Site-specific Digestion of Human IgA2m1

Site-specific digestion of human IgA2m1, generating homogeneous Fab and Fc fragments for high-resolution middle-level LC-MS characterization.

IgASAP Sub1+2 digests human IgA1 and IgA2 of allotype A2m1 at one specific site above the hinge. The above-hinge digestion results in the generation of Fab and Fc fragments, which allows for middle-level analysis.

The reaction is carried out under physiological conditions, and the specificity of IgASAP Sub1+2 prevents overdigestion of the substrates. To demonstrate the site-specificity of IgASAP Sub1+2, a commercially available recombinant human IgA2m1 was digested with IgASAP Sub1+2 Lyophilized overnight at 37°C, and compared to the undigested antibody.

Highly specific digestion of human IgA2A1 without the risk of overdigestion

To facilitate data interpretation, the samples were treated with PNGaseF Lyophilized under denaturing conditions for removal of all Fab and Fc N-glycans. The denatured samples were analyzed with LC-MS, and the acquired masses of the homogenous scFc and Fd fragments generated from IgASAP Sub1+2 hydrolysis confirm the single digestion site on human IgA2m1 at VTVPCP/VPPPPP (aa 102/103 of constant heavy chain acc. to Uniprot A0A0G2JMB2). The high-resolution mass data demonstrates the value of using middle-level analysis for characterization of the IgA2m1 antibody.

Middle-level analysis improves analysis of human IgA2m1

Site-specific digestion of IgA2m1. a) Schematic illustration of the workflow. Deconvoluted mass spectra of N-deglycosylated b) heavy chain (HC) of a commercially available recombinant IgA2m1 and the corresponding c) scFc fragment and d) Fd fragment after digestion using IgASAP Sub1+2 in an overnight reaction at 37°C. The samples were separated by reversed-phase chromatography (ACQUITY Premier Protein BEH C4, 300 Å, 1.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II).

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