HST-MRM-MS: A Novel High-Sample-Throughput Multiple Reaction

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HST-MRM-MS: A Novel High-Sample-Throughput Multiple Reaction Monitoring Mass Spectrometric Method for Multiplex Absolute Quantitation of Hepatocellular Carcinoma Serum Biomarker Hucong Jiang, Lei Zhang, Ying Zhang, Liqi Xie, Yi Wang, and Haojie Lu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00790 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Journal of Proteome Research

HST-MRM-MS: A Novel High-SampleThroughput Multiple Reaction Monitoring Mass Spectrometric Method for Multiplex Absolute Quantitation of Hepatocellular Carcinoma Serum Biomarker Hucong Jiang †, Lei Zhang†,‡, Ying Zhang†,‡, Liqi Xie†,‡, Yi Wang‡ and Haojie Lu†,‡,* † Shanghai Cancer Centre and Department of Chemistry, Fudan University, Shanghai 200032, P. R. China ‡ Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, P. R. China. *

To whom correspondence should be addressed. E-mail: [email protected]; Fax:

+86-21-54237961; Tel: +86-21-54237618

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ABSTRACT: Absolute quantification of clinical biomarkers by mass spectrometry (MS) has been challenged due to low sample-throughput of current multiple reaction monitoring (MRM) methods. To overcome this problem, in this work, a novel high-sample-throughput multiple reaction monitoring mass spectrometric (HST-MRM-MS) quantification approach is developed to achieve simultaneous quantification of 24 samples. Briefly, triplex dimethyl reagents (L, M, and H) and eight-plex iTRAQ reagents were used to label the N-termini and C-termini of the Lys C digested peptides respectively. The triplex dimethyl labeling produces three co-elute peaks in MRM traces, while the iTRAQ labeling produces eight peaks in MS2, resulting 24 (3*8) channels in a single experiment. HSTMRM-MS has shown good accuracy (R2>0.98 for absolute quantification), reproducibility (RSD 0.980. From Table S3 (Supporting Information), we found that the experimental amounts were smaller than expected values when the iTRAQ ratio was large especially in gradient f and gradient g (For example, the experimental amount of PAAT was about 2.50 ng in gradient g, however, the expected values was 7.08 ng). It suggested that the dynamic range of iTRAQ-labeling was compressed, as mentioned by Desouza et al.27 Additionally, the relative standard deviations (RSDs) of most channels at different concentration were less than 15%, indicating the good reproducibility of this method (Table S3). In addition, the triple references mixed at 1 : 1 : 1 were measured at ratio of 0.83 − 1.2 in most mixtures, which has demonstrated the accuracy of the results (Table S3). In order to assess the dynamic range of relative quantification based on the ratios of isotopic peak areas in MRM traces analysis and the ratios of reporter ion intensities in MS2 analysis, two mixtures of synthetic standard peptides were prepared. (I).The amounts of 16 ACS Paragon Plus Environment

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triplex dimethyl labeled peptides were set as (L : M : H = 0.5 : 1 : 0.5), and for each dimethyl group, the sample were divided into eight parts according to the ratio of iTRAQ (113 : 114 : 115 : 116 : 117 : 118 : 119 : 121 = 1 : 2 : 2 : 4 : 4 : 2 : 2 : 1), then labeled with eight-plex iTRAQ reagents, accordingly. (II). Expected ratio of dimethyl labeling was set as (L : M : H = 1 : 0.5 : 1), and the ratio of iTRAQ was set as (113 : 114 : 115 : 116 : 117 : 118 : 119 : 121 = 4 : 2 : 2 : 1 : 1 : 2 : 2 : 4). To ensure the accuracy of relative quantification results, the amount of every peptide was set in a concentration range closed to its endogenous abundance. Pooled unlabeled serum sample was also added as blank matrix background. In the MRM-MS analysis results of the two mixtures, the XICs of triplex MRM traces and the MS2 spectra of eight-plex reporter ions from the best transition (y4) of the standard peptide PAAT were shown as an example in Figure S3 (Supporting Information). For mixture (I), bar graphs showed that the measured ratio of the MRM traces is close to the expected value (0.5 : 1 : 0.5) for all the three target peptides and the RSDs of every peptide were less than 10% in all dimethyl types (Figure 4a1, 4b1, 4c1). This result indicated that the relative quantification obtained from the MRM traces are of good accuracy and reproducibility. The intensities of iTRAQ-113 and iTRAQ-121 were set as the denominator, and the calibration curves were made by comparing other iTRAQ labeling channels to the denominator. The results of every dimethyl type from every peptide showed a good linear response with R2 > 0.985 (Figure 4a2, 4b2, 4c2), which supported the 17 ACS Paragon Plus Environment


