Fig. 2: Sample assessment and the first steps of MS-based de novo sequencing. | Nature Communications

Fig. 2: Sample assessment and the first steps of MS-based de novo sequencing.

From: De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

Fig. 2

A Plasma titration for antibodies against the receptor-binding domain, RBD of Sars-Cov2. Three plasma samples (Subj. 1,2 and 3) were benchmarked against an internal in-house standard (C3) and a negative control. B Construct of the MS-based de novo assembly of “contig” around different CDRs (CDR1 left, CDR2 middle, and CDR3 right). The green regions are the framework region (FR) while the orange ones are the CDRs for the antigen-enriched polyclonal antibody. The proposed sequence is shown below the highlighted regions, and the bottom parts are short peptides corresponding to elements of the sequences. Green lines are peptides generated from the digestion with Chymotrypsin; light orange is LysC, blue is Trypsin, black is Pepsin, and dark orange is AspN. C Typical “middle-down” peptide allowing in this particular case, the assembly of a CDR2 with a CDR3 within the same chain. D Resulting peptides from the action of a given protease on a specific germline (IGHV3-53*01) under non-reduced conditions. The black arrows represent possible pepsin sites, the blue ones are peptides resulting from the digestion with trypsin, and the orange ones are peptides from the protease AspN. E, F Some disulfide-linked peptide analysis spectra. Both the experimental mass (Exp.) and the theoretical mass (Calc.) are indicated in both spectra. Those two peptides allow pairing each a specific CDR1 sequence with a specific CDR3 sequence. Source data are provided as a Source Data file.

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