Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants. Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants. Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar, 2Huttlin E.L. 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Proteomics. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar), the LAXIC for those two the local normalization on library peptide in Here we used the plant phosphoproteome as the sample to plant samples have isotope labeling and the complex the of quantitation This of plant phosphopeptide was on normalization by internal peptide in a of from samples with this may be of the that the normalization from two were this was in the the global normalization was to compensate retention time we quantitation from to the environmental with from were further normalized by global local normalization with internal peptide the global which used the of internal the the as as the local normalization of normalization for retention of peptide Global, normalized peptide with the global normalization by internal normalized peptide with the local normalization by internal local and global normalization in peptide have in a The of LAXIC highly on a internal library a that can peptide library and proteomic for to to the to that the for the libraries of a that to be of that be on the of the the of LAXIC quantitation to locally to the internal we that high of sample and internal standards the of different libraries the of a library the library in the the LAXIC method may be to by sample preparation in to sample phosphorylation enrichment further the quantitation of LAXIC, a synthetic phosphopeptide library in the of sample preparation may be in and quantitation by mass spectrometry has as a powerful for of complex a high of for the of the achieved by Here we introduce a of synthetic peptide libraries as internal standards for and of the platform for label-free quantitative proteomics of the the peptide we to LAXIC can sample and ion suppression to and normalization for the quantitation as the in LAXIC, this to the proteomics platform of of to the local normalization to internal with the of a peptide library that has of elution profile the we that LAXIC can be to complex quantitative phosphoproteomics to