An extensive survey of tyrosine phosphorylation revealing new sites in human mammary epithelial cells.
Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here, we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying antiphosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that a majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.
Heibeck
Tyler H
TH
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Richland, Washington 99352, USA.
Ding
Shi-Jian
SJ
Opresko
Lee K
LK
Zhao
Rui
R
Schepmoes
Athena A
AA
Yang
Feng
F
Tolmachev
Aleksey V
AV
Monroe
Matthew E
ME
Camp
David G
DG
Smith
Richard D
RD
Wiley
H Steven
HS
Qian
Wei-Jun
WJ
D000465:Algorithms; D020816:Amino Acid Motifs; D002460:Cell Line; D002853:Chromatography, Liquid; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D025941:Protein Interaction Mapping; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000032:analysis / Q000378:metabolism*
D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor