Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.

Journal of Proteome Research
Stacy D SherrodAmy-Joan Ham

Abstract

Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS(3) spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (≤15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22-31%) than SID (10-20%). Analyses using both method...Continue Reading

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Citations

Feb 6, 2014·Journal of the American Society for Mass Spectrometry·Michael S BeremanMichael J MacCoss
Aug 28, 2014·Nature Communications·Maria Stella RitortoMatthias Trost
Nov 12, 2013·Expert Review of Proteomics·Kai Pong Law, Yoon Pin Lim
Feb 5, 2013·Biochimica Et Biophysica Acta·Tieneke B M Schaaij-VisserConnie R Jiménez
Aug 8, 2014·Journal of Proteome Research·Vagisha SharmaBrendan MacLean
Mar 23, 2013·Biochemistry·Daniel C Liebler, Lisa J Zimmerman
Dec 20, 2014·Cell·Rommel A MathiasIleana M Cristea
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Oct 20, 2017·Journal of Experimental Botany·Himabindu Vasuki KilambiYellamaraju Sreelakshmi
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Jun 29, 2021·Frontiers in Molecular Neuroscience·Trong Khoa PhamMark O Collins
Jul 29, 2021·Journal of Proteome Research·Lilian R HeilMichael J MacCoss

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Methods Mentioned

BETA
electrophoresis
immunoprecipitation
affinity

Software Mentioned

pSRM
Skyline
Xcalibur

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