SCIENCE CHINA Chemistry • ARTICLES • · SPECIAL TOPIC · Mass Spectrometry Analysis

May 2014 Vol.57 No.5: 723–731 doi: 10.1007/s11426-013-5036-0

A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins LI WenWen1,2,3, WANG QuanHui4,5, CHEN JianJun2,3, ZHOU Jian2,3, ZHOU XinYu1,2,3 & XIE Peng1,2,3* 1

Department of Neurology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China 2 Chongqing Key Laboratory of Neurobiology, Chongqing 400016, China 3 Institute of Neuroscience, Chongqing Medical University, Chongqing 400016, China 4 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China 5 BGI-Shenzhen, Shenzhen 518083, China Received September 27, 2013; accepted November 12, 2013; published online November 25, 2013

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction: apolipoprotein H (APOH), apolipoprotein J (APOJ), apolipoprotein A4 (APOA4), apolipoprotein E (APOE), and apolipoprotein D (APOD). The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600, followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500. Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20% higher than corresponding non-labeled peptides. Through optimized transitions and parameters, we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins. The results indicated that the MRM signals of four target APOs were significantly increased after depletion, while the MRM signal of one APO, APOD, was decreased. Furthermore, the relative abundances of the five target APOs in healthy human plasma were stable, and the ranking of these proteins according to their MS responses changed slightly. Therefore, we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma. mass spectrometry, mTRAQ, MRM, apolipoprotein, APO, plasma

1 Introduction Apolipoproteins (APOs) play a well-established role in the transport and metabolism of lipids [1] and are becoming more important in both basic biological investigations and clinical biomarker studies. APOs have been implicated in a wide variety of disorders, including cardiovascular disease [2], cancer [3], neuropsychiatric disorders [4], diabetes [5], and inflammation [6] (Figure S1). Our lab’s work previously focused on plasma biomarkers for the diagnosis of *Corresponding author (email: [email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2013

neuropsychiatric disorders [7]. One previous study that employed a quantitative proteomic approach based on isobaric tags for relative and absolute quantitation (iTRAQ) and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) found that plasma APO levels were significantly perturbed in majorly depressed patients [8]. Moreover, 5 APOs––apolipoprotein H (APOH, beta2-glycoprotein 1), apolipoprotein J (APOJ, clusterin), apolipoprotein A4 (APOA4), apolipoprotein E (APOE), and apolipoprotein D (APOD)––have been associated with neuropsychiatric disorders and cognitive dysfunction [4, 9, 10]. We hypothesized that these 5 candidate APOs may serve as chem.scichina.com

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potential diagnostic and prognostic biomarkers of neuropsychiatric disease. In order to support APO biomarker development, improved laboratory-based quantitative methods for APOs are needed. Several studies have provided evidence that MS-based multiple-reaction monitoring (MRM) may be useful in the quantification of APOs in human serum and plasma. MRM, which has been established as the most promising approach for the simultaneous relative quantification of many known proteins in a single analysis without the cross-reacting interferences often found in multiplexed immunoassays (thereby negating the need for specific antibodies) [11], can be used to develop comprehensive biomarker panels with both high sensitivity and selectivity [12–14]. One specific method, MRM tags for relative and absolute quantitation (mTRAQ), uses MRM to target tryptic peptides from the protein of interest, and the non-isobaric tags quantify each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amounts of synthetic peptides tagged with one of mTRAQ labels, when used as quantification standards in a mixture with the opposingly labeled digested sample, finally determine the absolute amounts of the corresponding protein in the sample. The ability to label the sample and reference peptides with either one of two possible combinations is an inherent advantage of the mTRAQ method because it provides a means by which to verify the reported ratios [15]. However, with the notable exceptions of APOA1 and APOB, there are no currently available automated nephelometric immunoassay platforms that are able to measure multiple APOs in a single run [16]. In two previous studies, Kuzyk et al. [11] detected 45 serum proteins (including 9 APOs), and Kay et al. [17] analyzed 12 serum APOs (but excluded APOH and APOJ). Unfortunately, these approaches to multiplexed protein expression profiling did not fully cover all 5 candidate APOs. Therefore, it is necessary to develop and assess a novel multiplexed assay for the systematic detection and quantification of all 5 candidate APOs. In this study we developed and assessed a liquid chromatography multiple-reaction monitoring-mass spectrometric (LC-MRM/MS) method using mTRAQ stable isotope-labeled peptides as internal markers to systematically quantify the 5 clinically promising plasma APOs: APOH, APOJ, APOA4, APOE, and APOD. Such a method would be helpful in correlating APO levels with various neuropsychiatric disease states. The effects of depletion and non-depletion of highly abundant plasma proteins on MRM-MS sensitivity were also assessed.

