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Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma

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Abstract

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography–mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers.

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Abbreviations

AACT:

Alpha-1-antichymotrypsin

AFP:

Alpha-fetoprotein

AUC:

Area under the receiver operator characteristic curve

CID:

Collision-induced dissociation

CV:

Coefficient of variation

Fuc:

Fucose

HCC:

Hepatocellular carcinoma

Hex:

Hexose

HexNAc:

N-Acetylhexoseamine

LC:

Liquid chromatography

LLOQ:

Lower limited of quantitation

LOD:

Limited of detection

MS:

Mass spectrometry

NeuAc:

N-Acetylneuraminic acid

PRM:

Parallel reaction monitoring

ROC:

Receiver operator characteristic

TOF:

Time of flight

VTN:

Vitronectin

References

  1. Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science. 2001;291(5512):2364–9.

    Article  CAS  PubMed  Google Scholar 

  2. Goupille C, Hallouin F, Meflah K, Le Pendu J. Increase of rat colon carcinoma cells tumorigenicity by alpha(1-2) fucosyltransferase gene transfection. Glycobiology. 1997;7(2):221–9.

    Article  CAS  PubMed  Google Scholar 

  3. Yao X, McShane AJ, Castillo MJ. Quantitative proteomics in development of disease protein biomarkers. In: Issaq HJ, Veenstra TD, editors. Proteomic and metabolomic approaches to biomarker discovery. London: Academic; 2013. p. 259–78.

    Chapter  Google Scholar 

  4. Dalpathado DS, Desaire H. Glycopeptide analysis by mass spectrometry. Analyst. 2008;133(6):731–8.

    Article  CAS  PubMed  Google Scholar 

  5. Kurogochi M, Matsushista T, Amano M, Furukawa J, Shinohara Y, Aoshima M, et al. Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay. Mol Cell Proteomics. 2010;9(11):2354–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hong Q, Lebrilla CB, Miyamoto S, Ruhaak LR. Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring. Anal Chem. 2013;85(18):8585–93.

    Article  CAS  PubMed  Google Scholar 

  7. Benicky J, Sanda M, Pompach P, Wu J, Goldman R. Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease. Anal Chem. 2014;86(21):10716–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang N, Goonatilleke E, Park D, Song T, Fan G, Lebrilla CB. Quantitation of site-specific glycosylation in manufactured recombinant monoclonal antibody drugs. Anal Chem. 2016;88(14):7091–100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ruhaak LR, Kim K, Stroble C, Taylor SL, Hong Q, Miyamoto S, et al. Protein-specific differential glycosylation of immunoglobulins in serum of ovarian cancer patients. J Proteome Res. 2016;15(3):1002–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dallas DC, Martin WF, Hua S, German JB. Automated glycopeptide analysis—review of current state and future directions. Brief Bioinform. 2013;14(3):361–74.

    Article  CAS  PubMed  Google Scholar 

  11. Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics. 2012;11(11):1475–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gallien S, Bourmaud A, Domon B. A simple protocol to routinely assess the uniformity of proteomics analyses. J Proteome Res. 2014;13(5):2688–95.

    Article  CAS  PubMed  Google Scholar 

  13. Ronsein GE, Pamir N, von Haller PD, Kim DS, Oda MN, Jarvik GP, et al. Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics. J Proteome. 2015;113:388–99.

    Article  CAS  Google Scholar 

  14. Gallien S, Bourmaud A, Kim SY, Domon B. Technical considerations for large-scale parallel reaction monitoring analysis. J Proteome. 2014;100:147–59.

    Article  CAS  Google Scholar 

  15. Gallien S, Duriez E, Demeure K, Domon B. Selectivity of LC-MS/MS analysis: implication for proteomics experiments. J Proteome. 2013;81:148–58.

    Article  CAS  Google Scholar 

  16. Cohen Freue GV, Borchers CH. Multiple reaction monitoring (MRM): principles and application to coronary artery disease. Circ Cardiovasc Genet. 2012;5(3):378.

    Article  PubMed  Google Scholar 

  17. Zhaojing M, Veenstra TD. Mass spectrometry–based approach for protein biomarker verification. In: Issaq HJ, Veenstra TD, editors. Proteomic and metabolomic approaches to biomarker discovery. London: Academic; 2013. p. 407–24.

    Google Scholar 

  18. Sanda M, Pompach P, Brnakova Z, Wu J, Makambi K, Goldman R. Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease. Mol Cell Proteomics. 2013;12(5):1294–305.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics. 2002;1(11):845–67.

    Article  CAS  PubMed  Google Scholar 

  20. Hortin GL, Sviridov D. The dynamic range problem in the analysis of the plasma proteome. J Proteome. 2010;73(3):629–36.

    Article  CAS  Google Scholar 

  21. Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics. 2006;5(4):573–88.

    Article  CAS  PubMed  Google Scholar 

  22. Whiteaker JR, Zhao L, Anderson L, Paulovich AG. An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers. Mol Cell Proteomics. 2010;9(1):184–96.

    Article  CAS  PubMed  Google Scholar 

  23. Sano K, Asanuma-Date K, Arisaka F, Hattori S, Ogawa H. Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration. Glycobiology. 2007;17(7):784–94.

    Article  CAS  PubMed  Google Scholar 

  24. Hortin GL, Sviridov D, Anderson NL. High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of polypeptide abundance. Clin Chem. 2008;54(10):1608–16.

