoA1 molecules is 0.85 on HDL. ApoA1 and apoA2 also occur on all apoB-100 containing particles with increasing numbers of apoA1 of 0.5, 0.7 and 1.8 molecules per VLDL, LDL and Lp(a) particles respectively. However, the relative amount of apoA2 versus apoA1 is significantly lower compared to HDL (p < 0.05) resulting in apoA2 versus apoA1 ratios of 0.18, 0.01, and 0.16 for VLDL, LDL, and Lp(a) respectively. These significantly lower ratios indicate that the apoA1 molecules detected on apoB-100 containing particles are true components of the here purified particles and not due to an HDL contamination. The frequencies per apoA1 of all other apolipoproteins on HDL were closest to the frequencies measured on LDL with slightly higher abundances of apoC1 and apoC2 on HDL (Supplemental Table 1). Lipoproteinassociated non-apolipoproteins only occur at very low frequencies on all here analyzed lipoprotein classes. The most abundant amongst them are complement component C3 on Lp(a) (frequency of 0.22) and clusterin on VLDL (frequency of 0.12). Complement component C3 was only quantified on Lp(a) when considering more than six positive samples out of

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3.3. Comparison of protein frequencies between different 411 lipoprotein classes 412

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Looking at the protein frequency alone neglects the weight of the analyzed protein. However, simply looking at the protein mass misleadingly over pronounces the weight of large proteins such as apoB-100. The frequencies better represent the impact that proteins have on the function of each lipoprotein while the mass indicates their contribution to

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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Fig. 4 – Differences in fold change of protein frequencies per particle between (A) LDL versus VLDL, (B) Lp(a) versus VLDL and (C) Lp(a) versus LDL. For the comparison of protein frequencies on particles isolated from the same individuals (A) the fold change is expressed as the average and standard deviation of the log2 of individual frequency ratios. For the comparison of protein frequencies on particles isolated from different individuals (B and C) the fold change is given as the log2 of the average ratio of protein frequencies. Significant differences (*) of the median frequencies were tested using the Mann–Whitney U test (see Supplemental Table 2). 449 450 451 452 453 454 455 456

The focus of this study was to provide the frequencies of lipoprotein-associated proteins per particle for each lipoprotein class in healthy donors without cardiovascular disease. We used absolute quantification of proteins by mass spectrometry to elucidate the stoichiometry of apolipoproteins and associated proteins per lipoprotein particle. Since VLDL, LDL and Lp(a) contain exactly one molecule of apoB-100, the measured amount of apoB-100 reflects directly the amount of analyzed VLDL, LDL or Lp(a) particles. HDL does not contain such a unique protein backbone, but always contains apoA1 as its major protein component, however, at varying amounts. The most numerous HDL particles are the small ones containing two apoA1 molecules, whereas the largest HDL particles with 5 apoA1 molecules are relatively rare (personal communication with Jim Otvos, LipoScience Inc., USA). The measured amount of apoA1 was used to normalize the protein frequencies to quantitatively compare the amount

