Selectivity over coverage in de novo sequencing of IgGs

Abstract

Generating protein sequence ladders of the CDR3 variable regions of antibodies facilitates de novo sequencing by mass spectrometry.

Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation

“…An advantage of fragmenting proteins below 50 kDa in mass is that both the concomitantly formed low m / z c -ions and complementary high m / z z -ions can be isotopically resolved using high-resolution mass spectrometry (here 200000 at m / z 400). This allows us to further corroborate the observation—first evidenced by measurements on IgG 8 —that ECD without additional vibrational or electronic excitation does not lead to substantial cleavage of disulfide bridges in native immunoglobulins ( Figure 6 ).…”

“… 3 Consequently, IgA1 disulfide bonds constrain fragmentation to the region between the disulfide-stabilized Ig folds, which is smaller than its IgG1 counterpart and more comparable to the pattern observed for the IgG2, IgG3, and IgG4 subclasses. 8 …”

“…These disadvantages make of electron capture dissociation (ECD) an alternative choice. 5 − 8 Applied to denatured and native intact antibodies, 9 , 10 ECD results in significant backbone cleavages in both the LC and HC variable regions, primarily leading to the formation of ( c / z· ) fragment ions. Furthermore, since intramolecular disulfide bridges of native proteins are not frequently cleaved during ECD, 11 enhanced fragment formation is observed for sequence segments not involved in disulfide-bridged loops, e.g., for the segments covering the LC and HC CDR3s.…”

“…We further optimize ECD toward the de novo sequencing of IgA1 CDR3s and their downstream regions, generating clean fragment ion series composed solely of c -ions. 8 Using native mass spectrometry conditions, we achieve maximal separation between the precursor in the high m / z range and the informative fragment ions in the lower m / z range. We also show that, although IgA1 Fab isolation reduces the complexity of the spectrum and simplifies the precursor ion selection, simplified precursors are not required to obtain straightforward sequence reads from IgA1 CDR3 regions.…”

Human plasma IgG1 repertoires are simple, unique, and dynamic

“…The ETD mass spectra of the intact Fab yielded accurate masses of the light chain and Fd by cleavage of the interchain disulfide bond, thus providing direct information about the light-chain-heavy-chain pairing (Figure 3C, top spectra). In addition, these ETD spectra yielded extended sequence tags, covering informative parts of the CDR3 and framework (FR) 4 regions of both Fab chains, in a similar manner as previously reported by performing ETD or ECD of intact IgG molecules (den Boer et al, 2020;Fornelli et al, 2017;Shaw et al, 2020). Complementary, ETD spectra of the separated Fab chains yielded partial sequence information for the FR1, CDR1, FR2, CDR2, and the constant region of the selected clone (Figure 3C, bottom spectra and Figure S6).…”