Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry–based de novo sequencing

Abstract

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavilyO-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry–based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance toO-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies.

A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics

“…27 Stitch has been shown to enable the accurate reconstruction of monoclonal antibody sequences, as well as the sequencing of isolated Fab fragments from patient serum, M-proteins in monoclonal gammopathies, antibody light chains from urine, and the profiling of whole IgG from COVID-19 patient sera. [13][14][15]26 Sequence accuracies of ∼99% can be obtained, which is sufficient to reverse engineer functional antibody products. 21,22,28,29 Remaining sequencing errors stem in large parts from common mass coincidences of isobaric residues like leucine/isoleucine but also from incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions.…”

“…Additionally, B cells reside in the spleen, bone marrow, and blood, of which only the latter population can be easily sampled in human subjects. In contrast, mass spectrometry-based methods can probe specific antibody sequences directly from the secreted polypeptide product, thereby circumventing the need to sample the antibody-producing B-cell clone and providing a direct glimpse into the so-called serum compartment of the immunoglobulin repertoire. − …”

“…We have recently developed the software tool Stitch to perform template-based assembly of peptide sequences against the coding gene segments of antibodies available in immunogenetics databases . Stitch has been shown to enable the accurate reconstruction of monoclonal antibody sequences, as well as the sequencing of isolated Fab fragments from patient serum, M-proteins in monoclonal gammopathies, antibody light chains from urine, and the profiling of whole IgG from COVID-19 patient sera. − , Sequence accuracies of ∼99% can be obtained, which is sufficient to reverse engineer functional antibody products. ,,, Remaining sequencing errors stem in large parts from common mass coincidences of isobaric residues like leucine/isoleucine but also from incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions. Likewise, different combinations of amino acids, of potentially different lengths, can also coincide to the same mass (e.g., GGN, GAQ).…”

Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry – a perspective

“…First, using a bottom-up proteomics approach, antibody-derived peptides can be sequenced de novo from fragmentation spectra and assembled into full heavy/light chain sequences 12,16 . Sequence accuracy is such that functional monoclonal antibodies can be reconstructed from the input data, and several reports have described successful sequencing efforts of human serum, milk, and urine-derived antibodies [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] . Second, both hydrogen-deuterium exchange mass spectrometry and electron microscopy have been used to resolve a complex landscape of epitopes targeted by polyclonal antibody mixtures [33][34][35][36][37][38][39][40][41][42] .…”