2 Experimental 2.1 Immunoaffinity depletion of highly abundant plasma proteins This study was approved by the Ethical Committee of

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Chongqing Medical University, and samples were collected after written informed consent was obtained from the donors as previously described [7]. A set of 12 donors, consisting of both males and females, fasted for 12 h prior to blood collection. Blood was collected into 10 mL BD Vacutainer lavender tubes with 18 mg of K2-EDTA (reference number 367525, BD, Plymouth, UK). Immediately following blood collection, samples were centrifuged at 1500 g for 15 min at 22 °C to pellet the cells. The plasma was then divided into equal aliquots and immediately frozen at 80 °C until use. Samples were depleted of the 14 most highly abundant plasma proteins using the Human 14 Multiple Affinity Removal System MARS-Hu 14 (Hu14 4.6 × 50 mm, 5188– 6557, Agilent Technologies, Santa Clara, CA, USA). Briefly, 20 μL of plasma was diluted four times with Buffer A (Part #5185–5987, Agilent) to a final volume of 80 μL. Samples were filtered through 0.22 μm spin filters by centrifugation at 16000 g for 1 min and injected onto the depletion column using an Agilent 1260 HPLC system. Low abundant proteins were collected between 5.0–7.5 min and stored at 80 °C until analysis. Buffer A was used for the equilibration, loading, and washing steps. The column was regenerated after each sample with Buffer B (Part #5185-5988, Agilent) that was used for elution of the bound, highly abundant proteins from the cartridge. The experiment was conducted at room temperature according to the manufacturer’s protocol. The less-abundant proteins were concentrated and desalted using 5000 Da cutoff filters (Agilent 5185–5991). 2.2

Peptide selection for MRM analysis

The experimental workflow is displayed in Figure S2. Plasma samples were precipitated with acetone at 20 °C overnight, followed by ultrasound-assisted dissolution in 0.1% SDS and protein concentration determination using the Bradford assay. Proteins were then separated by 12% SDS-PAGE and stained with Coomassie Blue. The protein bands corresponding to the known molecular weights of the five APOs were excised and in-gel digested by trypsin overnight. The tryptic peptides were extracted from the gel particles by acetone nitrile (ACN), followed by vacuumdrying and reconstitution in 0.1% formic acid. Mass spectrometry analyses were performed on a TripleTOFTM 5600 System (AB Sciex, Foster City, CA, USA) packed with a Nanospray III source in information-dependent acquisition (IDA) mode. The ion spray voltage was set at 2.5 kV, the curtain gas and nebulizer gas at 30 psi and 15 psi, and the interface heater temperature at 150 °C. For collision-induced dissociation of all precursor ions, the sweeping collision energy was set at 35 ± 5 eV and the dynamic exclusion was set at half of the peak width (18 s). The acquired data were searched against the Uniprot Human database using Mascot (v. 2.3, Matrix Science,

