title: Sample Collection Method Bias Effects in Quantitative Phosphoproteomics.
abstract: Current advances in selective enrichment, fractionation, and MS detection of phosphorylated peptides allowed identification and quantitation of tens of thousands phosphosites from minute amounts of biological material. One of the major challenges in the field is preserving the in vivo phosphorylation state of the proteins throughout the sample preparation workflow. This is typically achieved by using phosphatase inhibitors and denaturing conditions during cell lysis. Here we determine if the upstream cell collection techniques could introduce changes in protein phosphorylation. To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, we compared different sample workflows by metabolic labeling and quantitative mass spectrometry on Saccharomyces cerevisiae cell cultures. We identified highly similar phosphopeptides for cells harvested in ice cold isotonic phosphate buffer, cold ethanol, trichloroacetic acid, and liquid nitrogen. However, quantitative analyses revealed that the commonly used phosphate buffer unexpectedly activated signaling events. Such effects may introduce systematic bias in phosphoproteomics measurements and biochemical analysis.
authors.affiliation: †Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kanshin
authors.affiliation: †Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.|‡Department of Medicine, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Tyers
authors.affiliation: †Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.|§Department of Chemistry, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D002853:Chromatography, Liquid; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins
keywords: Saccharomyces cerevisiae; osmotic shock; phosphoproteomics; quantitative proteomics; sample collection