Improvement of the Quantification Accuracy and Throughput for Phosphoproteome Analysis by a Pseudo Triplex Stable Isotope Dimethyl Labeling Approach

Abstract

Q uantification of specific phosphopeptides or phosphorylation sites is critical to better understand the role of reversible phosphorylation in cellular processes. Different approaches for phosphopeptide quantification have been proposed, including stable isotope labeling of amino acids in cell culture (SILAC), 1,2 isobaric tag for relative and absolute quantitation (iTRAQ), 3 and stable isotope dimethyl labeling. 4 Although in vivo labeling in animal models has been reported for SILAC, 5 its main use has been in cell culture experiments. In contrast, chemical labeling approaches such as iTRAQ can be readily used for tissues with the commercial isotopic labeling reagent kits. Alternatively, stable isotope dimethyl labeling, which leads to fast and complete reductive amination of peptides, 6 is a relatively simple and economic strategy for large scale quantitative analyses. To date, this approach has been used for comprehensive quantitative proteome analyses. 7À9The sophisticated quantitative results are indispensable to correctly understand the change of phosphorylation level on different physiological conditions. However, how to objectively evaluate the accuracy of the mass spectrometry (MS)-based quantification results is still in its infancy although some studies have paid attention to this question. 10À12 In quantitative proteomics, the changes of protein expression level are usually determined by averaging the ratios of corresponding isotope labeled peptide pairs derived from the same protein excluding spurious peptide ratios to eliminate their influences on protein quantification. As each protein can generate many peptides after digestion, this filtering strategy is effective in controlling the protein quantification accuracy. However, this assumption is not suitable for phosphoproteomics.11 The ratio for phosphopeptides obtained from the same protein will depend on the levels of the different protein isoforms and the activities of the different ABSTRACT: Accurately quantifying the changes of phosphorylation level on specific sites is crucial to understand the role of protein phosphorylation in physiological and pathological processes. Here, a pseudo triplex stable isotope dimethyl labeling approach was developed to improve the accuracy and the throughput of comprehensive quantitative phosphoproteome analyses. In this strategy, two identical samples are labeled with light and heavy isotopes, respectively, while another comparative sample is labeled with an intermediate isotope. Two replicated quantification results were achieved in just one experiment, and the relative standard deviation (RSD) criterion was used to control the quantification accuracy. Compared with the conventional duplex labeling approach, the number of quantified phosphopeptides increased nearly 50% and the experimental time was reduced by 50% under the same quantification accuracy. Combined with the automated online reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional separation system, a comparative pho...

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…In this study, we carried out a large-scale phosphoproteome profiling in the model plant Arabidopsis by applying the Ti 4+ -IMAC coupled online RP-SCX-RPLC [32,33]. This method has been successfully used for phosphoprotein profiling from animals [31][32][33] but has never been used in a plant system.…”

“…This method has been successfully used for phosphoprotein profiling from animals [31][32][33] but has never been used in a plant system. First, the Ti 4+ -IMAC was used for phosphopeptide enrichment, and then the online MudPIT equipped with a RP-SCX biphasic column was employed for phosphopeptide fractionation.…”

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

“…They also developed a pseudo-triplex stable isotope dimethyl labeling approach for high accuracy and throughput of comprehensive quantitative phosphoproteome analyses. These methodologies have great potential for application in high-throughput analyses of biological samples for screening and discovery of disease-specific biomarkers [38].…”

“…They developed a pseudo-triplex stable isotope dimethyl labeling approach coupled with an online RP-strong cation exchange-RP multidimensional separation system. The throughput and accuracy of the quantitative analysis were improved significantly by this strategy combined with the relative standard deviation criterion [38]. Finally, over 1800 phosphopeptides corresponding to 1918 unique phosphorylation sites were reliably quantified in a 42-hour online multidimensional analysis.…”

Analysis of human serum phosphopeptidome by a focused database searching strategy

“…Therefore, specific isolation of phosphopeptides is crucial for phosphorylation analysis. Ti (IV) based IMAC material was used for specific enrichment of phosphopeptides from protein digest [18,19,22,23]. Recently, an affinity material synthesized by modifying highly ordered mesoporous silica particles MCM-41 with titanium phosphonate (Ti (IV)-MCM-41) was demonstrated to have both size-exclusion capability of proteins and high specificity toward endogenous phosphopeptides [14].…”