Quantitation of the phosphoproteome using the library-assisted extracted ion chromatogram (LAXIC) strategy

Methods Mol Biol. 2014;1156:407-16. doi: 10.1007/978-1-4939-0685-7_27.

Abstract

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chromatography, Liquid / methods*
  • Mass Spectrometry
  • Phosphoproteins / chemistry*
  • Proteome*

Substances

  • Phosphoproteins
  • Proteome