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A Robust Multiplex-DIA Workflow Profiles Protein Turnover Regulations Associated with Cisplatin Resistance and Aneuploidy

ABSTRACT: Measuring protein turnover is essential for understanding cellular biological processes and advancing drug discovery. The multiplex-DIA mass spectrometry (DIA-MS) approach, combined with dynamic SILAC labeling (pulse-SILAC, or pSILAC), has proven to be a reliable method for analyzing protein turnover and degradation kinetics. Previous multiplex-DIA-MS workflows have employed various strategies, including leveraging the highest isotopic labeling channels of peptides to enhance the detection of isotopic MS signal pairs or clusters. In this study, we introduce an improved workflow that integrates a novel machine learning strategy and channel-specific statistical filtering, enabling dynamic adaptation to systematic variations in channel ratios throughout pSILAC and SILAC experiments. These functions were evaluated to significantly improve both the comprehensiveness and accuracy of protein turnover profiling. In addition, we developed Our integrative workflow was benchmarked on both 2-channel and 3-channel standard DIA datasets and across two mass spectrometry platforms, demonstrating its broad applicability. Applying this workflow to an aneuploid cancer cell model before and after developing cisplatin resistance, we discovered a remarkable negative correlation between transcript regulation and protein degradation for major protein complex subunits elucidating the proteome buffering mechanism in genome aneuploidy.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Fibroblast

DISEASE(S): Disease Free,Malignant Neoplasm Of Ovary

SUBMITTER: Barbora Salovska

LAB HEAD: Yansheng Liu

PROVIDER: PXD057632 | Pride | 2025-05-14

REPOSITORIES: Pride

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			Action	DRS
	20181024_A2780_Cis_Ctr_1.raw	Raw
	20181024_A2780_Cis_Ctr_2.raw	Raw
	20181024_A2780_Cis_Ctr_3.raw	Raw
	20181024_A2780_Nor_Ctr_1.raw	Raw
	20181024_A2780_Nor_Ctr_2rep.raw	Raw

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Project description:Understanding the mechanism of resistance in platinum-based regimens for the treatment of high-grade serous ovarian cancer (HGSOC) is important for identifying new therapeutic targets to improve the clinical outcome of ovarian cancer patients. Mass spectrometry-based proteomic strategy was applied to spheroidal cisplatin sensitive and resistant HGSOC generated cell lines in the absence and presence of cisplatin drug. A complete expressed HGSOC proteome and phosphoproteome was characterized in cisplatin sensitive and resistant HGSOC cell lines providing insight into the mechanism of resistance development. PCA analysis showed that phosphorylation of a few proteins provides better classification than the whole proteome of the cellular subtypes. Specifically, a distinctive phosphoproteomic signature between cisplatin sensitive and resistant cell lines in the absence of drug was observed. This same phosphoproteomic signature was observed in our cisplatin sensitive cell line in the absence and presence of drug, indicating a vital role for phosphorylation of proteins in resistance development to cisplatin. The most phosphorylated protein was sequestosome (p62/SQSTM1). Differential expressions of apoptosis by the prognostic factor ratio of Bcl-2/Bax and autophagy, known to be regulated by p62/SQSTM1, was validated in the proteome data and by western blot analysis. A significant increase in apoptosis in the presence of cisplatin was observed in only the sensitive cell line while autophagy revealed increased expression in the resistant relative to sensitive cell line. Furthermore, site specific phosphorylation on 20 modified residues of sequestosome was characterized. Elevated expression of phosphorylation of sequestosome in resistant HGSOC cell lines was validated with western blot analysis. Here, we propose phosphorylation of sequestosome to be a marker and key in cisplatin resistance development in HGOSC ovarian cancers by shuttling ubiquitinated proteins to the autophagy pathway and influencing down-regulation of apoptosis.

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A Robust Multiplex-DIA Workflow Profiles Protein Turnover Regulations Associated with Cisplatin Resistance and Aneuploidy

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