Protein Identification

If you wish to identify an unknown protein or to confirm that a protein sample is what you know or suspect it to be, then MS-based proteomics can provide several solutions.

1. Peptide Mass Fingerprinting (PMF):  This procedure, commonly used to identify low complexity protein samples (gel bands / simple mixtures), involves generation of an accurate mass spectrum of the peptide mixture resulting from the digestion of a protein by an enzyme (typically trypsin). The peptide fragments provide a characteristic 'fingerprint' that is highly specific for the protein under study. When the masses are searched using a database query tool such as Mascot™ it is often possible to identify the protein with a high degree of statistical significance. Typically (in our lab) this MS spectrum is generated using a MALDI-TOF instrument and the results are scored using the Mascot PMF tool.

Sample Requirements:  Samples are typically stained gel bands or simple mixtures.
Solution samples are best supplied in a fairly neutral pH buffer. Please avoid non-ionic detergents such as Tween or Triton in your solutions. Amounts of ~20 ng and above are routinely required for analysis. Low sample volumes (< 50 ul) are desirable.
Gel bands that are Coomassie blue-stained generally score significantly in Mascot. Faintly-stained bands, particularly those of small proteins <15 kDa, tend to score less well. Similarly with silver-staining (low ng); we find that results with Sypro Ruby™ staining are more consistent. Such samples may also benefit from MS/MS analysis (see below).
Excised gel bands may be submitted directly to us. Do not add additional liquid to your sample tubes.

2. MS/MS Analysis:  This can be viewed as a progression of mass fingerprinting. In MS/MS the digest peptides are both mass measured and then fragmented to generate ions characteristic of the parent peptide. This double analysis leads to significantly greater Mascot confidence scores. MS/MS analysis is a particularly powerful tool for analysis of both complex mixtures (coupled to HPLC - LC/MS/MS) and with modern MS instrumentation, analyzing low abundance samples (~low / sub-fmol range).  It can also be used to detect & characterise PTMs.

In our lab we offer LC/MS/MS analysis in a range of options depending on sample complexity:

  • Simple digests (gel bands) can be sprayed directly.
  • Medium complexity samples (5 - 20 components) can be analyzed with a simple 'trap & elute' LC/MS arrangement.
  • Highly complex samples (10s - 100s of components) require in-depth nano-LC/MS/MS analysis and perhaps additional steps for sample simplification.

The electrospray MS data that we generate is automatically searched and scored using both the Mascot MS/MS Ion Search Tool (Matrix Science) and the SEQUEST MS/MS Search Tool (Thermo).

Sample Requirements:  Sample preparation requirements are pretty much identical to those for PMF given above.
More complex samples for LC/MS/MS analysis are best supplied in pelleted form. You are advised to contact us to discuss sample preparation of complex samples & proteomes.
Again avoid using non-ionic detergents such as Tween or Triton in your solutions if possible.

Note: Edman N-terminal amino acid sequencing is another technique that can identify proteins. More details can be found here.

Costing, sample submission and ordering details are located here.