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London, UK). The parameter settings were configured as follows: allowance of up to one missed cleavage; carbamidomethylation of cysteine, N-terminal pyroglutamylation, and oxidation of methionine as variable modifications; peptide tolerance < 0.05 Da; and MS/MS tolerance < 0.05 Da. Reliably matched peptides should have met FDR < 0.01 and scored > 25. According to the sequence and mass spectral quality of the identified peptide in each APO, in addition to the reported criteria for MRM analysis, the candidate peptides for each APO were selected. 2.3

mTRAQ labeling and transition optimization

Plasma proteins were reduced in 10 mM DTT at 56 °C for 60 min, alkylated in 55 mM iodoacetamide in the dark for 60 min at room temperature, and digested with trypsin (50:1 protein to trypsin) overnight at 37 °C. Duplexed mTRAQ labeling was then conducted to quantify the abundances of APOs in the plasma samples. The synthesized peptides and protein digestions of the plasma samples were treated with mTRAQ reagents Δ00 and Δ08, respectively, and the labeled synthetic peptides with certain concentration were spiked in each plasma sample as internal standards. The collision energy (CE) voltages were optimized for each transition. The theoretical CE voltages for MRM ion pairs were first calculated using a generic formula (CE voltage = a × (precursor m/z) + b), in which the parameter pairs of a and b were set at 0.058 and 10 for singly charged ions and 0.044 and 9 for doubly charged ions. Under the MRM followed by data-dependent product ion scans (MRM-EPI) in acquisition mode, MRM peaks for each transition were acquired by setting the gradient CE voltage at a step of 1.0 eV, based upon the theoretical CE voltage. The optimal CE voltage was defined as the voltage that responded to the highest MRM signals for each transition as evaluated by MRMPilotTM (v. 2.1, AB SCIEX, Foster City, CA, USA). 2.4

LC-MRM/MS analysis of APOs in plasma

The LC-MRM/MS analysis was performed as previously reported [9]. Briefly, nanoLC separation of infused peptide solutions was performed with a nano Acquity Ultra Performance LC system with autosampler (Waters Assoc., Milford, MA, USA). The mobile phase consisted of Solvent A (0.1% aqueous formic acid) and Solvent B (98% acetonitrile with 0.1% formic acid). Peptides were separated on a nanocapillary column (XD-5017, 75 μm id, 3 μm particle diameter, 200 Å pore size, 15 cm length, Zhengdan, Beijing, China) at 300 nL/min, eluted with a gradient of 3%–10% Solvent B for 2 min, 10%–30% Solvent B for 8 min, and 30%–90% Solvent B for 3 min. The QTRAP 5500 with a nano-electrospray ionization source was used for LC-MRM/MS analysis with the fol-

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lowing parameters: ion spray voltage, 2800 eV; curtain gas, 30 psi; ion source gas, 20 psi; collision gas, high; interface heater temperature, 150 °C; declustering potential, 100; entrance potential, 10; and Q1 and Q3 resolution at 0.6–0.8 Da full width at half-height. MRM acquisition methods were constructed using three ion pairs per peptide with fragment-ion-specific CE voltages and retention times. 2.5

Data analysis

The data acquired with the MRM method were processed using Skyline (v. 1.4.0.4421), using the default settings for noise percentage and base-line subtraction window. The peak areas of Δ08-labeled transitions were normalized by those of Δ00. The amounts of the corresponding peptides were represented by the summed areas of the corresponding transitions, while the amounts of proteins were calculated by summing the values of the peptides. All the data were manually verified to ensure correct peak detection and accurate integration. The logarithmic values of the peak area of each APO were further used to describe the distribution of the APO abundances in the plasma samples.

3

Results and discussion

3.1 Selection and synthesis of APOs peptides for MRM analysis Selection of a peptide to represent a target protein in an MRM assay is a crucial step that dramatically affects the ultimate sensitivity and specificity of the assay and requires that the selected peptides are both detectable in every sample and reproducibly observed between sample preparations. When selecting a quantitative peptide, five critical factors should be taken into account: (1) the peptide length should range from six to twenty-five amino acid residues; (2) the peptide should be limited to a charge state of three or less; (3) the peptide should contain no missed cleavage sites; (4) amino acid sequences containing cysteines or methionines that are susceptible to chemical modification should be excluded; and (5) the amino acid sequence of the peptides must be unique to the target protein. In addition to these five theoretical criteria, peptides with good MS signals are critically important, and good signals can only be ascertained through their MS spectra. In order to accomplish this task, we mixed 12 plasma proteins, resolved them on a 12% SDS-PAGE gel, and conducted MS analysis. The gel bands representing four APO proteins according to their molecular weights (APOH: 38 kDa, APOJ: 52 kDa, APOA4: 45 kDa, APOE: 36 kDa, and APOD: 21 kDa) were excised and subjected to in-gel reduction, alkylation, and tryptic digestion. A proportion of the tryptic peptide mixture was analyzed by TripleTOFTM 5600 MS (AB SCIEX, Foster City, CA, USA). The peptide identification findings were further analyzed by

