Preserving the yeast proteome from sample degradation

Abstract

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95 degrees C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.

Quantitative Proteomics Targeting Classes of Motif-containing Peptides Using Immunoaffinity-based Mass Spectrometry

“…The pellets were dissolved in 10 ml ice-cold Milli-Q water and then centrifuged at 2000 × g (5 min at 4 °C). In order to preserve the samples from degradation (28), the pellets were resuspended in 1.5 ml cold 10% (v/v) TCA, transferred to 2 ml tubes and finally collected by centrifugation at 13,200 rpm for 1 min at 4 °C. Samples were snap-frozen in liquid nitrogen and stored at −80 °C until further use.…”

The Impact of Biosampling Procedures on Molecular Data Interpretation

“…However, cells in culture at the same time are far more closely connected to a stressful environment than cells in tissue biopsies, which are enclosed in the organizational structural remainder of the pre-sampling tissue, and therefore are likely to be more susceptible to even small perturbations. Extensive proteomic differences have been observed in a comparative BT study on yeast cells (28). Differences in culturing conditions and BTIs and BT methodology, such as the extent of time washing the cells, breaking up adherence by trypsination, biomechanical pressure effects, and limited access to oxygen during centrifugation, all combine to influence the degree to which the final sample represents the pre-sampling in vitro state.…”

“…This image is not as clear with larger proteins (Ͼ10 kDa). Proteomic profiles derived from SFIϩMCI neural tissue and in vitro yeast biosamples demonstrate an increase in low molecular weight proteins and a reduction in high molecular weight proteins (some of the former being fragments of the latter), a trend absent in SFI-handled biosamples from heart, liver, and pancreatic tissue (8,10,28). The neural tissue degradation profile is possibly a reflection of its high sensitivity to ischemia and/or the dissipation of ion gradients and subsequent rise in Ca 2ϩ concentration (causing an elevation of Ca 2ϩ protease activity) (77,82).…”

Effects of Heat Shock Treatment on Enzymatic Proteolysis for LC-MS/MS Quantitative Proteome Analysis

“…27 This results in protein denaturation in a reproducible and irreversible manner, facilitates trypsin cleavage, and prevents protein degradation, thereby leading to increased identification of protein and peptide IDs in plasma, tissue, neutrophils, and yeasts. 13,14,28 This is an interesting additional use of the instrument, which otherwise is mainly used to denature degradation enzymes irreversibly along with other proteins to preserve the proteome intact.…”