Computational protocol: Enhanced B-Cell Receptor Recognition of the Autoantigen Transglutaminase 2 by Efficient Catalytic Self-Multimerization

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Protocol publication

[…] TG2 modified with 5BP or biotin-QLPR (GL Biochem) or self-crosslinked TG2 fractionated by size exclusion chromatography was reduced with dithiothretiol and alkylated with iodoacetamide followed by overnight digestion with sequencing grade trypsin (ProMega). Tryptic digests were purified on homemade C18 microcolumns prior to separation by nano-LC connected to a quadrupole-Orbitrap (QExactive) mass spectrometer (ThermoElectron). Samples were separated on 25 cm C18 columns using a linear gradient from 5–50% methanol and 0.1% formic acid from 0–120 min. Mass spectra were acquired using Xcalibur 2.2 software performing single MS full-scan (300–1750 m/z, 70,000 resolution at m/z 200) followed by 10 data-dependent MS/MS scans. For identification of tryptic peptides modified with 5BP or biotin-QLPR, database search was performed by MaxQuant (1.5.1.2) using a fasta-file of the TG2 sequence as in-house database and 5BP or biotin-QLPR as variable glutamine and lysine modifications respectively. To compare the abundance of 5BP-modified glutamine residues or biotin-QLPR-modified lysine residues, the intensities of identified peptides for each modified residue were summed and divided by the total intensity of all modified residues. For identification of cross-linked peptide adducts, in-house Mascot database search was performed using Mascot Distiller Daemon and a fasta-file of the TG2 sequence as database, allowing for 6 miscleavages of trypsin. Unmatched spectra were exported as mascot generic files (.mgf). These files were uploaded for database search using Stavrox (3.4.11) to identify cross-linked peptide adducts where glutamine to lysine isopeptide cross-linking was added as cross-linker. From each fraction, peptide adducts that obtained a score above an FDR of 0.01 were grouped and adducts that were identified more than once (i.e. with more than one MSMS spectrum) are presented in . This table displays results from a representative analysis of one sample set and is representative of several independent experiments (reanalysis of the same samples, analysis of different digests of the same protein fractions and digestion of different cross-linked fractions) that gave comparable results with regards to targeted glutamine and lysine residues albeit with some variation in scoring and frequency of identified peptide adducts. Notably, peptides harboring cross-links within the same peptide, or peptides harboring more than one cross-link would not be identified by this method. Extracted ion chromatograms were generated in the Xcalibur Qual Browser. […]

Pipeline specifications

Software tools MaxQuant, Mascot Distiller, StavroX
Application MS-based untargeted proteomics
Diseases Celiac Disease