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MRMPilotTM (v. 2.1, AB SCIEX, Foster City, CA, USA) and filtered with a false-discovery rate (FDR) of less than 1%. Through this approach, the five candidate APOs were successfully identified. The unique peptides and their respective sequence coverages identified from each APO are listed in Table 1. The results of peptide selection were based on automated screening by MRMPilotTM (v. 2.1, AB SCIEX, Foster City, CA, USA) and manual inspection (Table 1). Twelve peptides identified as matching the five APOs and meeting the above criteria were selected, with at least two peptides per APO (except for APOD). On the basis of the amino acid sequence for the selected APO peptides, the peptides were synthesized by SciLight Biotechnology (Beijing, China). Synthetic peptides with a chemical purity of more than 95% were deemed suitable for quantification. 3.2

Optimized selection of MRM Q1/Q3 ion pairs

Creating the most sensitive MRM assay for a peptide requires judicious selection of the precursor ion charge state

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and fragment ions, combined with empirical tuning of the MS parameters. The signal intensity of an MRM Q1/Q3 ion pair for a peptide is determined by the combined ionization efficiency of the precursor peptide and how readily it can be dissociated into fragments. The MS/MS spectra of identified peptides from each APO were loaded into an MRMPilotTM (v. 2.1, AB SCIEX, Foster City, CA, USA), and the MRM transitions and the correspondent collision energy (CE) for each ion pair were computed in silico. The fragment ions of each peptide with the highest intensity were selected. Consequently, to optimize the ion transmission efficiency of each ion pair, the declustering potential (DP) was fine-tuned according to the computed value. To ensure the maximum MRM signal, the CE voltage was varied and the signal intensities from Q1/Q3 ion pairs for each peptide were ranked. The CE generating the most intense precursor and fragment ion pair was selected as the optimal value for MRM data acquisition. A single set of measurements for the CE optimization chromatograms acquired for the peptide LLPHANEVSQK-Δ00 is provided as an illustrative example in Figure 1; the MS signals were acquired under 12 CE

Table 1 Peptide selection and transition optimization Protein

Protein mass

No. a)

Peptide sequence b)

APOH (P02749)

39584

15

ATVVYQGER

Coverage (%) c) 61.1

EHSSLAFWK

APOJ (P10909)

53031

8

ASSIIDELFQDR

APOE (P02649)

45371

36246

29

14

IDQNVEELK

29.2

21547

2

Fragment Type

CE d)

511.8

652.3 751.4 850.4 664.4 751.4 838.5 587.3 922.4 1035.5 409.2 609.3 853.5 617.4 859.5 974.5 589.3 702.4 799.4 456.7 505.3 775.4 399.7 489.2 588.3 801.4 902.5 1015.5 1098.6 439.2 985.5

2+/y5 2+/y6 2+/y7 2+/y5 2+/y6 2+/y7 2+/b6 2+/y7 2+/y8 3+/y3 3+/y5 3+/y7 2+/y5 2+/y7 2+/y8 2+/y5 2+/y6 2+/y7 3+/y82+ 3+/y92+ 3+/y7 2+/y72+ 2+/y4 2+/y5 2+/y7 2+/y8 2+/y9 2+/y10 2+/b4 2+/y9

22.2 21.7 21.2 23.5 23.1 22.7 28.9 27.2 26.7 28.8 27.8 26.5 23.5 22.2 21.7 21.8 21.2 20.8 19.7 17.5 18.1 22.5 22.1 13.6 31.7 31.2 32.6 26.7 30.2 27.4