    Article  CAS  PubMed  Google Scholar 

  25. Lee HJ, Na K, Choi EY, Kim KS, Kim H, Paik YK. Simple method for quantitative analysis of N-linked glycoproteins in hepatocellular carcinoma specimens. J Proteome Res. 2010;9(1):308–18.

    Article  CAS  PubMed  Google Scholar 

  26. Hwang H, Lee JY, Lee HK, Park GW, Jeong HK, Moon MH, et al. In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. Anal Bioanal Chem. 2014;406(30):7999–8011.

    Article  CAS  PubMed  Google Scholar 

  27. Mayampurath A, Song E, Mathur A, Yu CY, Hammoud Z, Mechref Y, et al. Label-free glycopeptide quantification for biomarker discovery in human sera. J Proteome Res. 2014;13(11):4821–32.

    Article  CAS  PubMed  Google Scholar 

  28. Arrieta O, Cacho B, Morales-Espinosa D, Ruelas-Villavicencio A, Flores-Estrada D, Hernandez-Pedro N. The progressive elevation of alpha fetoprotein for the diagnosis of hepatocellular carcinoma in patients with liver cirrhosis. BMC Cancer. 2007;7:28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications. Clin Chim Acta. 2008;395(1–2):19–26.

    Article  CAS  PubMed  Google Scholar 

  30. Dela Rosa MA, Chen WC, Chen YJ, Obena RP, Chang CH, Capangpangan RY, et al. One-pot two-nanoprobe assay uncovers targeted glycoprotein biosignature. Anal Chem. 2017;89(7):3973–80.

    Article  CAS  Google Scholar 

  31. Kim KH, Lee SY, Hwang H, Lee JY, Ji ES, An HJ, et al. Direct monitoring of fucosylated glycopeptides of alpha-fetoprotein in human serum for early hepatocellular carcinoma by liquid chromatography-tandem mass spectrometry with immunoprecipitation. Proteomics Clin Appl. 2018;12(6):e1800062.

    Article  CAS  PubMed  Google Scholar 

  32. Ordonez NG, Manning JT Jr. Comparison of alpha-1-antitrypsin and alpha-1-antichymotrypsin in hepatocellular carcinoma: an immunoperoxidase study. Am J Gastroenterol. 1984;79(12):959–63.

    CAS  PubMed  Google Scholar 

  33. Ji ES, Hwang H, Park GW, Lee JY, Lee HK, Choi NY, et al. Analysis of fucosylation in liver-secreted N-glycoproteins from human hepatocellular carcinoma plasma using liquid chromatography with tandem mass spectrometry. Anal Bioanal Chem. 2016;408(27):7761–74.

    Article  CAS  PubMed  Google Scholar 

  34. Fu Q, Schoenhoff FS, Savage WJ, Zhang P, Van Eyk JE. Multiplex assays for biomarker research and clinical application: translational science coming of age. Proteomics Clin Appl. 2010;4(3):271–84.

    Article  CAS  PubMed  Google Scholar 

  35. Mazzara S, Rossi RL, Grifantini R, Donizetti S, Abrignani S, Bombaci M. CombiROC: an interactive web tool for selecting accurate marker combinations of omics data. Sci Rep. 2017;7:45477.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhu J, Warner E, Parikh ND, Lubman DM. Glycoproteomic markers of hepatocellular carcinoma—mass spectrometry based approaches. Mass Spectrom Rev. 2018.

  37. Yuan W, Sanda M, Wu J, Koomen J, Goldman R. Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease. J Proteome. 2015;116:24–33.

    Article  CAS  Google Scholar 

  38. Zhu J, Chen Z, Zhang J, An M, Wu J, Yu Q, et al. Differential quantitative determination of site-specific intact N-glycopeptides in serum haptoglobin between hepatocellular carcinoma and cirrhosis using LC-EThcD-MS/MS. J Proteome Res. 2018.

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Funding

This research was supported by the Creative Allied Project (CAP Research Grant No. NTM2371511) from National Research Council of Science and Technology, by the R&D Equipment Engineer Education Program from National Research Foundation of Korea (MSIP 2014R1A6A9064166), and by a Research Grant (No. T39710 to J.Y. K) from the Korea Basic Science Institute (KBSI).

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Authors and Affiliations

  1. Biomedical Omics Research Group, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongju, 28119, Republic of Korea

    Kwang Hoe Kim, Gun Wook Park, Ji Eun Jeong, Eun Sun Ji, Jin Young Kim & Jong Shin Yoo

  2. Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 34134, Republic of Korea

    Kwang Hoe Kim, Ji Eun Jeong, Hyun Joo An & Jong Shin Yoo

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  1. Kwang Hoe Kim
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  2. Gun Wook Park
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  3. Ji Eun Jeong
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  4. Eun Sun Ji
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  6. Jin Young Kim
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Correspondence to Jin Young Kim or Jong Shin Yoo.

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We obtained all serum samples from the Samsung Medical Center (Seoul, Korea) with Institutional Review Board approval. Informed consent was obtained from human volunteers participating according to ethical standards.

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The authors declare that they have no conflict of interest.

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Kim, K.H., Park, G.W., Jeong, J.E. et al. Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma. Anal Bioanal Chem 411, 3009–3019 (2019). https://doi.org/10.1007/s00216-019-01775-5

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  • DOI: https://doi.org/10.1007/s00216-019-01775-5

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