Figure 4.

Integrative de novo sequencing of the Fab clone ^24.4 1 _47,359.4 from donor F59 combining middle-down and bottom-up MS data

(A) Data analysis pipeline displaying the key steps in the de novo sequencing, namely, filtering of the germline database of light- and heavy-chain sequences, assembling of selected allelic variants with mass constraints, scoring of the assembled sequences by using middle-down MS data, iterative refining of the best scoring templates by using peptides in bottom-up MS, and benchmarking of the optimized mature sequences using data from both middle-down and bottom-up MS analysis.

(B) Alignment of the best matching germline IGLV amino acid sequence from the IMGT database (IGLV2-14^∗01) with the mature sequence that was determined for the light chain of the dominant clone (top box), the fragments from middle-down MS (middle box), and the peptides from bottom-up MS.

(C) Alignment of the best matching germline IGHV amino acid sequence from the IMGT database (IGHV3-9^∗01) with the mature sequence that was determined for the Fd of donor F59’s clone ^24.4 1 _47,359.4 (top box), the fragments from middle-down MS (middle box), and the peptides from bottom-up MS. CDR regions in top panels of (B) and (C) were annotated with reference to the closest matching IMGT sequence. Amino acids that were determined to be different in the mature ^24.4 1 _47,359.4 sequence are highlighted in red.