Automation of On-Resin Enrichment of S-Nitrosylated Proteins for Oxidized Cysteine-Selective CPILOT

Overview

Journal Vanderbilt Undergrad Res J

Date 2022 May 26

PMID 35615079

Authors

Andrew D Pumford

Albert B Arul

Katarena I Ford

Rena A S Robinson

Affiliations

Soon will be listed here.

Abstract

S-Nitrosylation (SNO) is a cysteine post-translational modification that increases with normal aging and is present in Alzheimer's disease and other aging-related illnesses. Detection of SNO-modified proteins can be challenging; however, we previously developed a robust quantitative proteomics approach termed "Oxidized Cysteine-Selective combined precursor isobaric labeling and isobaric tagging (OxcyscPILOT)" that allows for detection of endogenous SNO-modified proteins. OxcyscPILOT involves enrichment of SNO-modified proteins using a thiol-based resin. This enrichment is performed manually, and wash steps with the resin require numerous stages and buffer reagents. The goal of this study is to transfer the manual protocol to an automated liquid handler system in order to reduce wash steps, increase sample throughput, and minimize experimental error. In order to accomplish this, we evaluated the Biomek i7 liquid handler automated workstation and a Positive Pressure ALP (PPA) apparatus to conduct automated on-resin enrichment. Our findings provide starting pressure conditions for the use of PPA in an automated OxcyscPILOT proteomics workflow that could be transferred to other robotic liquid handling systems.

References

Forrester M, Hess D, Thompson J, Hultman R, Moseley M, Stamler J . Site-specific analysis of protein S-acylation by resin-assisted capture. J Lipid Res. 2010; 52(2):393-8. PMC: 3023561. DOI: 10.1194/jlr.D011106. View

Wang H, Qian W, Chin M, Petyuk V, Barry R, Liu T . Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment. J Proteome Res. 2006; 5(2):361-9. PMC: 1850945. DOI: 10.1021/pr0503681. View

Held J, Gibson B . Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes. Mol Cell Proteomics. 2011; 11(4):R111.013037. PMC: 3322581. DOI: 10.1074/mcp.R111.013037. View

Robinson R, Amin B, Guest P . Multiplexing Biomarker Methods, Proteomics and Considerations for Alzheimer's Disease. Adv Exp Med Biol. 2017; 974:21-48. DOI: 10.1007/978-3-319-52479-5_2. View

Couvertier S, Zhou Y, Weerapana E . Chemical-proteomic strategies to investigate cysteine posttranslational modifications. Biochim Biophys Acta. 2014; 1844(12):2315-30. DOI: 10.1016/j.bbapap.2014.09.024. View

Gu L, Robinson R . High-throughput endogenous measurement of S-nitrosylation in Alzheimer's disease using oxidized cysteine-selective cPILOT. Analyst. 2016; 141(12):3904-15. PMC: 4904844. DOI: 10.1039/c6an00417b. View

Arul A, Robinson R . Sample Multiplexing Strategies in Quantitative Proteomics. Anal Chem. 2018; 91(1):178-189. PMC: 6951796. DOI: 10.1021/acs.analchem.8b05626. View

Foster M, Hess D, Stamler J . Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med. 2009; 15(9):391-404. PMC: 3106339. DOI: 10.1016/j.molmed.2009.06.007. View

Robinson R, Evans A . Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging. Anal Chem. 2012; 84(11):4677-86. DOI: 10.1021/ac202000v. View

10.

Gygi S, Rist B, Gerber S, Turecek F, Gelb M, Aebersold R . Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 1999; 17(10):994-9. DOI: 10.1038/13690. View

11.

Murray C, Uhrigshardt H, OMeally R, Cole R, Van Eyk J . Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay. Mol Cell Proteomics. 2011; 11(2):M111.013441. PMC: 3277766. DOI: 10.1074/mcp.M111.013441. View

12.

Dyer R, Ford K, Robinson R . The roles of S-nitrosylation and S-glutathionylation in Alzheimer's disease. Methods Enzymol. 2019; 626:499-538. PMC: 6908309. DOI: 10.1016/bs.mie.2019.08.004. View

13.

Shiio Y, Aebersold R . Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry. Nat Protoc. 2007; 1(1):139-45. DOI: 10.1038/nprot.2006.22. View

14.

Gu L, Evans A, Robinson R . Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT. J Am Soc Mass Spectrom. 2015; 26(4):615-30. DOI: 10.1007/s13361-014-1059-9. View

15.

Gu L, Robinson R . A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML). Anal Bioanal Chem. 2016; 408(11):2993-3004. DOI: 10.1007/s00216-016-9307-4. View