Research Article

Systematic optimization of targeted and multiplexed MS-based screening workflows for protein biomarkers

Bioanalysis, Immunogenicity & Biomarkers, In-vitro/In-vivo Translation, Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA

Scientific Operations, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA

‡These authors contributed equally to this work

Search for more papers by this author

Zhuo Chen‡

Bioanalysis, Immunogenicity & Biomarkers, In-vitro/In-vivo Translation, Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA

‡These authors contributed equally to this work

Search for more papers by this author

Yun W Alelyunas

Scientific Operations, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA

Search for more papers by this author

Matthew E Szapacs

Bioanalysis, Immunogenicity & Biomarkers, In-vitro/In-vivo Translation, Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA

Search for more papers by this author

Mark D Wrona

Scientific Operations, Waters Corporation, 34 Maple Street, Milford, MA 01757, USA

Search for more papers by this author

Timothy W Sikorski

*Author for correspondence: Tel.: +1 610 270 4978;

E-mail Address: timothy.w.sikorski@gsk.com

Bioanalysis, Immunogenicity & Biomarkers, In-vitro/In-vivo Translation, Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA

Search for more papers by this author

Published Online:8 Mar 2022https://doi.org/10.4155/bio-2021-0245

View article

Abstract

Background: The capability of targeted MS-based methods to simultaneously measure multiple analytes with high selectivity and sensitivity greatly facilitates the discovery and quantitation of novel biomarkers. However, the complexity of biological samples is a major bottleneck that requires extensive sample preparation. Results: This paper reports a generic workflow to optimize surrogate peptide-based protein biomarker screening for seven human proteins in a multiplexed manner without the need for any specific affinity reagents. Each step of the sample processing and LC–MS methods is systematically assessed and optimized for better analytical performance. Conclusion: The established method is used for the screening of multiple myeloma patient samples to determine which proteins could be robustly measured and serve as potential biomarkers of the disease.

Keywords:

Papers of special note have been highlighted as: • of interest; •• of considerable interest

