11/19/2019 Data interpretation using Proteome Discoverer 2.2 Terminology: • PSM: peptide-spectrum match, a spectrum that matches to a peptide sequence • p-value: probability of observing an incorrect PSM with a given score or higher by chance, low p-value indicates a small probability of observing an incorrect PSM • FPR: false positive rate, proportion of incorrect PSMs above a certain score threshold over all incorrect PSMs • FDR: false discovery rate, expected proportion of incorrect predictions amongst a selected set of predictions. The fraction of incorrect PSMs within a selected set of PSMs above a given score threshold. • q-value: the minimal FDR threshold at which a PSM is accepted, transforming the FDR into a monotone function, increasing the score threshold will always lower the FDR and vice versa • PEP: posterior error probability (also called local FDR). Measures the significance of a single spectrum assignment with a specific PSM score. It is the probability of the PSM being incorrect, i.e. PEP of 0.01 means there is a 1% chance of the PSM being incorrect. P vs Q vs PEP-values information: https://bioinfomagician.wordpress.com/2013/10/14/p-vs-q-vs-pep-values-in-mass-spec-database- search/
Transcript
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Data interpretation using Proteome Discoverer 2.2 Terminology:
• PSM: peptide-spectrum match, a spectrum that matches to a peptide sequence • p-value: probability of observing an incorrect PSM with a given score or higher by
chance, low p-value indicates a small probability of observing an incorrect PSM • FPR: false positive rate, proportion of incorrect PSMs above a certain score threshold
over all incorrect PSMs • FDR: false discovery rate, expected proportion of incorrect predictions amongst a
selected set of predictions. The fraction of incorrect PSMs within a selected set of PSMs above a given score threshold.
• q-value: the minimal FDR threshold at which a PSM is accepted, transforming the FDR into a monotone function, increasing the score threshold will always lower the FDR and vice versa
• PEP: posterior error probability (also called local FDR). Measures the significance of a single spectrum assignment with a specific PSM score. It is the probability of the PSM being incorrect, i.e. PEP of 0.01 means there is a 1% chance of the PSM being incorrect.
Open the results file in Proteome Discoverer. File->Open Result. Choose the report you wish to view and click Open.
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Proteins tab The Proteins (filtered) tab will initially be shown. This will show the proteins that were found to have matches to the input spectra. Each protein will have a line number and a checkbox to allow selection of the protein for other features such as exporting the data. Clicking on the Field Chooser icon (top-left icon in the table header) allows you to choose which columns to display.
• Protein FDR confidence: level of confidence for the protein • Master: indicates whether the protein is the master protein of a protein group • Accession: unique identifier from the FASTA database • Description: name of the protein • Sum PEP score: protein score calculated as the negative logarithms of the PEP values of
the connected PSMs • Coverage: percentage of the protein sequence covered by the peptides • # Peptides: number of distinct peptide sequences in a protein group • # PSMs: total number of identified peptide sequences (peptide spectrum matches,
PSMs), including redundant peptides • # Unique Peptides: total number of peptide sequences unique to the protein group • # Protein Groups: number of protein groups the protein belongs to • # AAs: number of amino acids in the protein • MW [kDa]: molecular weight of the protein • calc. pI: pI of the protein • Score Mascot: sum of the scores of the individual peptides from the Mascot search. It is
the cumulative protein score based on summing the ion scores of the unique peptides identified. If a peptide is redundant, the highest-scoring peptides is used.
• # Peptides Mascot: number of distinct peptide sequences in a protein group from the Mascot search
• Score Sequest HT: sum of the scores of the individual peptides from the Sequest HT search. It is the sum of all the peptide Xcorr values above the specified score threshold. It is calculated by:
0.8 + peptide_charge X peptide_relevance_factor
Where peptide_relevance_factor is a parameter set in the Sequest HT node with a default value of 0.4
• # Peptides Sequest HT: number of distinct peptide sequences in a protein group from the Sequest HT search
Double-click on the protein line to open the Protein Identification Details window. This will show the coverage.
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The protein sequence is shown with the coverage of each peptide shown in either green, yellow or red (depending on the confidence level of the peptide). Modifications can also be shown, as well as PTMs.
Click on the ProteinCard tab to access more information about the protein.
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Protein Groups tab
This page will show all the protein groups found in the samples. Clicking on the Field Chooser icon (top-left icon in the table header) allows you to choose which columns to display.
• Protein Group ID: unique identifier for the protein group • # Proteins: number of proteins in the protein groupd • # Unique Peptides: total number of peptides unique to the protein group • # Peptides: total number of peptides identified from all included searches for the master
protein of the protein group • # PSMs: total number of PSMs identified from all included searches for the master
protein of the protein group • Group Description: the description for the master protein
Peptide Groups tab
This page will show all the peptide groups found in the samples. Clicking on the Field Chooser icon (top-left icon in the table header) allows you to choose which columns to display.
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• Sequence: the sequence of amino acids that compose the peptide. • Modifications: static and dynamic identifications found in this peptide • Quality PEP: posterior error probability is the probability that the observed PSM is
incorrect. Also called local FDR. • Quality q-value: q-value for the peptide group • # Protein Groups: number of protein groups in which this peptide is found • # Proteins: number of proteins in which this peptide is found • # PSMs: total number of peptide sequences for the protein, including redundant peptides • Master Protein Accessions: accession of the master protein • # Missed Cleavages: the number of cleavage sites in the peptide that the cleavage
reagent or enzyme did not cleave • Theo MH+[Da]: protonated, monoisotopic mass of the peptide • Confidence: Mascot and Sequest HT; confidence icon for the search; green for high
confidence, yellow for medium confidence, red for low confidence • Ions Score Mascot: the probability that the observed match is a random event. The score
is reported as -10*Log10(P), where P is the absolute probability. A higher score indicates less chance of it being incorrect.
• XCorr Sequest HT: scores the number of fragment ions that are common to two different peptides with the same precursor mass and calculates the cross-correlation score for all candidate peptides queried from the database.
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PSMs tab This page will display all the precursor ions that have been fragmented and give detailed information about the spectra.
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Double-click on the PSM to open the Peptide Spectrum Match Identification Details window.
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MS/MS Spectrum Info tab
Filtering results Choose View->Display Filter Show only the high confidence peptides.
1. Click the Peptide Groups Filter and Click “Add Property”. Choose “Confidence” from the dropdown menu.
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2. Set the level of confidence you want to filter on. 3. Click “Apply” 4. Switch back to the Proteins tab. The scores will now reflect the results using only the
high confidence peptides.
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Results Summary window (View->Summaries) The results summary window will show the parameters used to perform the search, including information about the raw file, spectrum parameters and the parameters specific to the search applications (database, modifications, etc.).
Instructions written using information from Thermo Scientific Proteome Discoverer Version 2.2 User Guide.
