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

ABSTRACT: 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. Here, we investigate the impact of protein extraction methodologies on proteomic analysis of S. cerevisiae, specifically, detergent-based lysis versus mechanical lysis with silica beads. We showcased this comparison by evaluating 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 4,700 proteins, covering approximately 80% of the yeast proteome. Hierarchical clustering and principal component analyses revealed that the choice of protein extraction method 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 the two SUMO E3 ligases deletion strains, implying potential redundant mechanisms of control for these proteins. These data underscore the importance of method selection at each single step of sample preparation in proteomic studies and enhances our comprehension of cellular adaptations to genetic perturbations.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Joao Paulo  

LAB HEAD: Joao A. Paulo

PROVIDER: PXD063969 | Pride | 2025-08-04

REPOSITORIES: Pride