697.4

667.3

62.3

544.3

LAPLAEDVR

492.3

LLPHANEVSQK

412.6

LGPLVEQGR

58

SELEEQLTPVAEETR

APOD (P05090)

Q3

552.8

EPQDTYHYLPFSLPHR

APOA4 (P06727)

Q1

NPNLPPETVDSLK

484.8

865.9

6.6

712.4

a) Identified unique peptide numbers, b) selected peptide sequence, c) sequence coverage, d) optimized CE.

mTRAQ-Δ00 labeled Q1 581.8

692.9

767.4

714.0

684.4

562.3

506.0

554.8

936.0

852.5

Q3 652.3 751.4 850.4 804.5 891.5 978.5 727.4 922.4 1035.5 409.2 609.3 853.5 757.4 999.5 1114.6 589.3 702.4 799.4 526.8 575.3 915.5 399.7 489.2 588.3 801.4 902.5 1015.5 1238.7 579.3 1125.6

CE d) 26.8 27.8 26.8 32.8 28.8 35.8 33.5 34.5 32.5 34.4 34.4 30.4 36.5 27.5 28.5 28.1 28.1 25.1 22.1 21.1 20.1 25.8 28.8 26.8 41.6 40.6 42.6 39.5 35.3 33.5

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Figure 1 Collision energy optimization chromatograms acquired for the peptide LLPHANEVSQK-Δ00. The red line corresponds to the predicted collision energy voltage. The collision energy voltage was then varied by a step size of 1.0 eV to compare the fragment ions and signal intensities under different collision energy voltages. Each colored (a) line and (b) bar represent the ion current for the transitions (506.03+/575.32+) selected for each collision energy voltage. The different collision energy voltages are represented in the figure legend as steps 10 to 10 (partial steps have been shown). Step -4 ([a] orange line, [b] orange bar) yielded the maximum ion peak area; therefore, the corresponding collision energy voltage (21.1 eV) was deemed to be the optimal collision energy voltage for this ion pair.

voltages varied by a step size of 1.0 eV centering around the median CE voltage of 25.1 eV. Comparison of the signal intensities clearly showed that the CE voltage of 21.1 eV yielded the maximal ion peak area. Therefore, 21.1 eV was deemed the optimal CE voltage for the ion pair 506.03+/575.32+. The optimal CE voltages for all ion pairs were determined with this procedure (Table 1). This relative quantification highlights the need for heavy-isotope-labeled internal standards that can normalize MRM peak areas across several samples to enable more reproducible, accurate quantifications [18]. Here, the mTRAQ-Δ00 labeled synthetic peptides served as the internal standards and were spiked into samples labeled with mTRAQ-Δ08 to normalize the MRM signals. Because these versions of the peptides differ in the isotopic content of their labels, they co-elute during the course of the LC separation by virtue of the mass differences between the labeled peptides and thereby enable the investigator to distinguish synthetic peptides and sample peptides. Importantly, the optimal CE voltage increased by an average ratio of 15%–20% in mTRAQ-labeled peptides (Table 1). With some peptides, such as the three ion pairs of SELEEQLTPVAEETR, the optimal CE voltage increased approximately 30% after mTRAQ labeling. This result suggests that it may be necessary to increase CE voltage in mTRAQ-labeled peptides. Moreover, the increments by which this voltage step-up should be performed may be dependent upon the characteristics of the peptides. 3.3 Comparison of the sensitivity of depleted plasma and non-depleted plasma After completing the MRM parameter optimization for the five APOs, we planned to detect them in the plasma sam-

