Format

Send to

Choose Destination
J Proteome Res. 2012 Jun 1;11(6):3467-79. doi: 10.1021/pr201240a. Epub 2012 May 17.

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

Author information

1
Jim Ayers Institute of Precancer Detection and Diagnosis and §Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.

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 methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry.

PMID:
22559222
PMCID:
PMC3368409
DOI:
10.1021/pr201240a
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center