TY - JOUR

T1 - TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC

AU - Engholm-Keller, Kasper

AU - Birck, Pernille

AU - Størling, Joachim

AU - Pociot, Flemming

AU - Mandrup-Poulsen, Thomas

AU - Larsen, Martin Rask

N1 - Copyright © 2012 Elsevier B.V. All rights reserved.

PY - 2012/10/22

Y1 - 2012/10/22

N2 - 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-¿ 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-¿ 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.

AB - 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-¿ 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-¿ 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.

KW - Animals

KW - Cell Line, Tumor

KW - Chemical Fractionation

KW - Chromatography, Liquid

KW - Insulinoma

KW - Interferon-gamma

KW - Phosphopeptides

KW - Phosphorylation

KW - Proteomics

KW - Rats

KW - Sensitivity and Specificity

KW - Signal Transduction

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Titanium

U2 - 10.1016/j.jprot.2012.08.007

DO - 10.1016/j.jprot.2012.08.007

M3 - Journal article

C2 - 22906719

VL - 75

SP - 5749

EP - 5761

JO - International Journal of Proteomics

JF - International Journal of Proteomics

SN - 2090-2166

IS - 18

ER -