Interactome and interface protocol (2IP): a novel strategy for high sensitivity topology mapping of protein complexes.
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.
Weerasekera
Rasanjala
R
Centre for Research in Neurodegenerative Diseases, University of Toronto, Ontario, Canada.
She
Yi-Min
YM
Markham
Kelly A
KA
Bai
Yu
Y
Opalka
Natacha
N
Orlicky
Stephen
S
Sicheri
Frank
F
Kislinger
Thomas
T
Schmitt-Ulms
Gerold
G
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D044783:F-Box Proteins; D007526:Isoelectric Point; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D044783:F-Box Proteins; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D014357:Trypsin; D003488:Cyanogen Bromide