Dataset Information

Shot-gun proteomic analysis of S Cerevisiae, Candidatus Accumulibacter and Clostridium sacch.

ABSTRACT: Shot-gun whole cell lysate proteomics data from pure and enriched microbes were de-novo sequenced using PEAKS de Novo. The obtained peptide sequence lists serves as a test data for the peptide-centric taxonomy database Unipept.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Clostridium Saccharoperbutylacetonicum Saccharomyces Cerevisiae (baker's Yeast) Candidatus Accumulibacter

SUBMITTER: Martin Pabst

LAB HEAD: Martin Pabst

PROVIDER: PXD016992 | Pride | 2021-03-15

REPOSITORIES: Pride

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Source:

			Action	DRS
	MP18112018_yeast_Y1_1uL_DDA01.raw	Raw
	MP18112018_yeast_Y1_1uL_DDA01_denovo_peptides.csv	Csv
	MP18112018_yeast_Y1_1uL_DDA02_denovo_peptides.csv	Csv
	MP18112018_yeast_Y2_1uL_DDA01.raw	Raw
	MP_AM27072018_S1SC_No1_DDA01.raw	Raw

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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

Shot-gun proteomic analysis of S Cerevisiae, Candidatus Accumulibacter and Clostridium sacch.

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