igm  (Genovis Inc)

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

doi: 10.1021/jasms.4c00094

Figure Lengend Snippet: Structural details of IgM. (A) The IgM protomer resembles somewhat the IgA protomer, although it is longer. The μ heavy chain is ∼576 amino acids long and includes a variable domain (Vμ ∼ 110 amino acids, represented in light blue), four distinct constant region domains (Cμ1, Cμ2, Cμ3, Cμ4, each ∼110 amino acids, depicted in dark blue), and a “tailpiece” of ∼20 amino acids. Each protomer contains 10 N-glycosylation sites (indicated with orange dots). The μ chains in each monomer are covalently linked with a disulfide bond at Cys337 (black line). Each light chain (variable domain VL = light purple, constant domain CL = dark purple) is disulfide bonded to the μ chain using Cys136 in the μ chain. IgMBRAZOR cuts specifically just below Cys337, between Thr343 and Ala344, in the stretch (...VPDQDT/AIRVFA...) (red dashed line). (B) Initially it was thought that IgM protomers form highly symmetric, starfish-like pentamers with C5 symmetry. The five IgM protomers are stabilized by interprotomer disulfide bridges (black lines). (C) Representative schematic of the textbook structure of J-chain coupled IgM. In this structure the C5 symmetry is reduced to C2 symmetry, as only two of the protomers are bound to the linking J-chain. Moreover, there is a wide gap between the two latter protomers, that can accommodate the AIM/CD5L protein. Whether this molecule still contains true C2 symmetry is debatable, as this requires the J-chain to be both symmetric and evenly localized in the gap.

Article Snippet: IgMBRAZOR was sourced from Genovis (Kävlinge, Sweden).

Techniques: Glycoproteomics

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

doi: 10.1021/jasms.4c00094

Figure Lengend Snippet: SEC-CDMS analysis of end products produced from IgMBRAZOR-digested IgM. (A) Schematic depiction of IgMBRAZOR-induced digestion of IgM. IgMBRAZOR liberates all five intact F(ab’) 2 subunits from the IgM precursor, co-yielding the truncated pentameric Fc core. (B) UV chromatogram from SEC-CDMS of fully processed IgM. The two final products, the Fc core (29.4%) and F(ab’) 2 (70.6%) subunits are baseline resolved, allowing MS parameters to be individually tuned for each analyte. A small peak marked by an asterisk (*) can be observed, which was found to originate from the IgMBRAZOR protease. The retention time for each species is labeled. (C–D) Mass histograms of the pentameric Fc core and F(ab’) 2 subunits, respectively, obtained by SEC-CDMS from a single 0.5 μg injection.

Article Snippet: IgMBRAZOR was sourced from Genovis (Kävlinge, Sweden).

Techniques: Produced, Labeling, Injection

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

doi: 10.1021/jasms.4c00094

Figure Lengend Snippet: Real-time kinetics monitoring of IgM digestion using SEC-CDMS. (A–D) Representative mass histograms from SEC-CDMS depicting the conversion of intact IgM to partially processed intermediate species. Individual species resulting from processive losses of F(ab’) 2 arms are identified by their decrease in mass. The signals for free F(ab’) 2 are omitted for clarity. (E) Plot of individual species abundances as a function of incubation time using 1 U (0.04 U/μg IgM) of IgMBRAZOR at 7 °C. Lower temperatures and enzyme loading was deliberately used in these experiments to decrease the rate of digestion. Each time point represents an independent SEC-CDMS injection. While the first 4 F(ab’) 2 arms are rapidly cleaved, the final fifth F(ab’) 2 arm appears somewhat resistant to cleavage. (F) Proposed mechanism of IgM digestion by IgMBRAZOR. Cleavage of the first four F(ab’) 2 moieties occurs quickly, leading to rapid formation of the [IgM – 4 × F(ab’) 2 ] species. The final fifth F(ab’) 2 is conformationally restricted and less accessible to IgMBRAZOR, relative to the other arms, likely due to proximity to the interacting J-chain. Prolonged incubation will eventually cleave the final F(ab’) 2 , yielding the fully processed Fc core ( Figure S1 ).

Article Snippet: IgMBRAZOR was sourced from Genovis (Kävlinge, Sweden).

Techniques: Incubation, Injection