Preserving the yeast proteome from sample degradation.
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.
Grassl
Julia
J
UCD Conway Institute of Biomolecular and Biomedical Research, School of Biomolecular and Biomedical Science, University College Dublin, Ireland.
Westbrook
Jules A
JA
Robinson
Aisling
A
Borén
Mats
M
Dunn
Michael J
MJ
Clyne
Rosemary K
RK
D015180:Electrophoresis, Gel, Two-Dimensional; D011309:Preservation, Biological / Q000379:methods*; D055550:Protein Stability; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014238:Trichloroacetic Acid / Q000737:chemistry
D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D014238:Trichloroacetic Acid