reliability of relative quantification results by comparing intensities of reporter ions. As shown in Figure S4 in the Supporting Information, the bar graphs and the calibration curves for relative quantification of mixture (II) also showed good accuracy, reproducibility and dynamic range. These results demonstrated that HST-MRM-MS strategy could achieve reliable and reproducible quantification not only in absolute quantification but also in relative quantification. Application for Protein Quantification in HCC Sera HST-MRM-MS strategy was further applied for protein quantitation of HCC serum samples. For each protein, one unique peptide was selected as the quantitation surrogate (Table 1), and 21 HCC serum specimens, 21 postoperative HCC serum specimens and 21 normal serum specimens were used. Because of the high-throughput of the method, all of the serum specimens could be quantitative analyzed in only three experiments, and each experiment was analyzed three times by MRM-MS. The absolute concentrations of the three proteins in every sample were calculated and shown in Table 2 (for details, see Table S4, Supporting Information) and are plotted in Figure 5. DISCUSSION Protein C3 is synthesized by liver hepatocytes. It plays a central role in the complement system and contributes to innate immunity.28 The amounts of C3 decreased slightly in HCC 18 ACS Paragon Plus Environment

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serum compared to normal human serum, and there was no significant difference between post-HCC serum and normal serum. It has been reported that C3 levels in plasma showed an overall trend of lower expression in those with liver cirrhosis (LC) compared to controls, and with a ‘‘bounce back’’ effect seen as the protein levels are somewhat restored to control levels in the HCC serum specimens.19 From our results, AAT expression in HCC serum and post-HCC serum was higher than that in normal human serum specimens. It was reported that AAT levels are marginally increased with LC followed by a marked upregulation with progression to HCC.19 Meanwhile, AAT is considered an acute-phase reactant as its plasma levels increase during the hostresponse to inﬂammation or tissue injury.29 Therefore, from the results of our experiments, postoperative HCC serum with high AAT level was reasonable. The function of HPX is scavenging the heme released or lost by the turnover of heme proteins such as hemoglobin and thus it protects the body from the oxidative damage that free heme can cause.30 In our study, HPX expression in HCC serum was decreased compared to normal human serum, and the decrease between postHCC serum and normal serum was slight. The trend for HPX expression was reported similar to that of C3 with its levels dropping from control to LC are somewhat restored to control levels in HCC.19,21 In summary, we developed a new HST-MRM-MS strategy combining triplex nonisobaric labeling (dimethyl) and eight-plex isobaric labeling (iTRAQ). Thus, this method expanded the throughput of MRM-MS to 24 channels in a single MRM-MS 19 ACS Paragon Plus Environment


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experiment. The new method showed good accuracy, reproducibility, linear response and finally successfully applied in evaluating amounts of three candidate biomarkers in HCC related serum specimens. With this method, we found the concentrations of C3 and HPX were decreased slightly in HCC serum compared to normal human serum, while the expressions of AAT were increased in HCC serum. In all, HST-MRM-MS shows a promising future in the quantitative proteomics for high-throughput verification of candidate biomarkers.


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FIGURES

Figure 1. Schematic illustration of UHT-MRM-MS strategy.



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Figure 2. (a) MRM trace and (b, c, d) MS2 spectra of endogenous target peptide PAAT (SVLGQLGITK). In the MRM trace (a), blue represents L-dimethyl, green represents M-dimethyl, and orange represents H-dimethyl. (b), (c) and (d) shows the MS2 spectra of reporter ions from triplex dimethyl labeling types L, M and H, respectively.


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Figure 3. Calibration curves of triplex dimethyl labeled synthetic standard peptides (a1) PC3-L, (a2) PC3-M, (a3) PC3-H, (b1) PAAT-L, (b2) PAAT-M, (b3) PAAT-H, (c1) PHPX-L, (c2) PHPX-M, and (c3) PHPXH, for absolute quantification. The average ratios were calculated and plotted with error bars of standard deviation based on three times analysis by MRM-MS.



