• Home
  • Search Results
  • SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

Journal of proteomics (2014-12-03)
Arminja N Kettenbach, Hiroyuki Sano, Susanna R Keller, Gustav E Lienhard, Scott A Gerber
ABSTRACT

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, ≥99.9%
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, 99.93%
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Supelco
Methanol, analytical standard
Sigma-Aldrich
Trifluoroacetic acid, suitable for HPLC, ≥99.0%
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Formaldehyde solution, ACS reagent, 37 wt. % in H2O, contains 10-15% Methanol as stabilizer (to prevent polymerization)
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Potassium chloride, ACS reagent, 99.0-100.5%
Sigma-Aldrich
Formic acid, reagent grade, ≥95%
Sigma-Aldrich
Potassium phosphate monobasic, ACS reagent, ≥99.0%
Sigma-Aldrich
Formaldehyde solution, for molecular biology, 36.5-38% in H2O
Sigma-Aldrich
DL-Dithiothreitol, ≥98% (HPLC), ≥99.0% (titration)
Sigma-Aldrich
Urea, ACS reagent, 99.0-100.5%
Sigma-Aldrich
Phosphoric acid, 85 wt. % in H2O, 99.99% trace metals basis
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, BioUltra, for molecular biology, pH 8.0, ~0.5 M in H2O
Sigma-Aldrich
Urea, powder, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
DL-Dithiothreitol, for molecular biology, ≥98% (HPLC), ≥99% (titration)
Sigma-Aldrich
Sodium cyanoborohydride, reagent grade, 95%
Sigma-Aldrich
Potassium chloride, for molecular biology, ≥99.0%
Sigma-Aldrich
Iodoacetamide, BioUltra