ples. However, we first had to resolve a serious issue: the necessity of depleting the most highly abundant plasma proteins. Although plasma is considered to be the most preferred biosample for proteomic studies that aim to identify relevant biomarkers for neuropsychiatric disease diagnosis and monitoring [19], there are challenges related to the wide dynamic range of protein concentrations and sample complexity. The most highly abundant plasma proteins, including albumin, immunoglobulins, transferrin, and macroglobulin, comprise approximately 99% of the total plasma protein content [20]. Biomarkers that play important roles in clinical diagnosis are typically less abundant, accounting for only 1% of the total plasma protein content. Conventional proteomic methods, such as LC-MS/MS, have substantial limitations in detecting less-abundant plasma proteins due to the masking effects of highly abundant plasma proteins and ion suppression in electrospray MS. Therefore, identification of less-abundant plasma proteins requires the depletion of highly abundant plasma proteins. Such depletion is also commonly recommended for MRM-based targeting proteomics. However, because APOs are a group of proteins of intermediate abundance in human plasma, it is unclear whether APOs are affected by highly abundant proteins and whether depletion of highly abundant plasma proteins is necessary for MRM analysis of APOs. Although multiple techniques have been applied to the reduction of sample complexity in human plasma, immunoaffinity-based depletion of highly abundant proteins remains the most specific technique for reducing the dynamic range of complex biological samples [21]. Although reports have suggested that multi-affinity removal system (MARS) column depletion of highly abundant plasma proteins can result in the concomitant removal of non-targeted lessabundant plasma proteins [22], MARS has been demon-

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strated to improve the intensity of less-abundant plasma proteins and to increase the resolution, specificity, efficacy, and repeatability relative to other methods. For our study, 12 plasma samples from healthy volunteers were collected. Each sample was split. Half was depleted of highly abundant plasma proteins and the other half

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was left non-depleted. The 12 samples were treated as biological replications and individually analyzed with label-free LC-MRM/MS. The results demonstrated that the peak area significantly increased for four APOs in the depleted plasma (Figure 2). Although the same amount of protein (1.3 μg) was used in both the depleted and non-

Figure 2 Comparative analysis of depleted versus non-depleted plasma samples. Liquid chromatography multiple-reaction-monitoring mass spectrometry (LC-MRM/MS) demonstrated that the peak area significantly increased for four apolipoproteins (apolipoprotein H, apolipoprotein J, apolipoprotein A4, and apolipoprotein E) in depleted plasma (top four panels), indicating that the detection sensitivity was significantly improved in the depleted plasma samples (name-D), while the peak area of apolipoprotein D significantly decreased in the depleted plasma samples (bottom panel).

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depleted samples, the average peak area in the depleted samples increased approximately tenfold for APOH, APOJ, APOA4, and APOE relative to the low mass signals observed in the corresponding non-depleted samples. This increase indicated that detection sensitivity was significantly improved in the depleted samples (Figure 2). However, the peak area of one APO protein, APOD, was found to be decreased in the depleted samples (Figure 2), possibly because of the non-specific loss of the non-targeted lessabundant plasma proteins that were removed along with the highly abundant plasma proteins during MARS pre-fractionation [22]. Accordingly, we deduced that there may be interactions between APOD and some of the 12 highly abundant proteins. However, these results could indicate that the highly abundant proteins affect the MS signals of the five APOs to a different extent; that is, there is a greater suppression of APOH, APOJ, APOA4, and APOE, and a lesser suppression of APOD. Although the peak area of APOD decreased more in the depleted samples than in the non-depleted ones, the MS signal was still high enough for MRM quantification of APOD’s relative abundance in plasma. The 12 samples showed consistent results with variations of less than 20%, which strongly suggests the reliability of the results. Even though MRM quantitation of proteins can be performed on simple tryptic digests of plasma without prior affinity depletion or enrichment [11] and removal of the highly abundant proteins raises experimental costs, MARS has the advantage of enhancing the detection sensitivity of target proteins. Therefore, depleted plasma samples were employed in the mTRAQ-based relative quantitative analysis of the five candidate APOs. 3.4 mTRAQ-based relative quantitative analysis of the five APOs We further evaluated the mTRAQ-based MRM approach to determine the relative abundance of the 5 APOs in plasma. The 12 depleted plasma samples from healthy volunteers and the synthetic peptides were labeled with the mTRAQ reagent, and the synthetic peptides served as internal standards. The amount of internal standards incorporated into samples should be suitable: if the amount is too low, the MS signal will be very weak; and if the amount is too high, the sample signal will be suppressed. Hence, the MRM signals of various amounts of standard peptides incorporated into a mixed plasma sample were detected and compared with the target peptide in the sample. The amount of standard peptide with the closest signal to the target peptide (specifically, 5 ng for all standard peptides) was chosen as the incorporated amount for the final MRM analysis. Three replicate injections for each sample were performed to assess the reproducibility of the mTRAQ-based MRM approach. Quantitation of the relative changes in the

