Proteomic identification of monoclonal antibodies from rabbit serum and analysis of FDR using differential cysteine labeling
Proteomic analysis of serum antibodies is challenging due to the non-germline origin of immunoglobulin (Ig) sequences and the complexity of Ig primary structure, with regions of hypervariable sequence interspersed within highly conserved framework sections resulting in an unusually high frequency of peptide-spectrum mis-identification using standard reference database search methods. Using a strategy of differential cysteine labeling with two alkylating reagents, we examined peptide-spectrum matches and decoy-based error modeling for the interpretation of antibody-derived mass spectral data.
Sample Processing Protocol
Immunoglobulin G was purified by protein A affinity chromatography from serum of a rabbit immunized with CCH protein. F(ab')2 fragments were generated by digestion with pepsin and antigen-specific F(ab')2 fragments were isolated by affinity chromatography using immobilized CCH protein on agarose beads. Proteins were denatured, reduced, and alkylated with either iodoacetamide (IAM) or iodoethanol (IodoEtOH), followed by digestion with trypsin. Tryptic peptides were analyzed by nanoLC-MSMS on an Orbitrap Velos. with data-dependent acquisition of 20 MS2 scans per MS1. Ions with charge >+1 were selected for CID, with monoisotopic precursor selection, charge state screening, and 45-s dynamic exclusion enabled.
Data Processing Protocol
Spectra were searched against a custom target protein sequence database consisting of IgG heavy chain and light chain variable genes determined by NextGen RNA sequencing (454) of donor rabbit PBMCs and BMPCs, rabbit protein-coding sequences from Ensembl (OryCun2.0), and common contaminants (MaxQuant). Decoy databases include shuffled and "ConJ" (heavy chain J-region sequence and Arg/Lys positions preserved, remainder of sequence was shuffled between cleavage sites). Searches performed by SEQUEST (Proteome Discoverer 1.4), with mass tolerances of 5 ppm (MS1) and 0.5 Da (MS2). Fully tryptic peptides (600-6000 Da) with up to 2 missed cleavage sites were allowed. Oxidation of methionine as dynamic modification and either cabamidomethyl (IAM) or ethanolyl (IodoEtOH) modification of cysteine as static modification were considered.
Daniel Boutz, University of Texas at Austin
Edward Marcotte, Center for Systems and Synthetic Biology Institute for Cellular and Molecular Biology Department of Molecular Biosciences University of Texas at Austin ( lab head )
Boutz DR, Horton AP, Wine Y, Lavinder JJ, Georgiou G, Marcotte EM. Proteomic Identification of Monoclonal Antibodies from Serum. Anal Chem. 2014 Mar 31 PubMed: 24684310