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isolated from different plasma samples. We therefore performed hierarchical clustering of protein frequencies on VLDL, LDL and HDL isolated from individual plasma samples (Supplemental Fig. 3). Protein frequencies on Lp(a) were analyzed as a separate group (Supplemental Fig. 4) because Lp(a) was isolated from different subjects. We then investigated the correlation of frequencies of those proteins that were grouped into clusters. Due to the low sample number we only found a small group of proteins on HDL that revealed correlating frequencies (R2 ≥ 0.9) throughout all eight samples. The measured frequencies of apoC2 and apoC3 both correlate to those of apoC1 with R2 values of 0.902 and 0.901 respectively. Furthermore there is a good correlation (R2 = 0.978) between the frequencies of apoM and apoF on all HDL samples. On apoB-100 containing particles we only found the correlation between apoB-100 and apo(a) on Lp(a) as expected and a weak correlation between apoC2 and apoC3 on VLDL (R2 = 0.892). It is noteworthy however, that the two LDL samples with the highest apoA1 frequencies of 1.81 and 2.28 are the only samples (P57 & P 64) with detectable amounts of PON1 on LDL. Those two subjects showed the highest triglyceride and LDL cholesterol values of all eight subjects (Table 1). Based on the small number of investigated samples we could not identify any protein frequency correlation between different lipoprotein particles. It is most likely that such correlations only occur in a certain group of individuals and not throughout all investigated subjects. In our cluster analysis we found two main groups of samples that were separated into cluster 1 (P44, P75, P92 and P95) and cluster 2 (P57, P62, P64 and P65). The two samples P62 and P65 which show very high protein frequencies of various apoCs on VLDL are clearly separated from all other samples. Also the two samples (P57 and P64) with PON1 detection and high apoA1 values on LDL were clustered together into a sub-cluster in cluster 2. However, the identification of signatures that can cluster subjects based on their protein frequencies of apolipoproteins and associated proteins on different classes of lipoprotein particles clearly requires higher sample numbers including healthy and CVD subjects.

eight as a significant number for quantification. PAF-AH showed similar frequencies on all apoB-100 containing particles with a slightly higher abundance on VLDL and PON1 was most abundant on HDL.

3.4. Comparison of the protein complements between individual samples We next asked the question if frequencies of some of the apolipoproteins and associated proteins correlate on particles

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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fragments must not include missed or non-tryptic cleavage sites, be of medium hydrophobicity and should not include methionine and cysteine which can be partially modified. For the same reason we have generally included only one peptide per protein since many of the apolipoproteins are small proteins that often yield only a single suitable tryptic peptide. We therefore previously tested and optimized the efficiency of the tryptic digestion for our lipoprotein samples [14]. The point of steady state of digestion was confirmed by absolute quantification of targeted peptides (data not shown) and in Lp(a) by an accurate ratio of 1 between apoB-100 and apo(a). The here targeted 17 proteins provide a good overview of the stoichiometry of apolipoproteins and associated proteins on the major lipoprotein classes VLDL, LDL and Lp(a) as well as protein amounts normalized to apoA1 on HDL. The stoichiometry or frequency of protein components per particle may provide a good indicator of their functional impact. For example van Dijk and co-workers showed that the rate of VLDL triglyceride lipolysis was inversely correlated with the apoE content in apoE−/−U LDL receptor−/− mice until a threshold of apoE was reached. Beyond that point more apoE had a detrimental effect on VLDL-triglyceride lipolysis by lipoprotein lipase (LPL) and actually resulted in severe hypertriglyceridemia [32]. It could also be used as an accurate measure to compare the composition of lipoprotein classes between different subjects. ApoB-100 is considered the major protein component on VLDL, LDL as well as Lp(a) in conjunction with apo(a). However, due to the large size of apoB-100 and also apo(a) on Lp(a), apolipoproteins and associated proteins may be underestimated in their abundance, e.g. 60 copies of apoC2 would be required to match the molecular mass of one apoB-100 molecule on VLDL. We found on average a 48 fold higher number of apoC2 molecules compared to apoB-100 on VLDL particles which underlines the important function of apoC2 for this class of lipoproteins. ApoC2 is the most abundant protein on VLDL followed by apoC3, apoC4 and apoC1. These four proteins are involved in triglyceride and cholesterol metabolism amongst other functions [33]. ApoC2 aids the metabolism of VLDL by activating LPL allowing for the hydrolysis of triglycerides and delivery of fatty acids to tissues. The deficiency phenotype for apoC2 is indistinguishable from LPL deficiency and is characterized by a significant elevation in triglyceride levels. ApoC3, on the other hand reduces the metabolism of VLDL particles by the liver through inhibition of LPL. Low apoC3 levels favor a healthier lipid profile with reduced triglyceride levels which accounts for apoC3's implication in CVD [34]. A few studies have investigated the importance of some of the apoCs on specific lipoprotein classes. Of particular interest are the observations that apoC1 when bound to HDL seems to inhibit CETP (cholesteryl ester transfer protein) activity in normolipidemic subjects whereas it loses part of this ability in dislipidemic patients [35]. Jensen and coworkers show that apoC3 on HDL may increase the risk of heart disease and demolish HDL's cardio protection [36]. Another study by the same group points out that it might be the balance between the apoC3 and apoE content on LDL which may be responsible for the progression of atherosclerosis and the occurrence of coronary events [37]. The average frequencies of apolipoproteins measured on the different lipoprotein particles are in good agreement with the limited published data that is available. A study

