Proteomics Facility

Director - David W. Speicher, Ph.D.
Location: Room 154
Tel: 215-898-3830/3181
Fax: 215-898-0664

Introduction

This facility provides mass spectrometry (MS) and sequence analysis of proteins and peptides at maximum sensitivity, as well as related services including in situ proteolysis of proteins in 1D or 2D gels and peptide mapping. The major applications of these resources fall into two groups, analyses of recombinant proteins and identification of novel proteins isolated from natural sources.

The primary purposes for analyzing recombinant proteins are to verify that the expected sequence is obtained, evaluate potential processing of the N-terminal or C-terminal regions, and determine the extent of post-translational processing that has occurred. The minimum characterization of recombinant proteins typically recommended is to perform an N-terminal sequence and a matrix-assisted laser desorption/ionization (MALDI) mass analysis of the intact protein. In some cases, it may also be necessary to analyze tryptic fragments of the protein by HPLC peptide mapping and MS to more precisely characterize and locate either post-translational or undesired artifactual modifications. Similarly, MALDI MS analysis is the most effective method for determining the integrity of synthetic peptides. Currently, MALDI MS can typically determine peptide masses to within <1 Dalton and protein masses to within <0.1% error (i.e., <25 Da for a 25K protein).

The most common goal when analyzing proteins isolated from natural sources is to identify the protein(s) associated with a particular biological function of interest. In nearly all cases, the protein of interest should be isolated using either 1D or 2D polyacrylamide gel electrophoresis (PAGE). Hence the PAGE step can be used as the final purification step. If >10 pmoles of the protein of interest can be isolated, the typical approach for protein identification is to: 1) digest the Coomassie blue stained band in the gel with trypsin; 2) separate the peptides by microbore reverse phase HPLC; 3) analyze at least 10 peaks by MALDI MS; and 4) select 1 or more of the longest peptides for Edman sequencing. In addition, about 10% of the sample can be analyzed in parallel by MALDI MS to attempt to identify the protein by MS mass fingerprint analysis. If less than 10 pmoles, but at least 100 femtomoles is isolated, the protein identification must be attempted by MS methods alone.