Multiplexed Phosphoproteomic Profiling Using Titanium Dioxide and Immunoaffinity Enrichments Reveals Complementary Phosphorylation Events.
A comprehensive view of protein phosphorylation remains an unmet challenge in the field of cell biology. Mass spectrometry-based proteomics is one of the most promising approaches for identifying thousands of phosphorylation events in a single experiment, yet the full breadth of the phosphoproteome has yet to be elucidated. In this article, we examined the complementarity of two methods for phosphopeptide enrichment based on either titanium dioxide (TiO2) enrichment or phosphorylation motif-specific immunoaffinity precipitation (IAP) with four different antibodies. Each method identified nearly 2000 phosphoproteins. However, distinct populations of phosphopeptides were observed. Despite quantifying over 10 000 unique phosphorylation events using TiO2and over 3900 with IAP, less than 5% of the sites were in common. Agreeing with published literature, the ratio of pS:pT:pY phosphorylation for the TiO2-enriched data set approximated 90:10:<1. In contrast, that ratio for the combined IAP data sets was 51:29:20. These differences not only suggest the complementarity between multiple enrichment methods but also emphasize their collective importance in obtaining a comprehensive view of the phosphoproteome.
Bluefin Biomedicine , Beverly, Massachusetts 01915, United States.
Anthony P
AP
Possemato
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Joao A
0000-0002-4291-413X
JA
Paulo
Bluefin Biomedicine , Beverly, Massachusetts 01915, United States.
Daniel
D
Mulhern
Bluefin Biomedicine , Beverly, Massachusetts 01915, United States.
Ailan
A
Guo
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Steven P
SP
Gygi
Bluefin Biomedicine , Beverly, Massachusetts 01915, United States.
Sean A
0000-0002-3356-4641
SA
Beausoleil
D045744:Cell Line, Tumor; D006801:Humans; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000096:biosynthesis* / Q000235:genetics; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D040901:Proteomics; D014025:Titanium / Q000737:chemistry
D010748:Phosphopeptides; D010750:Phosphoproteins; C009495:titanium dioxide; D014025:Titanium
SPS-MS3; TMT; TiO2; pY; phosphoproteome; phosphoproteomics; titanium dioxide