NEW! Hydrolysis of Flexible Linkers using GlySERIAS

June 14, 2022 | New Product, News |

GlySERIAS is a unique enzyme that digests flexible glycine-rich fusion protein linkers such as Gly4Ser and GlyxSery (GS) and polyglycine (G) linkers.
 
The enzyme enables separation of the individual domains of multi-functional fusion proteins to facilitate characterization and increase the understanding of these molecules. Middle-level analysis of fusion proteins serves to both reduce the overall sample complexity and allow for domain-specific identification and monitoring of post-translational modifications.
 

  • Unique enzyme digesting fusion proteins with flexible linkers
  • Improves resolution and reduces complexity of fusion protein analysis
  • A middle-level approach for characterization of fusion protein components

The fusion protein dulaglutide consists of two glucagon-like peptide-1 (GLP-1) molecules linked to an Fc region of human IgG4 via flexible GS linkers. To study the peptides and Fc region separately and thereby be able to identify specific PTMs, dulaglutide was digested with GlySERIAS at 37°C for 1 hour. To reduce the sample complexity, the Fc glycans were removed using the endoglycosidase GlycINATOR and the interchain disulfide bridges were reduced with DTT (Fig. 1a). 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 cleavage 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 attached (Fig. 1b, c). GLP-1 was present as two variants, with four and five glycine residues respectively. In addition, an oxidation modification on the GLP-1 peptide was identified. The Fc/2 subunit was present as three main variants: Fc/2 with one remaining serine residue, Fc/2 with a SG3 linker tail and Fc/2 with a SG4S linker tail. Triplicate digests showed repeatable results in the relative amount of the different Fc/2 variants obtained (Fig. 1d), despite GlySERIAS digesting at several sites simultaneously (Fig. 1e).

 

 
Digestion of dulaglutide using GlySERIAS. The flexible GS linker of dulaglutide was digested with GlySERIAS for 1 hour at 37°C under native conditions, and the Fc glycans were concurrently hydrolyzed using GlycINATOR 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 digest was performed in triplicate. a) Illustration of the sample preparation workflow. The samples were analyzed by reversed-phase LC-MS. b) Deconvoluted mass spectrum of the Fc/2 subunit. c) Deconvoluted mass spectrum 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.


 

 

 

 

GlySERIAS – Digestion of up to 2 mg fusion protein.