Mass spectrometry for the study of protein-protein interactions.
The identification of subpicomolar amounts of protein by mass spectrometry (MS) coupled with two-dimensional methods to separate complex protein mixtures is fueling the field of proteomics, and making feasible the notion of cataloging and comparing all of the expressed proteins in a biological sample. Functional proteomics is a complementary effort aimed at the characterization of functional features of proteins, such as their interactions with other proteins. Proteins comprise modular domains, many of which are noncatalytic modules that direct protein-protein interactions. Capturing proteins of interest and their interacting proteins by using high-affinity antibodies presents a simple method to prepare relatively simple protein mixtures easily resolved in one-dimensional formats. Individual or mixtures of proteins identified as stained bands in polyacrylamide gels are subjected to in situ digestion with the protease trypsin, and the extracted peptide fragments are analyzed by MS. The quality, quantity, and complexity of the tryptic digest, the species origin of the proteins, and the quality of the corresponding databases of genomic and protein information greatly influence the subsequent MS analysis in terms of degree of difficulty and methodological approach required to make an unambiguous protein identification. In this article we report the isolation of associated proteins from a complex cell-derived lysate by using an epitope-directed antibody. The protein pICLn engineered to carry an epitope tag was purified from cultured human embryonic kidney cells, and found to associate with a variety of proteins including the spliceosomal proteins smE and smG. By application of this general approach, the systematic identification of protein complexes and assignment of protein function are possible.
Figeys
D
D
MDS Proteomics Inc., 480 University Avenue, Toronto, Ontario, M5G 1V2, Canada.
McBroom
L D
LD
Moran
M F
MF
D000465:Algorithms; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry*; D011233:Precipitin Tests; D011506:Proteins / Q000737:chemistry*; D014162:Transfection / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin