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  Thursday, May 23, 2019 "C’est ce que nous pensons déjà connaître qui nous empêche souvent d’apprendre" Claude Bernard Kültz Lab · Intranet
CAMP Proteome DDA Profiling
Information for data interpretation
CAMP-DDA: Profiling data are based on online LCMS2-DDA. Data acquisition and processing software and workflows were previously reported (Kültz et al., 2013, MCP; Kültz et al., 2015, Proteomics). First-tier data-dependent acquisition results (unbiased discovery surveys) are analyzed by PEAKS-Q (BSI) in association with Mascot (Matrixscience) and X!Tandem (theGPM) for protein ID and label-free quantitation. Scaffold (Proteome Systems) is used for consolidation of spectral and ID data (Scaffold viewer).
CAMP-DIA: Results of second-tier targeted data-independent acquisition (DIA, hypothesis testing for independent validation of regulated proteins) are analyzed using MSMS spectral libraries that are generated in our lab in conjunction with freely distributed Skyline.
Summary page for protein identification and quantitation based on PEAKSQ p less 0.05 (incl. QC data)
  • Information about retention time deviation, mass error, and sample parameters pertinent to protein identification and label-free quantitation is displayed. A heat map showing proteins that are statistically significant betwen the two experimental groups, volcano plots for peptide and protein levels and other quality control charts are shown. Statistical significance for label-free quantitation is based on PEAKSQ -log10px10 greater than 13, corresponding to P less than 0.05.(Cox & Mann, Nature Biotechnol., 2008)
  • The most informative charts on the summary page are 1) the heatmap, which includes the accession numbers of the proteins that significantly differ between the two conditions, and 2) the protein level volcano plot, which shows the distribution and statistical significance (-log10px10) of protein abundance differences for each protein identified and quantified in the two samples.
  • Note that the heat map color scale and the order of arrangement of samples in the heat map is identical to that of the thumbnail heatmap images shown in the PEAKS-Q protein LFQ profiles and the corresponding detail pages containing the peptide profiles (PEAKS icon link ).
  • Also note that the heat map color is based on a log2 scale.
Summary page for protein identification and quantitation based on BH-corrected ANOVA p less 0.05 (incl. QC data)
  • Information about retention time deviation, mass error, and sample parameters pertinent to protein identification and label-free quantitation is displayed. A heat map showing proteins that are statistically significant betwen the two experimental groups, volcano plots for peptide and protein levels and other quality control charts are shown. Statistical significance significance for label-free quantitation is based on BH-corrected (Benjamini & Hochberg, J. Royal. Stat. Soc., 1995) ANOVA (Welch, Biometrika, 1951) P less than 0.05.
  • The most informative charts on the summary page are 1) the heatmap, which includes the accession numbers of the proteins that significantly differ between the two conditions, and 2) the protein level volcano plot, which shows the distribution and statistical significance (-log10px10) of protein abundance differences for each protein identified and quantified in the two samples.
  • Note that the heat map color scale and the order of arrangement of samples in the heat map is identical to that of the thumbnail heatmap images shown in the PEAKS-Q protein LFQ profiles and the corresponding detail pages containing the peptide profiles (PEAKS icon link ).
  • Also note that the heat map color is based on a log2 scale.
The PEAKS-Q protein LFQ profiles page displays data for all proteins.
  • A table of proteins with sample-specific and group-specific heatmaps for each protein is displayed for two experimental groups.
  • Clicking on an accession number for any protein listed in that table opens a detail page with peptide-level information for that protein.
  • The key for the heatmap intensity scale and sample/ group order is the same as that provided in the PEAKS-Q heatmap and LCMS quality control data page (previous link) - please refer to the first link to retrieve it.
  • A white (missing) box indicates that the corresponding protein/ peptide was either not identified or less abundant than the intensity threshold for reliable quantitation in the corresponding sample.
The protein table containing data for complying with MIAPE and Mol.Cell.Proteomics standards.
  • The column headers in the Excel table have the following meanings:
  • Protein Group: Proteins with ambigous sets of peptides (e.g. isoforms) are grouped into sets to remove ambiguity. If this number is identical for two proteins then it means that they share a common set of peptides
  • Accession: NCBI protein or UniProt accession number.
  • Significance (-10lgP): P value for statistical significance in protein abundance. For details on how this value is calculated refer to the PEAKS-Q manual and tutorials at the BSI website.
  • Coverage (%): Portion of the protein sequence that is covered by unambigously identified peptides.
  • #Peptides: Number of peptides identified for that protein.
  • #Unique: Number of peptides identified that are unique for that protein.
  • PTM: Whether the protein contains a PTM in at least one corresponding peptide.
  • [Sample Name] Area: The XIC integral (label-free quantitative value) for each biological replicate of that protein.
  • Sample Profile (Ratio): The relative protein abundance in each sample separated by a colon.
  • [Group Name] Area: The average XIC integral for each experimental group.
  • Group Profile (Ratio): The relative protein abundance of the averages of each experimental group separated by a colon.
  • Avg. Mass: Molecular mass of the protein (in Dalton).
  • Description: Protein annotation.
The peptide table containing data for complying with MIAPE and Mol.Cell.Proteomics standards.
  • The column headers in the Excel table have the following meanings:
  • All column headers with same name as shown above for the protein table have the same meanings.
  • Sequence: peptide sequence.
  • Unique: Whether the peptide is unique to a single protein.
  • Quality: The spectral (MSMS) quality score for the peptide.
  • m/z: mass over charge ratio of the peptide.
  • RT: retention time of the peptide (referring to when - during what part of the gradient - it is eluted from the column and injected into the mass spectrometer).
  • z: peptide charge.
  • Normalized Area: XIC integral for the peptide normalized against the corresponding total ion chromatogram (TIC) integral to account for possible slight differences in sample loading.
  • Start: Sequence position in the protein at the peptide start.
  • End: Sequence position in the protein at the peptide end.
  • Used: Whether the peptide was used for automatic quantitation of the corresponding protein. Note that PEAKS-Q uses the first three unique peptides with the highest average area for calculating the abundance of the corresponding protein. Note that this table allow customized inclusion or selection of additional peptides
  • PTM: Whether the peptide contains a posttranslational modification. The position of the PTM is shown in the peptide sequence and the nature of the PTM can be deduced from the mass difference indicated in the peptide sequence by using UniMod.
The Normalization factor for quantitative comparison of the spectra.
  • The Normalization factor has already been incorporated into the data shown as heatmaps and in the tables. It is provided only for information and quality control purposes.
  • The normalization factor has been calculated by integrating the entire base peak chromatogram (BPC) of each sample with DataAnalysis 4.2 (Bruker Daltonics).
The Scaffold file containing data, metadata, and a Materials & Methods paragraph for publication.
  • For private datasets (when a user is logged in), the extension of the downloaded file scaffold.cfm needs to be changed to scaffold.sf3 before opening the file in Scaffold Viewer.
  • Scaffold allows comprehensive and detailed inspection of data, metadata, and all spectra for identified peptides.
  • Scaffold Viewer is free and can be downloaded at Proteome Software website.
  • Please refer to the Scaffold Users Guide for instructions on how to use this software.
  • Scaffold can also be used to derive crude quantitaive estimates based on spectral counts and presence/absence. However, the label-free quantitation done with PEAKS-Q, which is based on extracted ion chromatogram (XIC) integrals, is more precise.
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Disclaimer The content presented on this site is for information purposes only. The author(s) and the University of California are not liable for any inaccuracies or losses resulting from the use of this site. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation, National Institutes of Health, or any other supporting agency. Kültz Lab. © 2002 - 2019.