Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC

• Published in: Proteomics (Weinheim) 2005-09, Vol.5 (14), p.3589-3599
• Main Authors: Gevaert, Kris, Staes, An, Van Damme, Jozef, De Groot, Sara, Hugelier, Koen, Demol, Hans, Martens, Lennart, Goethals, Marc, Vandekerckhove, Joël
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Line; Chromatography, Affinity - methods; Combined fractional diagonal chromatography; Fundamental and applied biological sciences. Psychology; Hepatocytes - chemistry; Hepatocytes - metabolism; Humans; Miscellaneous; Molecular Sequence Data; Non-gel proteomics; Peptide Mapping; Phosphoproteins - chemistry; Phosphorylation; Protein phosphorylation; Proteins; Proteomics
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200401217

We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+‐immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed‐phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non‐phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split‐differential 16O –18O labeling. The method was validated with alpha‐casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190 phosphorylated peptides from 152 different proteins. This dataset includes 38 novel protein phosphorylation sites.