Selective enrichment and identification of cross-linked peptides to study 3-D structures of protein complexes by mass spectrometry.
Chemical cross-linking of protein complexes combined with mass spectrometry is a powerful approach to obtain 3-D structural information by revealing amino residues that are in close spatial proximity. To increase the efficiency of mass spectrometric analysis, we have demonstrated the selective enrichment of cross-linked peptides from the 350 kDa protein complex RNA polymerase (RNAP) from Bacillus subtilis. Bis(succinimidyl)-3-azidomethyl glutarate was used as a cross-linker along with an azide-reactive cyclooctyne-conjugated resin to capture target peptides. Subsequently released peptides were fractionated by strong cation exchange chromatography and subjected to LC-MS/MS. We mapped 10 different intersubunit and 24 intrasubunit cross-links by xComb database searching supplied with stringent criteria for confirmation of the proposed structure of candidate cross-linked peptides. The cross-links fit into a homology model of RNAP. Cross-links between β lobe 1 and the β' downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the α subunits suggest conformational flexibility. The analytical strategy presented here can be applied to map protein-protein interactions at the amino acid level in biological assemblies of similar complexity. Our approach enables the exploration of alternative peptide fragmentation techniques that may further facilitate cross-link analysis.
Swammerdam Institute for Life Sciences, Mass Spectrometry of Biomacromolecules, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Hansuk
H
Buncherd
Merel A
MA
Nessen
Niels
N
Nouse
Sacha K
SK
Stelder
Winfried
W
Roseboom
Henk L
HL
Dekker
Jos C
JC
Arents
Linde E
LE
Smeenk
Martin J
MJ
Wanner
Jan H
JH
van Maarseveen
Xiao
X
Yang
Peter J
PJ
Lewis
Leo J
LJ
de Koning
Chris G
CG
de Koster
Luitzen
L
de Jong
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D030562:Databases, Protein; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D012321:DNA-Directed RNA Polymerases