Evaluating Protein Extraction Techniques for Elucidating Proteomic Changes in Yeast Deletion Strains.

Abstract

Background: Alterations in protein abundance profiles in yeast deletion strains are frequently utilized to gain insights into cellular functions and regulatory networks, most of which are conserved in higher eukaryotes.

Methods: This study investigates the impact of protein extraction methodologies on the whole proteome analysis of S. cerevisiae, comparing detergent-based lysis versus mechanical lysis with silica beads. We evaluated the proteomic profiles of wild-type and two yeast deletion strains, siz1Δ and nfi1Δ (siz2Δ), which are SUMO E3 ligases. Combining isobaric TMTpro-labeling with mass spectrometry using real-time search MS3, we profiled over 4700 proteins, covering approximately 80% of the yeast proteome.

Results: Hierarchical clustering and principal component analyses revealed that the choice of protein extraction method significantly influenced the proteomic data, overshadowing the genetic variances among these strains. Notably, the detergent-based lysis showed superior performance in extracting proteins compared to mechanical lysis. Despite minimal proteomic alterations among strains, we observed consistent changes regardless of the lysis strategy in proteins such as Ino1, Rep1, Rep2, Snz1, and Fdh1 in both SUMO E3 ligase deletion strains, implying potential redundant mechanisms of control for these proteins.

Conclusion: These findings underscore the importance of method selection at each step of sample preparation in proteomic studies and enhance our comprehension of cellular adaptations to genetic perturbations.