Shotgun cross-linking analysis for studying quaternary and tertiary protein structures.
We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.
Lee
Young Jin
YJ
Proteomics Core Facility, Genome Center, and Molecular and Cellular Biology, University of California, Davis 95616, USA. yojlee@ucdavis.edu
Lackner
Laura L
LL
Nunnari
Jodi M
JM
Phinney
Brett S
BS
D000465:Algorithms; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry; D003627:Data Interpretation, Statistical; D034281:Dynamins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D003432:Cross-Linking Reagents; D010455:Peptides; D045304:Cytochromes c; D034281:Dynamins