Sample Collection Method Bias Effects in Quantitative Phosphoproteomics

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.