Immunoglobulin V-gene usage among human serum IgA with or without specificity to the autoantigen transglutaminase 2
To study V-gene usage among antigen-specific and non-antigen-specific serum IgA antibodies in celiac disease patients, purified antibody fractions were subjected to proteomic analysis. Patients either had eastablished, untreated disease with severe intestinal inflammation or "potential" celiac disease characterized by presence of antibodies but no or mild inflammation. Quantification of the fraction of antibodies using different IGHV segments revealed that antibodies specific to the autoantigen transglutaminase 2 (TG2) displayed a bias toward usage of IGHV5-51 in agreement with previous observations. The fraction of antibodies using IGHV5-51 correlated with the degree of intestinal inflammation, indicating that production of hallmark IGHV5-51 anti-TG2 antibodies coincides with onset of clinical celiac disease.
Sample Processing Protocol
Total IgA was purified from 1 ml of serum obtained from 12 celiac patients using Peptide M-agarose. The TG2-specific fraction was obtained by incubation with biotin-TG2 coupled to streptavidin-agarose. The flow-through was saved as the non-TG2-specific fraction, whereas the TG2-specific fraction was eluted from the beads with 20 mM HCl after extensive washing. Both the TG2-specific fraction and the TG2 flow-through fraction were reduced with DTT and alkylated with iodoacetamide before they were digested with trypsin overnight. Peptides were purified on C18 and analyzed using an Easy nLC1000 nano-LC system connected to a quadropole Orbitrap (Q Exactive) mass spectrometer equipped with a nanoelectrospray ion source.
Data Processing Protocol
LC-MS/MS data were analyzed with MaxQuant software (version 184.108.40.206) by searching against a database containing germline amino acid sequences of all human immunoglobulin genes. The database was generated from sequences obtained from the International ImMunoGeneTics Information System (IMGT).
Iversen R, Roy B, Stamnaes J, Høydahl LS, Hnida K, Neumann RS, Korponay-Szabó IR, Lundin KEA, Sollid LM. Efficient T cell-B cell collaboration guides autoantibody epitope bias and onset of celiac disease. Proc Natl Acad Sci U S A. 2019 PubMed: 31285344