| Autor: |
Comamala G; Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark., Krogh CC; Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark., Nielsen VS; Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark., Kutter JP; Microscale Analytical Systems Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark., Voglmeir J; Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China., Rand KD; Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. |
| Abstrakt: |
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing post-translational modifications (PTMs), such as disulfide bonds and glycosylations, present challenges to HDX-MS, as disulfide bond reduction and deglycosylation is often required to extract HDX information from regions containing these PTMs. In-solution deglycosylation with peptide-N4-( N -acetyl-β-d-glucosaminyl)-asparagine amidase A (PNGase A) or PNGase H + combined with chemical reduction using tris-(2-carboxyethyl)phosphine (TCEP) has previously been used for HDX-MS analysis of disulfide-linked glycoproteins. However, this workflow requires extensive manual sample preparation and consumes large amounts of enzyme. Furthermore, large amounts of TCEP and glycosidases often result in suboptimal liquid chromatography-mass spectrometry (LC-MS) performance. Here, we compare the in-solution activity of PNGase A, PNGase H + , and the newly discovered PNGase Dj under quench conditions and immobilize them onto thiol-ene microfluidic chips to create HDX-MS-compatible immobilized microfluidic enzyme reactors (IMERs). The IMERS retain deglycosylation activity, also following repeated use and long-term storage. Furthermore, we combine a PNGase Dj IMER, a pepsin IMER, and an electrochemical cell to develop an HDX-MS setup capable of efficient online disulfide-bond reduction, deglycosylation, and proteolysis. We demonstrate the applicability of this setup by mapping the epitope of a monoclonal antibody (mAb) on the heavily disulfide-bonded and glycosylated sema-domain of the tyrosine-protein kinase Met (SD c-Met). We achieve near-complete sequence coverage and extract HDX data to identify regions of SD c-Met involved in mAb binding. The described methodology thus presents an integrated and online workflow for improved HDX-MS analysis of challenging PTM-rich proteins. |
