A Comparative Study of Synthetic Winged Peptides for Absolute Protein Quantification

Overview

Journal Sci Rep

Specialty Science

Date 2021 May 26

PMID 34035340

Citations 3

Authors

Eliska Benesova

Veronika Vidova

Zdenek Spacil

Affiliations

Soon will be listed here.

Abstract

A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.

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References

Brun V, Dupuis A, Adrait A, Marcellin M, Thomas D, Court M . Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics. 2007; 6(12):2139-49. DOI: 10.1074/mcp.M700163-MCP200. View

Bronsema K, Bischoff R, van de Merbel N . Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2012; 893-894:1-14. DOI: 10.1016/j.jchromb.2012.02.021. View

Lowenthal M, Liang Y, Phinney K, Stein S . Quantitative bottom-up proteomics depends on digestion conditions. Anal Chem. 2013; 86(1):551-8. DOI: 10.1021/ac4027274. View

Faria M, Halquist M, Yuan M, Mylott Jr W, Jenkins R, Karnes H . An extended stable isotope-labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC-MS/MS quantification. Biomed Chromatogr. 2015; 29(11):1780-2. DOI: 10.1002/bmc.3471. View

Kushnir M, Rockwood A, Roberts W, Abraham D, Hoofnagle A, Meikle A . Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem. 2013; 59(6):982-90. PMC: 4016991. DOI: 10.1373/clinchem.2012.195594. View

Ludwig K, Schroll M, Hummon A . Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies. J Proteome Res. 2018; 17(7):2480-2490. PMC: 9319029. DOI: 10.1021/acs.jproteome.8b00235. View

Lin Y, Zhou J, Bi D, Chen P, Wang X, Liang S . Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins. Anal Biochem. 2008; 377(2):259-66. DOI: 10.1016/j.ab.2008.03.009. View

Arnold S, Stevison F, Isoherranen N . Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation. Anal Chem. 2015; 88(1):746-53. PMC: 4817721. DOI: 10.1021/acs.analchem.5b03004. View

Neubert H, Muirhead D, Kabir M, Grace C, Cleton A, Arends R . Sequential protein and peptide immunoaffinity capture for mass spectrometry-based quantification of total human β-nerve growth factor. Anal Chem. 2012; 85(3):1719-26. DOI: 10.1021/ac303031q. View

10.

Guizado T . Analysis of the structure and dynamics of human serum albumin. J Mol Model. 2014; 20(10):2450. DOI: 10.1007/s00894-014-2450-y. View

11.

Anderson N, Anderson N . The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics. 2002; 1(11):845-67. DOI: 10.1074/mcp.r200007-mcp200. View

12.

Cao J, Gonzalez-Covarrubias V, Covarrubias V, Straubinger R, Wang H, Duan X . A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver. Anal Chem. 2010; 82(7):2680-9. PMC: 2883886. DOI: 10.1021/ac902314m. View

13.

Fernandez Ocana M, James I, Kabir M, Grace C, Yuan G, Martin S . Clinical pharmacokinetic assessment of an anti-MAdCAM monoclonal antibody therapeutic by LC-MS/MS. Anal Chem. 2012; 84(14):5959-67. DOI: 10.1021/ac300600f. View

14.

Brun V, Masselon C, Garin J, Dupuis A . Isotope dilution strategies for absolute quantitative proteomics. J Proteomics. 2009; 72(5):740-9. DOI: 10.1016/j.jprot.2009.03.007. View

15.

Scott K, Turko I, Phinney K . Quantitative performance of internal standard platforms for absolute protein quantification using multiple reaction monitoring-mass spectrometry. Anal Chem. 2015; 87(8):4429-35. DOI: 10.1021/acs.analchem.5b00331. View

16.

Fernandez Ocana M, Neubert H . An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum. Anal Biochem. 2010; 399(2):202-10. DOI: 10.1016/j.ab.2010.01.002. View

17.

Shuford C, Walters J, Holland P, Sreenivasan U, Askari N, Ray K . Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised. Anal Chem. 2017; 89(14):7406-7415. DOI: 10.1021/acs.analchem.7b00858. View

18.

Jiang H, Zeng J, Titsch C, Voronin K, Akinsanya B, Luo L . Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum. Anal Chem. 2013; 85(20):9859-67. DOI: 10.1021/ac402420v. View

19.

Siepen J, Keevil E, Knight D, Hubbard S . Prediction of missed cleavage sites in tryptic peptides aids protein identification in proteomics. J Proteome Res. 2007; 6(1):399-408. PMC: 2664920. DOI: 10.1021/pr060507u. View

20.

Gillette M, Carr S . Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods. 2012; 10(1):28-34. PMC: 3943160. DOI: 10.1038/nmeth.2309. View