Protein Identification, by tryptic digestion and mass spectrometry

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 block the protein's thiols with iodoacetamide and digest the protein in-gel with trypsin. Peptides are extracted with combinations of acid and organic solvents, concentrated by evaporation, and subjected to mass spectrometry.

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.
For Q-TOF data, the measured monoisotopic masses of peptides and their daughter ions are used to interrogate Swissprot or NCBI-NR databases using Micromass ProteinLynx Global Server software. The hit list is inspected to identify significant matches, usually with at least two peptides. Typically, 5 to 15 matching peptides are found. Often, significant matches to homologues can be found in cases where the true protein is not represented in the database. If no significant match is found, automated sequence assignment coupled with BLAST searching may be performed.
ION-TRAP data are processed with Bruker DataAnalysis and BioTools then searched over the in-house MASCOT-database, 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.
If the data are used as part of a scientific publication, due reference to the APC should be made.

Biomolecule mass measurement, by mass spectrometry

The masses of molecules of interest (proteins/peptides, oligosaccharides, lipids or nucleic acids) can be determined with high accuracy using mass spectrometry. The size of a known protein or peptide can be confirmed or the extent of its modification (proteolytic, post-translational or chemical) determined. Mass profiles of biological fluids can be made.

The sample should be in soluble form and free of detergents. Desalting by reversed phase chromatography (on-line or off-line) is often desirable.

Several of the APC's mass spectrometers can be used.

Electrospray: generates multiple charge states of proteins

Electrospray Q-TOF2 (Waters)
Ion-Trap (Bruker HCT ultra ION-Trap)
accurate to within 50-100 ppm for proteins
resolution near theoretical limit of isotopic envelopes.

MALDI: generates singly or doubly charged forms of most proteins

MALDI TOF/TOF (Bruker ultraflex III)
accurate to within 150-300 ppm for proteins
resolution ¼ - ½ of the theoretical limit of isotopic envelopes
can be highly sensitive
can generate profiles from biological fluids in a single analysis
highly accurate for the measurement of peptides of mass < 4 kDa.

Sample is introduced into electrospray instruments by direct injection or reversed phase LC.
Sample is mixed with matrix on a target plate and may be desalted on the target.

Electrospray: mass(es) are calculated by mathematical transformation of the m/z ion series.
MALDI: Masses of the analytes are normally observed directly.

A written report is provided, describing the analysis undertaken and mass(es) measured. Where appropriate, comparisons with calculated masses are made. Raw data are in proprietary formats and are not included with the report. Data are archived by the APC.
If the data are used as part of a scientific publication, due reference to the APC should be made.