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5 APOs in each sample was determined using MultiQuantTM (v. 2.0.2, AB SCIEX, Foster City, CA, USA). The peak area of 08-labeled peptides were normalized by Δ00 and then logarithmically scaled. It was found that the average coefficients of variation (CVs) of ApoH, ApoJ, ApoA4, ApoE, and ApoD for the triplicate injections of each sample were 7.0%, 3.1%, 4.2%, 4.0%, and 12.1%, respectively, which suggests that the results are highly reproducible. Moreover, when comparing the peak areas of the 5 APOs among the 12 samples, the relative abundances of the 5 APOs in healthy human plasma were stable (Figure 3; corresponding peak areas are displayed in Figure S3), which implies the reproducibility of the approach and, more importantly, the relative stability of APOs in healthy individuals. Notably, the rank order of the 5 APOs in healthy plasma samples was APOH, APOJ, APOA4, APOE, and APOD, which is mainly due to the different MS responses of selected peptides for each protein. This finding prompts the hypothesis that the rank order of the 5 APOs can be applied as a diagnostic signature for diseased plasma. Lee et al. [23] have previously verified mTRAQ’s utility in relative quantification using internal standard peptides and plasma samples. As known amounts of synthetic peptides are tagged with mTRAQ labels, this approach has the benefit of increased specificity by virtue of the differences in precursor and product ion masses as well as absolute quantification. The ability to dramatically increase specificity and analytical precision has been enabled by the more sensitive QTRAP 5500 (AB SCIEX, Foster City, CA, USA), which displays a higher incidence of Q1 charging on account of its larger orifice. In addition, this approach also allows the simultaneous quantification of the relative protein abundances in two samples by one injection. MRM analysis based on at least two peptides with multiple transitions from the same protein is believed to be more

Figure 3 Relative concentrations of the five candidate apolipoproteins. The relative plasma concentrations of the five candidate apolipoproteins (apolipoprotein H, apolipoprotein J, apolipoprotein A4, apolipoprotein E, and apolipoprotein D) were found to be stable.

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accurate than analysis based on one unique peptide alone [16]. As described above, a single, unique, optimized peptide with multiple transitions was used to quantify plasma APOD. Our previously published study that compared the quantitative results based on one and two peptides per protein [9] demonstrated that, except for rare individual cases, most differences were consistent. Thus, the selection of peptide and ion pairs for APOD can be used for relative quantification, and the abundance differences observed among samples will not be altered through the use of either one or more peptides. There are several limitations to this study. First, the primary objective of this study was to assess a detection method for the five selected APOs in human plasma. Therefore, because the approach described herein is a relative quantification method for the five selected APOs, the plasma analytes included in this study are not comprehensive. Second, this study was limited to analyzing the expression of the 5 APOs sampled from 12 healthy volunteers. Before these 5 APOs can be used for diagnostic and/or prognostic purposes in neuropsychiatric disorders, these 5 APO candidates must be assessed in a larger cohort that includes samples from healthy individuals and demographically matched neuropsychiatrically disordered patients.

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4 Conclusions In this study, we developed and assessed a rapid, sensitive, multiplexed LC-MRM/MS assay for the relative quantification of five clinically promising plasma APOs: APOH, APOJ, APOA4, APOE, and APOD. Our findings indicated that the relative levels of the five candidate APOs in healthy human plasma were stable. The mTRAQ-MRM/MS-based approach developed here is highly reproducible in healthy human plasma and may be of future use in clinical biomarker development.

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16 We thank Dr. N.D. Melgiri for editing and proofreading the manuscript. This work was supported by the National Basic Research Program of China (2009CB918300) and the National Natural Science Foundation of China (31271189 and 81101009). 1 2

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