Proteomics in the PMI involves mass spectrometry-based characterization of the protein complement of a biological sample. The biological sample can be a bacterial culture, a plant tissue, fungal mycelia, or a community containing multiple organisms. The primary workflow is to extract the proteins from the biological sample, digest those proteins into smaller peptides using trypsin or another proteolytic enzyme, and then analyze those peptides using nanoscale high-performance liquid chromatography interfaced with tandem mass spectrometry (LC-MS-MS). Computer analysis of the resulting data provides identification of the peptides and proteins in the biological sample, and semi-quantitative estimates of their abundances. Comparative experiments highlight proteins that are different across samples (e.g. treatment versus control.)
Step 1: Experiment information about sample, collaborator, experimenter, etc.
Sample generated by/received from
Date samples received
Cell or tissue type
URL(s) or protein fasta file(s) for sample species
Other information (replicates, miscellaneous information from collaborator, …)
Step 2: Sample Preparation- extract proteins from sample(s), and digest the proteins enzymatically to make smaller peptides that can be analyzed by mass spectrometry
Sample Processing date
Sample Processing Protocol (Word document to be uploaded if new, or chosen from pick list if previously uploaded)
Note: if an experiment involves several samples, the remaining items in Step 2 could be in a spreadsheet.
Results of total protein amount estimation
Protein estimation reagent used
Estimate of protein amount
Results of Peptide amount estimation (optional)
Peptide estimation reagent used
Estimate of peptide amount
Step 3: LC-MS-MS Analysis: HPLC separation of peptides from Step 2, followed by tandem mass spectrometry of each peptide to produce partial amino acid sequence information.
Back column details (length, packing material)
Amount of sample loaded onto back column
Back column wash method
Front column details (tip type, length, packing material)
Mass spectrometer (choose from a pick list)
HPLC system (choose from a pick list)
Mass spectrometer method file(s) (upload)
Mass spectrometer sequence file(s) (upload)
XCalibur software version (Xcalibur is the ThermoScientific software for controlling mass spectrometer data acquisition)
Step 4: Data Analysis: automated matching of experimental tandem mass spectra with predicted tandem mass spectra from all possible peptides from the sample to produce a list of peptides. Infer proteins from the list of identified peptides. Compare identified proteins across different samples/treatments/controls in the experiment. Analyze identified proteins for functional classes and metabolic pathways in which they are involved.
Person performing search
Search software and version (pick list: Sequest, Myrimatch, DBDigger)
Search output file names/locations, or upload, depending on whether search performed by Manesh Shah or locally
Parameter file for search (upload)
Post-search filtering software and version (pick list: DTASelect, IDPicker)