Detection of crosslinks within and between proteins by LC-MALDI-TOFTOF and the software FINDX to reduce the MSMS-data to acquire for validation.
Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Institute for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
Christopher A G
CA
Söderberg
Wietske
W
Lambert
Sven
S
Kjellström
Alena
A
Wiegandt
Ragna Peterson
RP
Wulff
Cecilia
C
Månsson
Gudrun
G
Rutsdottir
Cecilia
C
Emanuelsson
D007553:Isotope Labeling; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D046912:Multiprotein Complexes; D010455:Peptides; D021122:Protein Subunits; D011506:Proteins