Proteomic identification of anti-tetanus toxoid antibodies from human serum (HD1)
Proteomic identification and characterization of antibodies comprising the serological response to antigen can provide unique insight into the functional dynamics of adaptive immunity. We have developed a novel method to overcome the technical challenges which previously limited the direct analysis of immunoglobulin proteins in serum, as demonstrated by the identification of human anti-tetanus toxoid (TT) immunoglobulin G (IgG) proteins following booster vaccination. We analyzed the serum IgG repertoire across four time-points corresponding to pre-vaccination, 7 days, 3 months, and 9 months post vaccination. Antigen-specific antibodies were affinity purified against immobilized TT protein and sequenced by bottom-up nanoLC-MS/MS. Interpretation of mass spectra required a custom reference database of IgG heavy and light chain variable sequences determined by NextGen RNA sequencing of the donor's circulating plasmablasts and memory B cells following booster vaccination.
Sample Processing Protocol
IgG was purified from serum with protein G, and F(ab')2 fragments were generated by digestion with pepsin. Anti-tetanus toxoid F(ab')2 fragments were isolated by affinity chromatography, denatured, reduced, alkylated, and digested with trypsin for analysis by nanoLC-MSMS on an Orbitrap Velos Pro. Data-dependent acquisition was utilized, with up to 20 MS2 scans collected per MS1 (100K resolution). Precursor ions of charge >+1 were fragmented by CID. Monoisotopic precursor selection, charge state screening, and 45-s dynamic exclusion were 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 human donor plasmablasts and memory B cells. The database was supplemented with IgG constant region sequences (IGMT) and human protein-coding sequences from Ensembl (release 64), as well as common contaminants (MaxQuant). Searches were 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 permitted. Oxidation of methionine as dynamic modification and cabamidomethyl modification of cysteine as static modification were considered. High confidence PSMs (<1% FDR) were determined by Percolator.
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(s) : 24684310
Lavinder JJ, Wine Y, Giesecke C, Ippolito GC, Horton AP, Lungu OI, Hoi KH, DeKosky BJ, Murrin EM, Wirth MM, Ellington AD, Dörner T, Marcotte EM, Boutz DR, Georgiou G. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):2259-64 PubMed(s) : 24469811