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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.

ABSTRACT: 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.

SUBMITTER: Zelter A

PROVIDER: S-EPMC2917471 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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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.

Zelter Alex A Hoopmann Michael R MR Vernon Robert R Baker David D MacCoss Michael J MJ Davis Trisha N TN

Journal of proteome research 20100701 7

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 m ...[more]

PMID: 20476776

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Project description:Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.

Dataset Information

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.

Publications

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.

Similar Datasets

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