News

Instructions for preparation and submission of samples for protein identification

Submitting samples contained in  Coomassie-stained and silver-stained SDS-PAGE gel slices 

Care should be taken while running the gel and cutting out bands to minimize contamination with keratin. All reagents should be of good quality, and all equipment used for running and staining the gel should be pre-cleaned to get rid of any dust. The gel should be handled with gloves only. We accept gel slices stained with Coomassie, fluorescent dyes such as SYPRO Ruby, or MS-compatible silver stain (no glutaraldehyde used). Please call us at 913-522-7611 prior to submitting silver-stained gel slices to discuss whether destaining or other treatment is needed. 

Samples should be submitted with a picture of the gel taken before band excision. The bands that are being sent for analysis should be clearly marked on the picture.

Gel slices should be placed in 1.5 ml plain, non-lubricated Eppendorf tubes. The slices can be moist, but any excess liquid should be removed. Please make sure that the tubes are labeled clearly and that the sample identification number or name on the tube matches the number or name on the sample submission form.

Eppendorf tubes containing gel slices can be sent in a regular padded envelope without ice if shipped by overnight mail. Please send a picture of the gel and the sample submission form in the same envelope as the sample. 

Submitting samples in solution

If submitting protein samples in solution, please call us at 913-522-7611 prior to sending samples to discuss your project and to receive a quote.  We ask that our customers run part of the sample on SDS-PAGE gel, stain it with Coomassie and send us the gel image and a completed sample submission form by e-mail or fax (555-555-5555). After we receive all the information about your sample, we will be able to tell you whether your sample will need additional purification, concentration, buffer exchange or other treatment before it can be sent for analysis.

Protein solutions should be shipped on dry ice.

Sorry, we do not accept radioactive samples.

Please call 913-522-555 or email with any questions.

Technology used for protein identification

Nano-LC/MS/MS technique has become the standard method for protein identification when sensitivity and accuracy of the analysis are needed, especially when many proteins are present in a sample.

The processing of the sample starts with proteolytic digestion, typically with trypsin. The resulting peptide mixture is concentrated on a peptide trap column and washed to get rid of salts and other impurities. Then the peptides are separated on a microcapillary C18 reverse-phase chromatography column. We use PicoFrit columns that enable direct spray of the eluting peptides from the tip of the column into the mass spectrometer, eliminating post-column losses.

Full MS and MS/MS spectra are acquired by the LCQ Deca XP Plus ion trap mass spectrometer (ThermoFinnigan). MS/MS spectra are obtained after precursor peptide ions are isolated and fragmented inside the ion trap of the mass spectrometer and the resulting series of of daughter ions are detected in a tandem mass spectrometry experiment. In a typical 90 min analysis of a sample, about 400 MS and 1200 analytical MS/MS spectra can be acquired, which is sufficient for identification of hundreds of peptides. The detection of low-abundance peptides is facilitated by the dynamic exclusion function of the instrument. It temporarily puts a parental mass on the exclusion list after its MS/MS spectra is acquired and lets the instrument collect MS/MS spectra of the less intense components that otherwise would not be examined. The sequences of the parent peptides are inferred by matching the MS/MS spectra to protein sequence databases. This analysis is performed by the TURBOSEQUEST software.

Protein Identification Service

Currently, we offer protein identification by tandem mass spectrometry. We utilize nano-LC/MS/MS technique, which is one of the most sensitive and reliable methods available now. It allows identification of proteins at low fmol levels and enables identification of numerous proteins in complex protein mixtures.

Our protein identification service includes complete sample processing: we perform reduction, alkylation, and in-gel trypsinization, followed by peptide extraction and separation on a microcapillary reverse-phase column. Peptides are eluted and electrosprayed directly into an LCQ Deca XP Plus ion trap mass spectrometer. Full MS spectra as well as MS/MS spectra are acquired, and the data is analyzed by TURBOSEQUEST software. The results of this analysis are e-mailed to the customer 2-4 business days from the day we accept the sample.

To discuss analysis of mixtures of proteins in solution or other types of projects please call 913-522-5555 or email us.

Our prices are comparable to the lowest prices offered by university core facilities.

-