References

1. Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin. Pharmacol. Ther. 69(3), 89–95 (2001).
MedlineGoogle Scholar
2. Haab BB, Paulovich AG, Anderson NL et al. A reagent resource to identify proteins and peptides of interest for the cancer community: a workshop report. Mol. Cell. Proteom. 5(10), 1996–2007 (2006).
Medline CASGoogle Scholar
3. Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteom. 1(11), 845–867 (2002).
Medline CASGoogle Scholar
4. Domon B, Aebersold R. Mass spectrometry and protein analysis. Science 312(5771), 212–217 (2006).
Medline CASGoogle Scholar
5. Gillette MA, Carr SA. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 10(1), 28 (2013).
Medline CASGoogle Scholar
6. Hüttenhain R, Malmström J, Picotti P, Aebersold R. Perspectives of targeted mass spectrometry for protein biomarker verification. Curr. Opin. Chem. Bio. 13(5–6), 518–525 (2009).
Medline CASGoogle Scholar
7. Han X, Aslanian A, Yates JR III. Mass spectrometry for proteomics. Curr. Opin. Chem. Bio. 12(5), 483–490 (2008).
Medline CASGoogle Scholar
8. Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell. Proteom. 5(4), 573–588 (2006).
Medline CASGoogle Scholar
9. 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. Proteom. 9(1), 184–196 (2010).
Medline CASGoogle Scholar
10. Addona TA, Abbatiello SE, Schilling B et al. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nature Biotechnol. 27(7), 633–641 (2009).
Medline CASGoogle Scholar
11. Anderson NL, Anderson NG, Haines LR, Hardie DB, Olafson RW, Pearson TW. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). J. Proteome Res. 3(2), 235–244 (2004).
Medline CASGoogle Scholar
12. Burkhart JM, Schumbrutzki C, Wortelkamp S, Sickmann A, Zahedi RP. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J. Proteomics. 75(4), 1454–1462 (2012).
Medline CASGoogle Scholar
13. Ferńndez Ocaña M, James IT, Kabir M et al. Clinical pharmacokinetic assessment of an anti-MAdCAM monoclonal antibody therapeutic by LC-MS/MS. Anal. Chem. 84(14), 5959–5967 (2012).
MedlineGoogle Scholar
14. Carr SA, Abbatiello SE, Ackermann BL et al. Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach. Mol. Cell. Proteomics 13(3), 907–917 (2014). • A comprehensive guide for targeted MS assay categorization based on the assay purpose and the required analytical characteristics.
Medline CASGoogle Scholar
15. Furukawa Y, Kikuchi J. Molecular pathogenesis of multiple myeloma. Int. J. Clin. Oncol. 20(3), 413–422 (2015).
Medline CASGoogle Scholar
16. Plumb RS, Johnson KA, Rainville P et al. UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation. Rapid Commun. Mass Spectrom. 20(13), 1989–1994 (2006).
Medline CASGoogle Scholar
17. Strader MB, Tabb DL, Hervey WJ, Pan C, Hurst GB. Efficient and specific trypsin digestion of microgram to nanogram quantities of proteins in organic-aqueous solvent systems. Anal. Chem. 78(1), 125–134 (2006).
Medline CASGoogle Scholar
18. Kusebauch U, Deutsch EW, Campbell DS, Sun Z, Farrah T, Moritz RL. Using PeptideAtlas, SRMAtlas, and PASSEL: comprehensive resources for discovery and targeted proteomics. Curr. Protoc. Bioinform. 46(1), 13.25.11–13.25.28 (2014).
Google Scholar
19. Chen Z, Alelyunas YW, Wrona MD, Kehler JR, Szapacs ME, Evans CA. Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones. Bioanalysis 11(13), 1275–1289 (2019). •• Real-world example indicating the benefits of microflow ultra performance liquid chromatography and high-resolution MS on quantitative bioanalysis.
CASGoogle Scholar
20. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 75(13), 3019–3030 (2003).
Medline CASGoogle Scholar
21. Kloepfer A, Quintana JB, Reemtsma T. Operational options to reduce matrix effects in liquid chromatography – electrospray ionisation–mass spectrometry analysis of aqueous environmental samples. J. Chromatogr. A 1067(1–2), 153–160 (2005).
Medline CASGoogle Scholar
22. Gangl ET, Annan M, Spooner N, Vouros P. Reduction of signal suppression effects in ESI-MS using a nanosplitting device. Anal. Chem. 73(23), 5635–5644 (2001).
Medline CASGoogle Scholar
23. Van Hoof N, Courtheyn D, Antignac JP et al. Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers. Rapid Commun. 19(19), 2801–2808 (2005).
Medline CASGoogle Scholar
24. Guo B, Li C, Deng Z et al. A new method for measurement of (-)-sophocarpine, a candidate therapeutic for viral myocarditis, in plasma: application to a toxicokinetic study in beagle dogs. Rapid Commun. 19(19), 2840–2848 (2005).
