Reproducible Digestion of a GS-linked Fusion Protein

Robust and reproducible digestion of dulaglutide linkers, separating GLP-1 peptides from the Fc for domain-specific LC-MS analysis of protein modifications

The fusion protein dulaglutide consists of two glucagon-like peptide-1 (GLP-1) molecules linked to the Fc region of human IgG4 via flexible GS linkers.

To study the peptides and Fc region separately, and thereby identify domain-specific PTMs, dulaglutide was digested with GlySERIAS Lyophilized at 37°C for 1 hour. To reduce the sample complexity, the Fc glycans were removed using the endoglycosidase GlycINATOR Lyophilized and the interchain disulfide bridges were reduced with DTT.

Middle-level analysis enables domain-specific localization of modifications

Analysis of the sample by reversed-phase LC-MS showed that the peptides were completely removed from the Fc region upon linker digestion using GlySERIAS. The multitude of glycine residues in the linker offers many different potential digestion sites for GlySERIAS, which is why both the Fc/2 and the GLP-1 peptide were detected as several variants with different numbers of glycine and serine residues still attached.

Efficient digestion of flexible linkers using GlySERIAS

In addition, an oxidation of the GLP-1 peptide was identified. Triplicate digests showed reproducible results in the relative amount of the different Fc/2 variants obtained, despite GlySERIAS digesting at several sites simultaneously.

Reproducible hydrolysis improves confidence in data

Replicate digestion of dulaglutide using GlySERIAS Lyophilized. The flexible GS linkers of dulaglutide were digested with GlySERIAS Lyophilized for 1 hour at 37°C under non-denaturing conditions, and the Fc glycans were concurrently hydrolyzed using GlycINATOR Lyophilized to reduce sample complexity. To stop the GlySERIAS reaction, 1 mM ZnCl2 was added. The interchain disulfide bonds were reduced with 20 mM DTT for 30 minutes at 37°C. The digestion was performed in triplicate. a) Illustration of the sample preparation workflow. The samples were analyzed by reversed-phase LC-MS. b) Deconvoluted mass spectra of the Fc/2 subunit. c) Deconvoluted mass spectra of the GLP-1 peptide. d) Relative amount of the identified Fc/2 variants, displaying the mean value between the triplicate digests and error bars representing the standard deviation. The digestion products were separated by reversed-phase chromatography (BioResolve™ RP mAb Polyphenyl, 450 Å, 2.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II). e) Schematic image of the flexible linker, connecting the GLP-1 peptide to the Fc/2 subunit, and the identified digestion sites.

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