BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics

Evgeniy V. Petrotchenko, Kunhong Xiao, Jennifer Cable, Yiwen Chen, Nikolay Dokholyan, Christoph H. Borchers

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking re-agents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein β-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.

Original languageEnglish (US)
Pages (from-to)273-286
Number of pages14
JournalMolecular and Cellular Proteomics
Volume8
Issue number2
DOIs
StatePublished - Feb 1 2009

Fingerprint

Proteomics
Peptides
Proteins
Mass spectrometry
Gels
Cytology
Arrestin
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
G-Protein-Coupled Receptors
Tandem Mass Spectrometry
Electrophoresis
Isotopes
Amines
Cell Biology
Digestion
Mass Spectrometry
Esters
Software
Fluorescence
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Petrotchenko, Evgeniy V. ; Xiao, Kunhong ; Cable, Jennifer ; Chen, Yiwen ; Dokholyan, Nikolay ; Borchers, Christoph H. / BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics. In: Molecular and Cellular Proteomics. 2009 ; Vol. 8, No. 2. pp. 273-286.
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BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics. / Petrotchenko, Evgeniy V.; Xiao, Kunhong; Cable, Jennifer; Chen, Yiwen; Dokholyan, Nikolay; Borchers, Christoph H.

In: Molecular and Cellular Proteomics, Vol. 8, No. 2, 01.02.2009, p. 273-286.

Research output: Contribution to journalArticle

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AU - Petrotchenko, Evgeniy V.

AU - Xiao, Kunhong

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AB - Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking re-agents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein β-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.

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