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of targeted proteins relative to apoA1 but does not reflect the actual number of HDL particles. A suitable method to isolate highly purified native lipoproteins was crucial to the success of our analysis. The most commonly used method for the isolation of lipoproteins is density ultracentrifugation [24,28]. However the high ionic strength and shear forces during prolonged centrifugation times may result in the dissociation of apolipoproteins and associated proteins [29]. Whereas FPLC alone as an alternative method [30] results in lipoproteins co-purified with plasma proteins, as noted by Kontshu and Chapman [29]. The here used combination of short density centrifugation steps with isocratic FPLC yielded pure lipoprotein fractions [14]. Agarose gel electrophoresis showed clean bands for each of the different purified lipoproteins and the FPLC chromatograms confirmed a single peak at the expected position for the respective lipoprotein classes. However, since these methods are of lower sensitivity for the detection of contaminating lipoproteins than the here used absolute protein quantification by mass spectrometry we used the actual mass spectrometry data to evaluate the purity of lipoprotein fractions. We could exclude any cross-contamination by Lp(a) by the negative detection of apo(a) molecules in all other lipoprotein fractions. The absence of other apoB-100 containing lipoproteins in the Lp(a) fraction was confirmed by the exact ratio of apoB-100 versus apo(a) of 1. A cross-contamination with other apoB-100 containing particles would result in a higher amount of apoB-100 compared to apo(a) [14]. A relevant cross contamination with HDL would mostly alter the numbers of apoA1 and apoA2 molecules measured on apoB-100 containing particles and would be indicated by an apoA2 versus apoA1 ratio close to 0.85 as determined for HDL in our and other studies. A recent consensus statement on HDL [31] describes that 65% of the protein mass on HDL is composed of apoA1 and 15% of apoA2. Considering the molecular weights of both proteins without signal peptide this translates into a frequency of 0.80 apoA2 per apoA1, which is in a very close agreement with our data. The relative amount of apoA2 however, was significantly reduced in all apoB-100 containing lipoproteins and we measured average apoA2/apoA1 ratios below 0.2. This firstly excludes a relevant contamination with HDL and secondly confirms a significant presence of apoA1 on VLDL and Lp(a) particles. We also did not detect any apoB-100 or apo(a) in the HDL fraction confirming the purity of HDL particles. There is no unique protein or protein ratio to discriminate between VLDL and LDL. However, VLDL shows a characteristic protein profile with very high average frequencies of all apoCs and apoE which is not reflected in LDL where we measured rather low frequencies of all apolipoproteins. This is consistent with the described changes in the iTRAQ profile during the maturation of LDL from VLDL [9] and further indicates the purity of our VLDL and LDL fractions. We targeted 17 of the most abundant apolipoproteins and associated proteins (as shown in Table 2) on VLDL, LDL, Lp(a) and HDL isolated from individual subject. We did not include apoA4, apoA5 and apoL1 in our analysis because no suitable tryptic peptide that would allow an accurate quantification could be detected in our shotgun proteomics data (data not shown). Stable isotope labeled peptides that mimic tryptic

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Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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This study is the first to quantitatively characterize the individual protein cargo of VLDL, LDL, Lp(a) and HDL isolated from individual healthy subjects and provides an overview of the distribution pattern of frequencies of targeted apolipoproteins and associated proteins on lipoprotein particles. Our data identifies distinct differences between all four classes of lipoproteins and reveals sample specific dynamics, which when analyzed in larger cohorts may hold important information to gain further insights into lipoprotein complexity and quantitative alterations of protein profiles associated with CVD or diabetes. The here conducted AQUA approach could translate into a tool for the discovery of better candidate biomarkers/protein signatures to diagnose lipoprotein-related CVD conditions. Our AQUA peptide mixes tailored to the different lipoproteins are not limited to the number of added peptides and more peptides targeting different protein species can be included.

