Phosphorylation analysis of primary human T lymphocytes using sequential IMAC and titanium oxide enrichment.
T lymphocytes mediate cellular and humoral defense against foreign bodies or autoantigens. An understanding of T-cell information processing furthers studies of the immunological response. We describe a large-scale phosphorylation analysis of primary T cells using a multidimensional separation strategy, involving preparative SDS-PAGE for prefractionation, in-gel digestion and sequential phosphopeptide enrichment using IMAC and TiO2. A total of 281 phosphorylation sites (197 of high confidence, Ascore > 15), mapping to 204 human gene sequences, were identified by LC-MS(n) analysis in an LTQ linear ion trap. Subsequently, we created the LymPHOS database (http://lymphos.org), which links mass spectrometric peptide information to phosphorylation sites and phosphoprotein sequences.
CSIC/UAB Proteomics Laboratory, IIBB-CSIC, IDIBAPS, Facultad de Medicina, Campus UAB, 08193 Bellaterra, Spain. montserrat.carrascal@gmail.com
Montserrat
M
Carrascal
David
D
Ovelleiro
Vanessa
V
Casas
Marina
M
Gay
Joaquin
J
Abian
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology / Q000379:methods*; D006801:Humans; D007107:Immune System; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D013601:T-Lymphocytes / Q000378:metabolism*; D014025:Titanium / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D010455:Peptides; D010748:Phosphopeptides; C009495:titanium dioxide; D014443:Tyrosine; D014025:Titanium