Dataset Information

Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.

ABSTRACT: A common problem encountered when performing large-scale mass spectrometry proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). M. pneumoniae cultures were grown in defined media, and their proteomes were analyzed by mass spectrometry. An initial database search with a curated M. pneumoniae protein database left around 78% of the acquired spectra without an assignment. After re-analysis of the data with the PEAKS software and a larger protein database, the percentage of non-assigned spectra was reduced to 27%, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33413 of spectra with assigned amino acid sequences, could not be mapped in any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to post-translational modifications and 4% to protein variants. We found that deamidation of Asn affected preferentially to integral membrane proteins and that about 1% of the peptides had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or Arg/Lys at the C-terminus. Thus, in an ideal system, we maximized the level of assignment to 73% of the spectra (51453 out of 70040 initial acquired spectra).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Francesco Mattia Mancuso

LAB HEAD: Eduard Sabidò

PROVIDER: PXD002779 | Pride | 2016-01-05

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	140325_S_MLLS_04_01.raw	Raw
	140325_S_MLLS_04_01_NCBI_Mpneumoniae.pep.xml	Pepxml
	140325_S_MLLS_04_02.raw	Raw
	140325_S_MLLS_04_02_NCBI_Mpneumoniae.pep.xml	Pepxml

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Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.

Lluch-Senar Maria M Mancuso Francesco M FM Climente-González Héctor H Peña-Paz Marcia I MI Sabido Eduard E Serrano Luis L

Proteomics 20160201 4

A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common cont ...[more]

PMID: 26702875

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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.

Project description:Mycoplasma pneumoniae is a significant cause of pneumonia, particularly in children, the elderly and the immunocompromised, and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model pathogen where extensive ‘omics’ studies have been conducted to gain insight into how minimal genome self-replicating organisms function. Here we conducted an N-terminome study to determine which proteins are targets of processing events in M. pneumoniae. We identified 6,973 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Nearly half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events which predominately target negatively charged residues in P1′ (D and E) with lysine or serine/alanine in P2′ and P3′ positions. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Mycoplasma pneumoniae culture conditions The M. pneumoniae (strain M129) cells were cultured in modified Hayflick’s medium at 37 °C in tissue culture flasks using established culture conditions63. Modified Hayflick’s medium contained 21 g PPLO broth base without crystal violet, 5 g of D-glucose, 4 mL of 0.5% phenol red, 100 mL of liquid yeast extract (150 g/L), 200 mL heat-inactivated horse serum (56 °C, 30 min) supplemented with 1 g ampicillin (Sigma, A5354, St. Louis, MO, USA) per litre. M. pneumoniae cells were harvested at mid-log phase by washing adherant cells 3 times with PBS and collected using cell scaping.

Dataset Information

Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.

Dataset's files

Publications

Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.

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