A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification.

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database.

Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum.

A set of 19 heat shock proteins (Hsp) was observed - by subtractive two-dimensional gel electrophoresis - to be induced when Bradyrhizobium japonicum, the nitrogen-fixing root-nodule symbiont of soybean, was temperature up-shifted from 28 degrees C to 43 degrees C. Up-regulated protein spots were excised from multiple two-dimensional gels. The proteins were concentrated using a funnel-gel device before being blotted onto poly(vinylidene difluoride) membranes for digestion with trypsin before MS and tandem MS analysis or for Edman sequence determination.

An algorithm for the identification of proteins using peptides with ragged N- or C-termini generated by sequential endo- and exopeptidase digestions.

We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, 'Tag searching', in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, 'Rag Tag' searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation.

Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry.

Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements.