Proteomics Workflow

PMI Workflow – Proteomics

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.

  1. Sample generated by/received from
  2. Date samples received
  3. Sample Information
  4. Sample species/component
  5. Cell or tissue type
  6. Sample mass
  7. URL(s) or protein fasta file(s) for sample species
  8. Other information (replicates, miscellaneous information from collaborator, …)
  9. Tag/ID number/barcode
  10. Proteomics experimenter

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

  1. Sample Processing date
  2. Sample Processing Protocol (Word document to be uploaded if new, or chosen from pick list if previously uploaded)
  3. Note: if an experiment involves several samples, the remaining items in Step 2 could be in a spreadsheet.
  4. Results of total protein amount estimation
  5. Protein estimation reagent used
  6. Estimate of protein amount
  7. Protein digestion
  8. Enzyme type
  9. Enzyme amount
  10. Digestion conditions
  11. Results of Peptide amount estimation (optional)
  12. Peptide estimation reagent used
  13. 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.

  1. Back column details (length, packing material)
  2. Amount of sample loaded onto back column
  3. Back column wash method
  4. Front column details (tip type, length, packing material)
  5. Mass spectrometer (choose from a pick list)
  6. HPLC system (choose from a pick list)
  7. Mass spectrometer method file(s) (upload)
  8. Mass spectrometer sequence file(s) (upload)
  9. 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.

  1. Person performing search
  2. Search software and version (pick list: Sequest, Myrimatch, DBDigger)
  3. Search output file names/locations, or upload, depending on whether search performed by Manesh Shah or locally
  4. Parameter file for search (upload)
  5. Post-search filtering software and version (pick list: DTASelect, IDPicker)
  6. Filtered results file names/locations
  7. Statistical/comparative analysis results file names/locations
  8. Functional analysis of identified proteins
  9. Description of methods
  10. Results file
  11. Metabolic pathways for identified proteins
  12. Description of methods
  13. Results file