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Figure 4. Bar graphs of triplex dimethyl labeling types (L : M : H = 1 : 0.5 : 1) of labeled synthetic standard peptides (a1) PC3, (b1) PAAT, and (c1) PHPX, and the dynamic range of relative quantification of MS2 of labeled synthetic standard peptides (a2) PC3, (b2) PAAT, and (c2) PHPX. The average was calculated and plotted with error bars of standard deviation based on three times analysis by MRM-MS. (Blue represents L-dimethyl, green represents M-dimethyl, and orange represents H-dimethyl)


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Figure 5. Concentration distribution of C3, AAT and HPX in HCC serum, postoperative HCC serum and normal serum (μg/μL serum).



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TABLES Table 1. Transitions Used for MRM Analysis.

Proteins

Peptides

Label

Precursor

Charge

Product

Charge

type

(Q1)

state (Q1)

(Q3)

state (Q3)

CE (V)

1005.67 (y6) L

696.41

3+

807.54 (y4)

1+

31.50

1+

31.50

1+

31.50

1+

37.10

1+

37.10

1+

37.10

1+

35.00

1+

35.00

1+

35.00

694.45 (y3) Complement C3

RIPIEDGSGEVVLSRK

（C3）

( PC3 )

1005.67 (y6) M

697.75

3+

807.54 (y4) 694.45 (y3) 1005.67 (y6)

H

699.09

3+

807.54 (y4) 694.45 (y3) 1133.72 (y8)

L

674.43

2+

1020.64 (y7) 722.47 (y4)

Alpha-1-antitrypsin （AAT）

1133.72 (y8) SVLGQLGITK ( PAAT )

M

676.44

2+

1020.64 (y7) 722.47 (y4) 1133.72 (y8)

H

678.45

2+

1020.64 (y7) 722.47 (y4) 1133.72 (y8)

L

724.05

3+

1020.64 (y7) 722.47 (y4)

Hemopexin（HPX）

SGAQATWTELPWPHEK ( PHPX )

1133.72 (y8) M

725.39

3+

1020.64 (y7) 722.47 (y4) 1133.72 (y8)

H

726.73

3+

1020.64 (y7) 722.47 (y4)


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Table 2. Concentration of C3, AAT and HPX in HCC serum, postoperative HCC serum and normal serum (μg/μL serum).

Serum type (μg/μL) Protein

HCC

Post-HCC

Normal

C3

0.92±0.47

1.20±0.55

1.27±0.54

AAT

0.81±0.40

1.33±0.70

0.65±0.27

HPX

0.41±0.13

0.77±0.23

0.91±0.38



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ASSOCIATED CONTENT Supporting Information: The following supporting information is available free of charge at ACS website http://pubs.acs.org Table S1. Clinical features of the patients and healthy volunteers. (PDF) Table S2. The theoretical amount of three peptide for the calibration mixtures of all dimethylation types. (PDF) Table S3. The actual amounts of peptides and RSDs for absolute quantitation. Table S4. Detailed concentration of C3, AAT and HPX. (PDF) Figure S1. MALDI TOF MS spectra of the labeling efficiency and specificity of triplex N-terminal dimethylation. (PDF) Figure S2. MALDI-TOF MS1 and MS2 spectra of the each labeling steps of standard peptide. (PDF) Figure S3. The MRM traces and MS2 spectra of the labeled standard peptide. Figure S4. Bar graphs and the calibration curves for relative quantification of mixture (II). (PDF) AUTHOR INFORMATION Corresponding Author


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*Tel: +86 21 54237618. E-mail: [email protected]. ORCID Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The work was supported by the National Key Research and Development Program of China (2017YFC0906600 and 2016YFA0501303), NSF (Grants 21335002 and 31670835), the Ph.D. Programs Foundation of Ministry of Education of China (20130071110034), Key Laboratory of Glycoconjugates Research Ministry of Public Health and Shanghai Projects (Eastern Scholar, 15JC1400700 and B109), Special Project on Precision Medicine under the National Key R&D Program Program (SQ2017YFSF090210).



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