Interactome and interface protocol (2IP): A novel strategy for high sensitivity topology mapping of protein complexes

• Published in: Proteomics (Weinheim) 2007-11, Vol.7 (21), p.3835-3852
• Main Authors: Weerasekera, Rasanjala, She, Yi-Min, Markham, Kelly A, Bai, Yu, Opalka, Natacha, Orlicky, Stephen, Sicheri, Frank, Kislinger, Thomas, Schmitt-Ulms, Gerold
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cross-Linking Reagents; Cyanogen Bromide; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; Electrophoresis, Gel, Two-Dimensional; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; F-Box Proteins; Fundamental and applied biological sciences. Psychology; Isoelectric Point; Mass spectra; Miscellaneous; Models, Molecular; Molecular Sequence Data; Molecular Weight; Multiprotein Complexes - chemistry; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Mapping; Protein Interaction Mapping; Protein structure characterization; Proteins; Proteomics - methods; Quadrupole-time of flight; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Sequence Homology, Amino Acid; SKP Cullin F-Box Protein Ligases - chemistry; SKP Cullin F-Box Protein Ligases - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Two-dimensional gel electrophoresis; Ubiquitin-Protein Ligase Complexes - chemistry; Ubiquitin-Protein Ligase Complexes - genetics; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200700688

A few well-characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low-resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2-D electrophoresis. The side-by-side comparison of a thus generated and a 2-D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In-gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1-Cdc53/Cullin-F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software.