Characterizing Proteins from 2-DE Gels by Internal Sequence Analysis of Peptide Fragments: Strategies for Microsample Handling

For years, proteolytic digest and peptide purification were essential tools for the biochemist wishing to determine the primary structure of a protein. The strategies changed with the availability of a polypeptide gas-phase sequencer and the advent of
DNA
recombinant technology. However, blocked N-termini and changing trends in molecular cloning techniques, such as
PCR
, brought back “internal” sequencing as well, a term coined by Aebersold et al. in their seminal paper on the
in situ
micropreparative digestion of electroblotted proteins (
1
). Since then, many practical improvements of the original digest recipe and alternative approaches have been proposed (
2
). Considerable effort has also been expended to interface in situ digestion with micro-liquid chromotography (LC), peptide sequencers, and mass spectrometers. The ability to generate a set of specific peptides (e.g., tryptic), together with recent advances in biopolymer mass spectrometry provide a novel approach to protein identification. Accurate masses of several protein fragments compose a “peptide mass fingerprint,” theoretically sufficient for unambiguous searching of sequence repositories (
3
). It is therefore expected that enzymatic proteolysis will become even more widely used in the future.