Medline CASGoogle Scholar
25. Quintana JB, Rodil R, Reemtsma T. Suitability of hollow fibre liquid-phase microextraction for the determination of acidic pharmaceuticals in wastewater by liquid chromatography–electrospray tandem mass spectrometry without matrix effects. J. Chromatogr. A 1061(1), 19–26 (2004).
Medline CASGoogle Scholar
26. Lien GW, Chen CY, Wang GS. Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations. J. Chromatogr. A 1216(6), 956–966 (2009).
Medline CASGoogle Scholar
27. Zhou W, Yang S, Wang PG. Matrix effects and application of matrix effect factor. Bioanalysis 9(23), 1839–1844 (2017).
CASGoogle Scholar
28. Chiva C, Ortega M, Sabido E. Influence of the digestion technique, protease, and missed cleavage peptides in protein quantitation. J. Proteome Res. 13(9), 3979–3986 (2014).
Medline CASGoogle Scholar
29. Siepen JA, Keevil EJ, Knight D, Hubbard SJ. Prediction of missed cleavage sites in tryptic peptides aids protein identification in proteomics. J. Proteome Res. 6(1), 399–408 (2007).
Medline CASGoogle Scholar
30. Hervey WJT, Strader MB, Hurst GB. Comparison of digestion protocols for microgram quantities of enriched protein samples. J. Proteome Res. 6(8), 3054–3061 (2007).
Medline CASGoogle Scholar
31. Waas M, Bhattacharya S, Chuppa S et al. Combine and conquer: surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins. Anal. Chem. 86(3), 1551–1559 (2014).
Medline CASGoogle Scholar
32. Freije JR, Mulder PP, Werkman W et al. Chemically modified, immobilized trypsin reactor with improved digestion efficiency. J. Proteome Res. 4(5), 1805–1813 (2005).
Medline CASGoogle Scholar
33. Ludwig KR, Schroll MM, Hummon AB. Comparison of in-solution, FASP, and S-Trap based digestion methods for bottom-up proteomic studies. J. Proteome Res. 17(7), 2480–2490 (2018).
Medline CASGoogle Scholar
34. Leon IR, Schwammle V, Jensen ON, Sprenger RR. Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol. Cell. Proteomics 12(10), 2992–3005 (2013).
Medline CASGoogle Scholar
35. Tanca A, Abbondio M, Pisanu S, Pagnozzi D, Uzzau S, Addis MF. Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue. Clin. Proteomics. 11(1), 28 (2014).
MedlineGoogle Scholar
36. Twomey JD, Brahme NN, Zhang B. Drug-biomarker co-development in oncology – 20 years and counting. Drug Resist. Updat. 30, 48–62 (2017).
MedlineGoogle Scholar
37. Tan J, Chen S, Huang J et al. High PD-1 and Tim-3 expression concurrent with exhausted CD4⁺ and CD8⁺ T cells in bone marrow compared with peripheral blood from patients with multiple myeloma. Blood 130(Suppl. 1), 4391 (2017).
Google Scholar
38. Shah N, Chari A, Scott E, Mezzi K, Usmani SZ. B-cell maturation antigen (BCMA) in multiple myeloma: rationale for targeting and current therapeutic approaches. Leukemia 34(4), 985–1005 (2020).
MedlineGoogle Scholar
39. Bandow JE. Comparison of protein enrichment strategies for proteome analysis of plasma. Proteomics 10(7), 1416–1425 (2010). • A nice review of the most popular protein enrichment strategies for protein analysis.
Medline CASGoogle Scholar
40. Björhall K, Miliotis T, Davidsson P. Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics 5(1), 307–317 (2005).
MedlineGoogle Scholar
41. Betancourt LH, De Bock PJ, Staes A et al. SCX charge state selective separation of tryptic peptides combined with 2D-RP-HPLC allows for detailed proteome mapping. J. Proteomics. 91, 164–171 (2013).
Medline CASGoogle Scholar
42. Shi T, Fillmore TL, Sun X et al. Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum. PNAS 109(38), 15395–15400 (2012).
Medline CASGoogle Scholar
43. Pu J, An B, Vazvaei F, Qu J. Enrichment of protein therapeutics and biomarkers for LC–MS quantification. Bioanalysis 10(13), 979–982 (2018).
CASGoogle Scholar

Vol. 14, No. 6

Supplemental Materials

Metrics

Downloaded 158 times

History

Received 3 November 2021

Accepted 17 February 2022

Published online 8 March 2022

Published in print March 2022

Information

Keywords

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.4155/bio-2021-0245

Acknowledgments

The authors appreciate the suggestions on the manuscript from Ian Edwards and Kelly Doering.

Financial & competing interests disclosure

This research was supported by GlaxoSmithKline (GSK) Research and Development and Waters Corporation. All authors are employees of GSK or Waters Corporation, who may be eligible for stock options or have stock ownership. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

PDF download