Mass spectrometry (MS) and immunoassay identification of CLL mAb-binding antigens. (A) Purification and identification of I83/IGHV3-30_M IgM-specific proteins by MS. FM55_M2 melanoma cell extract was affinity purified by I83 IgM–protein G beads. The photographs show parallel lanes from SDS-PAGE (left panel), one transferred for Western blot and one lane was Coomassie stained. A single 54-kDa protein was found in Western blot. The Coomassie-stained gel show 3 major proteins, 54 kDa, 39 kDa, and 17 kDa. These were excised and digested with trypsin and analyzed by ESI-MS/MS. The doubly charged molecular ion with m/z = 786.4, obtained from the 54-kDa antigen, was selected and subjected to ESI-MS/MS. Peptide sequencing by ESI-MS/MS identified the I83 IgM-specific antigen (54 kDa) as vimentin and the protein of 39 kDa as aldolase A. The detected b (N-terminal) and y (C-terminal) fragment ions are shown in the spectrum, and the complete peptide sequences are shown above each spectrum with the numbers corresponding to the b and y ions. (B) Purification and identification HG3/IGHV1-2_UM IgM specific antigen. (C) Vimentin-ELISA performed on I83/IGHV3-30_M IgM and Abs from 48-hour ex vivo CLL cultures. (D) Binding of Wa/IGHV3-30.3_UM/subset-32 CLL IgM to S pneumoniae polysaccharides in luminex assay. Multiplex beads were coated with 1 of 7 pneumococcal capsular polysaccharides. The median fluorescent intensity (MFI) of IgM binding to each of 7 serotype-coated multiplex beads is shown against 4 doubling dilutions.