2014
DOI: 10.1021/pr500893m
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Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

Abstract: Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of … Show more

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Cited by 289 publications
(264 citation statements)
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“…Proteolytic digestion products were desalted on a Sep-Pak C 18 cartridge (Waters, Milford), lyophilized for 2 days and enriched in phosphopeptides by consecutive incubations with TiO 2 beads (38 -39). Half sample of each fraction was directly analyzed by LC-MS/MS whereas the remaining halves were pooled together and further fractionated using high pH fractionation prior MS analysis (40).…”
Section: Reagentsmentioning
confidence: 99%
“…Proteolytic digestion products were desalted on a Sep-Pak C 18 cartridge (Waters, Milford), lyophilized for 2 days and enriched in phosphopeptides by consecutive incubations with TiO 2 beads (38 -39). Half sample of each fraction was directly analyzed by LC-MS/MS whereas the remaining halves were pooled together and further fractionated using high pH fractionation prior MS analysis (40).…”
Section: Reagentsmentioning
confidence: 99%
“…However, recent protocols either avoid this step (Soufi et al 2015) or use alternative peptide fractionation methods, such as high-pH reversed-phase chromatography (Batth et al 2014). If you are applying an alternative prefractionation protocol, proceed to Step 15 for TiO 2 enrichment of peptide fractions.…”
Section: Performing Scx Chromatographymentioning
confidence: 99%
“…In addition, because of their low abundance and ionization properties (34), few phosphopeptides can be quantified by direct MRM analysis of neat cellular lysate, and enrichment is required. Although this can be achieved using the multidimensional biochemical fractionation workflows (10,33,35), these workflows require specialized expertise/instrumentation, are low in throughput, and are cost-prohibitive as a workhorse assay platform for the community. We have greatly simplified sample preparation, reduced assay costs, and improved throughput by coupling immunoaffinity enrichment of peptides with quantification by MRM (28).…”
Section: Selection Of Analytes and Development Of Assaymentioning
confidence: 99%