Protein identification: guidelines for sample submission
Overview of workflow
The protein identification service is provided for the analysis of protein samples from gel bands from 1D SDSPAGE or gel spots from 2D-PAGE gels. In-gel reduction alkylation trypsin digestion is applied to samples and the
extracted tryptic peptides are analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS)
followed by database searching to obtain amino acid sequence.
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) “In-gel digestion for mass spectrometric characterization of proteins
and proteomes” Nat Protoc 1(6): 2856-2860. PMID: 17406544
Granvogl B, Gruber P, Eichacker LA. (2007) “Standardisation of rapid in-gel digestion by mass spectrometry” Proteomics 7(5): 64254. PMID: 17340585
Granvogl B, Plöscher M, Eichacker LA. (2007) “Sample preparation by in-gel digestion for mass spectrometry-based proteomics”
Anal Bioanal Chem 389(4): 991-1002. PMID: 17639354
Examples of applications for protein identification:
 Identifying unknown proteins from 1D SDS-PAGE or 2D PAGE gels.
 Verification of expression product.
 Discovery of interacting partners after immuno-precipitation (IP).
Sample preparation
 Run your 1D or 2D gels. E.g. Tris-Glycine, 4-20%, Bis-Tris 10% MOPS or MES buffer.
 Recommended amount of protein sample loaded onto the gel:
i. For a 20kD protein, load 50 ng to 150 ng for identification.
ii. For a 80kD protein, load 200 ng to 0.5ug for identification.
iii. For mixtures of proteins, estimate loading amount so that each gel band has about 1ug of proteins.
 To reduce contamination of keratins, always wear gloves when handling gels, use dedicated clean glassware,
staining trays for gels and avoid dust which contains human keratin.
 Stain your gels with Coomassie blue, SYPRO Ruby or “MS friendly” silver stain (please check with
manufacturer’s protocol).
 Cut out the gel spots/bands using a clean blade; wash between samples to avoid cross-contamination
 The gel spots/bands should not be greater than 0.25 cm2, about this size “
” for a 1mm thick gel.
 Place the samples into a natural colored 1.5mL micro-centrifuge tubes (separate tubes for each gel spot/band)
and cover gel pieces with about 50-200µL ddH2O. Please do not submit samples in colored tubes.
 Clearly label tubes with Ultra-Fine Point permanent Sharpie marker. Label tubes with PI’s initials and number
them consecutively. (Ex: PI-01, PI-02, etc.)
Sample submission
 Fill out sample submission form http://www.BioMS.pitt.edu/ProteinID_SubmissionForm.pdf
 Samples with completed submission form can be dropped off at the BST3 (3501 Fifth Ave Pittsburgh, PA
15260). Please contact the BioMS lab by email bioms@pitt.edu or lab phone 412-383-5937 // 412 648-1811
to arrange a time for drop off. BioMS Center Lab is on the 9th floor, you will need to stop at the security desk in
the lobby and call the lab phone or have the security guard call and one of the lab members will come down to
meet you and pick up the samples.
 Samples are processed weekly starting on Monday afternoon. Samples received after Monday noon will be
processed the following week.
 Cost: $80/gel band billed monthly to the 32 digit account code. Please make sure you are registered on the
BioMS website in order for us to invoice your lab.
Report
• Results are emailed to investigators on the following Monday with instructions on how to view the data.
Apr-14