Affinity mass spectrometry-based approaches for the analysis of protein-protein interaction and complex mixtures of peptide-ligands.
Combined applications of affinity purification procedures and mass-spectrometric analyses (affinity mass spectrometry or affinity-directed mass spectrometry) have gained broad interest in various fields of biological sciences. We have extended these techniques to the purification and analysis of closely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptides. We also show that affinity mass spectrometry allows the mapping of sequential binding motifs of two interacting proteins. Using the ActA/Mena protein-protein complex as a model system, we show that we can selectively purify Mena-binding peptides from a tryptic digest of ActA. The results from this assay are compared to data sets obtained earlier by classical methods using synthetic peptides and molecular genetic experiments. As a further expansion of affinity mass spectrometry, we have established an internally standardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the monoclonal tubulin-specific antibody YL1/2 are determined in terms of half-maximal competition.
Rüdiger
A H
AH
Department of Cell Biology, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, Braunschweig, D-38124, Germany. AMRudiger@aol.com
Rüdiger
M
M
Carl
U D
UD
Chakraborty
T
T
Roepstorff
P
P
Wehland
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C102176:Enah protein, mouse; D008024:Ligands; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010455:Peptides; C072980:actA protein, Listeria monocytogenes