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Systematic Evaluation of the Impact of Lysis Buffer on the Cellular Proteome and Glycosylation Modifications

ABSTRACT: In this study, the effects of four commonly used lysis buffers (sodium dodecyl sulfate (SDS), guanidine hydrochloride (GuHCl), urea (UA), and Mammalian Protein Extraction Reagent (MPER)) on proteome and N-glycosylation modification analysis, providing valuable reference for optimizing experimental workflows in proteomics and glycoproteomics research. Proteins were digested into peptides using the Filter-Aided Sample Preparation (FASP) protocol, followed by enrichment of glycopeptides via Hydrophilic Interaction Liquid Chromatography (HILIC). Proteomic data were analyzed with MaxQuant, while glycoproteomic data were processed using pGlyco3 and Panda software.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yang Zhao

PROVIDER: PXD067587 | iProX | Thu Aug 21 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:The continuing ‘desire’ in obtaining more quantitative and detailed information from cellular proteomics experiments in regards to proteome coverage and protein modifications asks for a systematic investigation of the protein extraction and digestion protocols, in particular when working with unicellular organisms with a strong cell wall such as the model yeast S. cerevisiae. The selection of a suitable sample preparation workflow is crucial to obtain in-depth proteome coverage, as the use of certain sample preparation protocols may bias the protein identification or induce (unexpected) peptide modifications. Here, we made an extensive comparison of preparation workflows commonly applied to S. cerevisiae. Workflows were compared on the basis of identified MS/MS spectra, peptide sequences and number and type of modifications using both restricted (Peaks) and unrestricted (TagGraph) database search approaches. The proteome coverage was mainly affected by the sample collection approach, while it was maximized using a FASP. Extensive reagent specific peptide modifications were detected when using formic acid, but also when using acetone. Such artefacts split the analyte mass signals and generate additional chemical noise that may also elute differently compared to the native peptide. The use of both an unrestricted and restricted database search increased identification rates significantly and resulted in the identification of approximately 70% of the MS2 spectra for the best protocol. The unidentified spectra were assessed by their de novo sequencing score. This confirmed that those spectra consisted by a majority of very low quality spectra, insufficient to match to database sequences. However, a small fraction of the unidentified spectra showed high quality, which presumably derive from unknown sequence variants not present in the database. This study demonstrates the high importance of the sample preparation workflow and the obtained results will guide researchers in the field to optimize sample preparation procedures.

Dataset Information

Systematic Evaluation of the Impact of Lysis Buffer on the Cellular Proteome and Glycosylation Modifications

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