Proteomics

Peptide Mapping by RP HPLC

Proteins can be chemically or enzymatically digested and then the fragments separated by RP C to produce one type of peptide map. The HPLC map (chromatogram) itself cannot be used to identify a protein, but fractions can be collected, then one or more of the fragments can be sequenced. The digest can be done with any of a wide variety of enzymatic or chemical methods. The most common choices are trypsin (which enzymatically cleaves on the C-terminal side of Arg and Lys residues) or BrCN (which chemically cleaves on the C-terminal side of Met residues). Our service includes the digest of your choice, and separation and collection of the fragments by HPLC. Separated fragments can then be characterized by N-terminal sequencing or other means.

Mass Spectral Peptide Mapping

CBI also offers peptide mapping by MALDI-TOF/MS and/or by ES/MS. This can be done on solution phase samples as well as on proteins extracted from electrophoresis gels. In this method, the protein is digested while still in the acrylamide gel slice, and can be done with less material than required for Edman degradation. This method is, however, not compatible with samples transferred to PVDF. After the in-gel digest, the complete digest mixture is subjected to linear MALDI-TOF/MS which produces a spectrum showing the masses of all of the fragments. The set of masses can be used in a database search to identify the protein, or if the sequence is known, the observed masses can be compared to the set of predicted masses. Our service includes the digest, linear MALDI-TOF analysis, and a search of the database.

If peaks of sufficient ion intensity are seen in the mass spectrum, selected mass peaks can be subjected to post source decay (PSD)/collision induced decay (CID) fragment analysis with and without treatment for chemical assisted fragmentation (CAF). The PSD/CID/CAF spectrum reveals a series of peptide fragments differing in masses corresponding to single amino acid residues. This data can be interpreted to give the amino acid sequence of up 25 residues. Alternatively, ES/MS/MS will reveal the primary amino acid sequence of mass species observed in the Total Ion Count chromatogram.

Data base searching can be by mass and/or by sequence. All usual data bases, UniProt, PIR, GenEmbl, are resident on CBI's in-house servers and all are updated on a calendar quarter basis.



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Commonwealth Biotechnologies, Inc. 601 Biotech Drive, Richmond, VA 23235 800-735-9224 (800-RELY-CBI) FAX: 804-648-2641 email: [email protected]