The very first time you receive your Excel file(s) summarizing the search results, you might feel confused. No worries, based on my experience, you are not alone! This post will guide you through.
You should receive twice as many files as the number of samples you submitted. Your sample is always injected after a blank run. This is done to account for potential carryover from previous sample injections, which is unavoidable in a service facility environment. Thus, “14-01-29-blank-01” precedes your first sample, “14-01-29-TNL1-02” and so on. I may also include my standard data so that you could see what type of data is obtained using a pure standard. Blanks, samples, your controls, and my standards are always run using the same instrument parameters.
UniprotKB protein description. Provides the name of the protein exclusive of the identifier that appears in the Accession column.
The number of identified proteins in the protein group of a master protein. Proteins are grouped based on sequence homology and/or isoforms as explained below.
The number of peptide sequences unique to a protein group.
The number of distinct peptide sequences in the protein group.
The calculated parameters of the protein based on the amino acid sequence in the FASTA database used to generate the report. The Proteome Discoverer application calculates the molecular weight without considering post-translational modifications. If you have separated proteins by molecular weight by PAGE, you can use the protein’s molecular weight as a rough constraint to estimate whether it is reasonable to identify a particular protein in a certain fraction that was analyzed.
Next, expand the sheet by clicking on [+] which opens the column parameters for the associated peptides.
A top level confidence achieved with the peptide sequence: high confidence, medium confidence, or low confidence. I send you only the high-confidence data, unless instructed otherwise.
The sequence of amino acids that compose the peptide.
The number of protein groups in which this peptide is found.
MS/MS-based proteomics studies are based on peptides. However, deducing protein identities from a set of identified peptides could be difficult because of sequence redundancy, such as the presence of proteins that have shared peptides. These redundant proteins are automatically grouped and are not initially displayed in the search results report.
The proteins within a group are ranked according to the number of peptide sequences, the number of PSMs, their protein scores, and the sequence coverage. The top-ranking protein of a group becomes the master protein of that group. By default, only the master proteins are displayed on the Proteins page.
One master protein that is identified by a set of peptides that are not included (all together) in any other protein group.
All proteins that are identified by the same set or a subset of those peptides.
The # Proteins column on the Proteins and Peptides pages of the results report displays the number of identified proteins in the protein group of a master protein.
The unique identifiers (accessions) of all master proteins from all protein groups that include this peptide sequence. Since I normally group proteins by selecting the “Consider Leucine and Isoleucine as Equal” option, this column also lists identifiers from master proteins that may include this specific peptide sequence. The identifiers displayed in the Protein Group Accessions column are the same as those displayed in the Accession column on the Proteins page.
The static and dynamic modifications identified in the peptide. I always use iodoacetamide for Cys alkylation, and this static modification will be in your search results as ‘Carbamidomethyl’ unless you modified your Cys residues with a different reagent. Met oxidation and Asn and Gln deamidation are common dynamic modifications.
The charge state of the peptide, z (z is always greater than 1 as set during the MS analysis).
Calculated m/z of the peptide with z = 1. It should be “MH+ [m/z]”, not [Da].
The peptide’s retention time during chromatographic separation.
The number of cleavage sites in a peptide sequence that a cleavage reagent (enzyme) did not cleave. This number excludes cases where an amino acid (e.g. Pro) inhibits the cleaving enzyme (e.g. trypsin).
If you prefer the original user guide, I have included it for your reading pleasure.
This entry was posted in Data and tagged coverage, data, Excel report, probability, protein group, protein identification, PSM, results, score, unique peptides, Xcorr on January 29, 2014 by Tania.