IF you have a Coomassie-stained band from 1D SDS-PAGE, simply follow these instructions:

1. Place the excised band in an Eppy tube with about 100µl of de-staining solution or water.

2. Complete a sample submission form in the GSL, and label your tube according to the GSL ID#.

(Required information: OUC, account and phase, your book-keepers name) 

3. Place tube and completed form in the boxes provided.

We shall then proceed with in-gel digestion - method can be found on our FAQs page. 

Your results will be e-mailed to you in the form of an Excel sheet. If this does not match your requirements, or you are unsure, read on........ 

Basics of protein identification are simple: Proteins are digested and the resulting peptides are analyzed by mass spectrometers to obtain their fragmentation (or sequence) data. There are three different types of MS data that can be used in a database search. They are (1) molecular weights of tryptic peptides that can be used for Peptide Mass Mapping, (2) a combination of mass data and partial amino acid sequence that can be used for Sequence Tag, and (3) tandem mass spectrometry data (uninterpreted) that are used for MS/MS fragmentation ion search.

It is option (3) that is available within our facility.

Most proteins submitted to us are gel purified, which are submitted to in-gel digestion with trypsin. The digest is then analyzed by LC-ESI-MS/MS in a LTQ linear ion trap mass spectrometer (Thermo, San Jose, CA). Database search is against fasta databases held within the lab, these can be either organism specific, user submitted, or Uniprot-TREMBL. Searches can also be made against nucleotide databases (genomic or cDNA), including those created with our own GS FLX.

We also accept in-solution and dried samples.  Identification typically requires less than a picomole, i.e., 50 nanograms for a 50kDa protein.

Not all proteins are digested equally well by trypsin. For example, highly hydrophobic and aggregated proteins, and extensively glycosylated proteins are not digested well. Trypsin may not be the ideal enzyme for very small proteins (<10 kDa) or some very acidic proteins that are lacking or have a low occurrence of basic amino acids (Lys and Arg) in the sequence. When trypsin fails to digest the protein, or the specific sequence sought is unsuited to tryptic digestion, we use alternate digestion protocols as an additional effort: this is typically by either chymotrypsin or Glu-C (V8 protease).

Questions?, ask Norm

P.S. If you did not use gloves throughout your sample preparation, your results can be found here . 

It is often possible to identify several hundred proteins within a sample on a single LC-MS/MS run. This may be of particular value if you are interested in 'spotting the difference' between protein profiles across a number of samples (treatments). The data obtained by this approach is non-quantitative in nature, but rapidly pinpoints gross differences / similarities between samples. This is often a good staging point prior to iTRAQ and SILAC experiments, and gives a good indication of how many proteins the iTRAQ or SILAC approach can quantify.

Results will be supplied in a Scaffold format, a free viewer can be downloaded here.

Data is typically distributed via Velocity - details here.