Isotope signatures allow identification of chemically cross-linked peptides by mass spectrometry: a novel method to determine interresidue distances in protein structures through cross-linking.
Knowledge of protein structures and protein-protein interactions is essential for understanding of biological processes. Recent advances in protein cross-linking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we report a novel method to rapidly and accurately identify cross-linked peptides based on their unique isotope signature when digested in the presence of H(2)(18)O. This method overcomes the need for specially synthesized cross-linkers and/or multiple MS runs required by other techniques. We validated our method by performing a "blind" analysis of 5 proteins/complexes of known structure. Side chain repacking calculations using Rosetta show that 17 of our 20 positively identified cross-links fit the published atomic structures. The remaining 3 cross-links are likely due to protein aggregation. The accuracy and rapid throughput of our workflow will advance the use of protein cross-linking in structural biology.
Zelter
Alex
A
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Hoopmann
Michael R
MR
Vernon
Robert
R
Baker
David
D
MacCoss
Michael J
MJ
Davis
Trisha N
TN
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D003432:Cross-Linking Reagents; D007782:Lactoglobulins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D000596:Amino Acids; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007782:Lactoglobulins; D010103:Oxygen Isotopes; D010446:Peptide Fragments; D011506:Proteins; C496337:Bard1 protein, rat; D044767:Ubiquitin-Protein Ligases; D012259:Ribonuclease, Pancreatic; D009113:Muramidase