Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry
- 18 November 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 8 (1), 133-141
- https://doi.org/10.1021/pr800381w
Abstract
Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multiphosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.Keywords
This publication has 33 references indexed in Scilit:
- Phosphoproteomic approaches to elucidate cellular signaling networksCurrent Opinion in Biotechnology, 2006
- Improved Immobilized Metal Affinity Chromatography for Large-Scale Phosphoproteomics ApplicationsJournal of Proteome Research, 2006
- Protein phosphorylation in signaling – 50 years and countingTrends in Biochemical Sciences, 2005
- Immunoaffinity profiling of tyrosine phosphorylation in cancer cellsNature Biotechnology, 2005
- The mouse kinome: Discovery and comparative genomics of all mouse protein kinasesProceedings of the National Academy of Sciences of the United States of America, 2004
- Phosphoregulators: Protein Kinases and Protein Phosphatases of MouseGenome Research, 2003
- The Protein Kinase Complement of the Human GenomeScience, 2002
- A Mass Spectrometry-based Proteomic Approach for Identification of Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific AntibodiesMolecular & Cellular Proteomics, 2002
- The origins of protein phosphorylationNature, 2002
- The regulation of protein function by multisite phosphorylation – a 25 year updateTrends in Biochemical Sciences, 2000