TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC.
Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Kasper
K
Engholm-Keller
Pernille
P
Birck
Joachim
J
Størling
Flemming
F
Pociot
Thomas
T
Mandrup-Poulsen
Martin R
MR
Larsen
D000818:Animals; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D007340:Insulinoma / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium / Q000737:chemistry*
D010748:Phosphopeptides; C009495:titanium dioxide; D007371:Interferon-gamma; D014025:Titanium