Detecting Cross-Linked Peptides by Searching against a Database of Cross-Linked Peptide Pairs

Abstract

Mass spectrometric identification of cross-linked peptides can provide valuable information about the structure of protein complexes. We describe a straightforward database search scheme that identifies and assigns statistical confidence estimates to spectra from cross-linked peptides. The method is well suited to targeted analysis of a single protein complex, without requiring an isotope labeling strategy. Our approach uses a SEQUEST-style search procedure in which the database is comprised of a mixture of single peptides with and without linkers attached and cross-linked products. In contrast to several previous approaches, we generate theoretical spectra that account for all of the expected peaks from a cross-linked product, and we employ an empirical curve-fitting procedure to estimate statistical confidence measures. We show that our fully automated procedure successfully reidentifies spectra from a previous study, and we provide evidence that our statistical confidence estimates are accurate.

αB-crystallin interacts with and prevents stress-activated proteolysis of focal adhesion kinase by calpain in cardiomyocytes

“…The MS/MS data were acquired selecting the top three precursors with 1 s MS/MS acquisition time, selecting precursor ions of charge 42 þ . The raw data were processed using Mascot Distiller 2.4.3 (Matrix Science) and the processed spectra were used for crosslinked peptide identification using Crux search for xlinks 41 and XLinker Identifier 42 softwares. Crosslinked identification was performed using the following parameters: 0.1 Da tolerance for both MS and MS/MS, hydrolysed DSS for lysines and oxidation for methionine as variable modifications, iodoacetamide as fixed modification on cysteines and three missed cleavages, trypsin digestion.…”

Intermolecular condensation products formed during the pyrolysis of peptides

“…Further evidence of N-terminus to Cterminus condensation was provided by the pyrolysis of a standard N-acetylated peptide containing an internal arginine group, which did not form any detectable crosslinked product. Peptide crosslinking during pyrolysis of proteins and peptides has been shown to occur at pyrolysis temperatures between 180 and 300 • C. Even though sequence information of the individual peptides involved in the condensation reaction is preserved, caution must be taken in analyzing sequence data derived from MS/MS analysis of condensed peptides as these result from permutated combinations of the reacting peptides, resulting in the scrambling of sections of the original peptide sequence [14]. The implementation of a multistage tandem MS analysis (e.g., MS 3 ) could circumvent this shortcoming by analyzing shorter fragments of the condensed peptide that would accurately correspond to a sequence of the original peptide and/or protein.…”

Conformational and functional studies of a cytosolic 90 kDa heat shock protein Hsp90 from sugarcane

“…Peptides were fractionated using in an oasis cartridge and eluted with different concentrations of acetonitrile and analyses were performed by LC-MS/MS using a Synapt mass spectrometer (Waters Co.). Crosslinked peptides were identified by using Crux-for-Xlinks [38] and Xlink-Identifier [39] software followed by manual validation. The digestion profile was used to identify cross-linked lysines, indicating that they were within 24 A of each other (Ca distances).…”