BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics.
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 reagents. 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 beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
Evgeniy V
EV
Petrotchenko
Kunhong
K
Xiao
Jennifer
J
Cable
Yiwen
Y
Chen
Nikolay V
NV
Dokholyan
Christoph H
CH
Borchers
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D005453:Fluorescence; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology*; D007554:Isotopes; D008027:Light; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D017483:Receptors, Vasopressin / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000071557:beta-Arrestins
D019390:Arrestins; D003432:Cross-Linking Reagents; D006877:Hydroxamic Acids; D007554:Isotopes; C400001:N-biphenylsulfonylphenylalanine hydroxamic acid; D010748:Phosphopeptides; D017483:Receptors, Vasopressin; D000071557:beta-Arrestins; D012260:Ribonucleases; C022542:ribonuclease S