Protein Identification by MS/MS (Peptide Sequencing)
The masses of peptides produced by the tryptic digestion of proteins are measured by mass spectrometry with high accuracy (known as a peptide mass fingerprint). Selected peptides are fragmented inside the mass spectrometer (tandem MS) to produce daughter ions, the masses of which relate to the peptide's sequence. The combined data are matched against protein sequence databases to determine the protein's identity.
- Protein from 1D or 2D gels, or otherwise purified.
- The protein's thiols may be blocked with iodoacetamide or acrylamide prior to SDS-PAGE.
- Coomassie stains are preferable although some silver stains may be used. To minimise contamination by human keratins, wear gloves when handling samples and keep gel tanks clean and covered.
Typically, we reduce the protein's thiols and modify them with iodoacetamide. The protein is then digested in-gel with trypsin. Peptides are extracted with a combination of acid and organic solvents, concentrated by evaporation, and subjected to mass spectrometry.
Several of the APC's mass spectrometers can be used.
LC ESI MS/MS: peptides are resolved by reversed phase nanoLC into the ION-TRAP via a nanoelectrospray ion source. Peptide ions are fragmented by Collision-Induced Dissociation or Electron Transfer Dissociation.
Ion Trap: nanoLC (Agilent Technologies), Captive Spray and Amazon ETD ion trap (Bruker Daltonics);
Orbitrap: nanoLC 3000 RSLC, LTQ Orbitrap XL ETD (both Thermo Fisher Scientific): for high resolution and high mass accuracy precursor spectra in combination with fast MS/MS analysis
Q-TOF: nanoLC 3000 RSLC, impact HD orthogonal Q-TOF (Bruker Daltonics): high resolution and high mass accuracy mass spectra (MS and MS/MS) for label-free experiments.
MALDI TOF/TOF (Ultraflex III and UltrafleXtreme, Bruker Daltonics): a peptide mass fingerprint and, typically, three MS/MS spectra (produced by Laser-Induced Dissociation) are acquired.
ION-TRAP and Q-TOF data are processed with Bruker DataAnalysis and BioTools then searched over the in-house MASCOT-database, matching against SwissProt, MSDB or NCBI databases.
Orbitrap data are processed using Proteome Discoverer, then searched over the in-house Mascot-database or sequest database, matching against SwissProt, MSDB or NCBI databases.
MALDI TOF/TOF data are simplified to lists of monoisotopic masses and usually analysed using an in-house MASCOT server, matching against SwissProt, MSDB or NCBI databases.
A written report is provided, describing the analysis undertaken and outlining the evidence for a protein's identification. If no hit is obtained, an indication of the possible reasons may be included. The report is normally sent by email, as PDF. Raw data are in proprietary formats and are not included with the report. Data are archived by the APC for at least 12 months or according to customer's requirements.