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conducted by Sun et al. used iTRAQ to investigate up- and down regulation of proteins between pooled VLDL and LDL samples [9]. They reported lower levels for PON1, apoM, albumin (ALB) and apoF compared to higher levels for all apoCs, apoE, serum amyloid A4 (SAA4) and apo(a) on VLDL versus LDL with apoA1, apoA2 and apoL unchanged. We showed similar trends for most of the proteins in our study, but lower levels of apoA1 and apoA2 on LDL. We also did not detect apo(a) in either one of these lipoprotein classes. Interestingly, the frequencies we observed for apoC1, apoC2 and apoC3 are indeed decreasing from VLDL to LDL but are still higher on Lp(a) compared to LDL which may be suggestive of pathways through which Lp(a) is formed from apoB-100 containing particles in circulation. The display of the lipoproteins by SDS-PAGE confirms distinct protein profiles for each of the lipoprotein classes and indicates individual variations in the quantity of certain apolipoproteins. Overall, the most significant differences between all lipoprotein classes were seen in the absolute amount of all apoCs. Furthermore, we found a correlation between the frequencies of apoC1, apoC2 and apoC3 on HDL even in the small number of analyzed samples. Two other proteins which showed differences between the lipoprotein classes are apoE and apoF. Vaisar et al. reported a range of 750–800 μg (apoA1) and 10–20 μg (apoE) per mg of pooled HDL, which corresponds to 26.7–28.5 pmol (apoA1) and 290–580 fmol (apoE). This translates into frequencies per apoA1 molecule of 0.01–0.02, which is close to our average frequency of 0.07. ApoE mediates cholesterol metabolism and it is essential for the normal catabolism of triglyceride-rich lipoproteins. With an average frequency of 4.96 it is significantly enriched on VLDL. ApoF is reported to be an inhibitor of CETP [38] and it has been shown that compositional changes that alter the surface-to-core lipid ratio of LDL are responsible for apoF binding and activation [39]. However its physiological function and involvement in lipoprotein metabolism is not fully understood. In our study apoF was significantly higher on Lp(a) compared to all other lipoproteins. It is most likely that the stoichiometry of all apoCs together with apoE and apoF (plus other proteins and factors) and not simply their presence or absence might play an important role for lipoprotein metabolism such as the maturation of LDL from VLDL or the clearance of cholesterol and cholesterol esters from the periphery through HDL or provide more insights into the still largely unknown functionality of Lp(a). Our results fall within the range of quantification previously described for some apolipoprotein species on different lipoprotein particles. Slight discrepancies are to be explained by the different isolation protocols, quantification methods, instrumental equipment and sample characteristics. Overall the comparison proves that our method is detecting a true snapshot of the analyzed lipoprotein species. However, comparing the average values does not reflect the strong sample specific variations that we can see between the different individuals. The quantitative protein makeup varies dramatically between individuals and might hold important information for the discovery of protein signatures indicative for certain conditions and alterations of lipoprotein profiles.

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The authors thank Rebecca Oskam-Schmidt for recruiting blood donors and taking of blood samples and Allan Carne for assisting in SDS-PAGE of lipoproteins. Funding for this study was obtained from the Otago School of Medical Sciences Dean's Bequest funds (TK) and the Otago Medical Research Foundation (AvZ, grant AG 305). AvZ is supported by a research fellowship from the New Zealand Heart Foundation (Grant No 1467).

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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

879

Please cite this article as: von Zychlinski A, et al, Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.04.030

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