Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels

摘要

The completion of numerous genomes and the advancement of mass spec-trometric analysis have enabled proteins of interest from complex biological mixtures to be identified at lower levels and considerably faster than using classical edman chemical sequencing. The approach is to enzymatically or chemically cleave the protein into smaller peptide fragments and analyze the digest / cleavage products using either high mass accuracy matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) (1) or tandem mass spectrometry (MS/MS) utilizing either MALDI or Electrospray ionization (ESI) sources (2). Following the mass spectral acquisition, proteins of interest are identified in-silico by matching the corresponding accurate peptide mass data (peptide mass fingerprinting) and the peptide fragment data (ion matching) to the theoretically digested and fragmented peptides in protein databases (3). For both of these types of mass spectrometric analyses, as well as for digests destined for edman chemical sequencing, it is important to obtain a high efficiency of digestion, with peptides in the 8 to 30 residue lengths preferable. Although several different approaches may be taken to cleave proteins, one of the most common is to digest the protein enzymatically with a relatively specific protease such as trypsin or lysyl endopeptidase. Since final purification is often dependent on SDS-PAGE, cleavage procedures that can either be carried out in the poly-acrylamide gel matrix (4, 5) or that may be used on samples that have been blotted from SDS polyacrylamide gels onto PVDF (6) or nitrocellulose (6, 7) membranes are extremely useful. The proteins are usually stained with Coomas-sie blue, or digestion compatible silver stains (used with polyacrylamide gels) prior to excision and proteolytic digestion. The resulting peptides are separated by reverse-phase HPLC or UPLC (ultra high pressure or ultra performance liquid chromatography), and in most instances, the LC system is directly attached to a mass spectrometer for real time protein identification analysis. Relatively straightforward solution and in-gel digestion procedures, including robotic digestion, that have been used extensively in the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University will be described in this chapter, whereas a procedure suitable for in situ digestion of SDS-PAGE blotted proteins is described in Chapter 101.