Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.

Peptide-N 4-(N-acetyl-β-glucosaminyl) asparagine amidases (PNGases, N-glycanases, EC 3.5.1.52) are indispensable tools in releasing N-glycans from glycoproteins. So far, only a limited number of PNGase candidates are available for the structural analysis of glycoproteins and their glycan moieties. Herein, a panel of 13 novel PNGase H + candidates (the suffix H + refers to the acidic pH optimum of these acidobacterial PNGases) was tested in their recombinant form for their deglycosylation performance. One candidate (originating from the bacterial species Dyella japonica) showed superior properties both in solution-phase and immobilized on amino-, epoxyand nitrilotriacetate resins when compared to currently acidic available PNGases. The high expression yield compared to a previously described PNGase H + , broad substrate specificity, and good storage stability of this novel N-glycanase makes it a valuable tool for the analysis of protein glycosylation.

The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here we present the characterization of an endo-xanthanase from Paenibacillus sp. 62047. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone, capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. 3-D structure determination reveals a complex multi-modular enzyme in which a catalytic (α/α)6 barrel is flanked by an N-terminal "immunoglobulin-like" (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains which have very few homologs in sequence databases and, at least, one of which functions as a new xanthan-binding domain, now termed CBM84. The solution phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g. removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing posttranslational 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.

1Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method 2 to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional 3 HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We 4 have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated 5 protein regions. Here we have investigated the use of the acidic glycosidase PNGase H + , which has a pH optimum 6 at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our 7 results show that PNGase H + retains high deglycosylation activity at HDX quench conditions. When used in an 8 HDX-MS workflow, PNGase H + allowed the extraction of HDX data from all five glycosylated regions of the serpin 9 α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H + are capable of similar deglycosylation 10 performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody Trastuzumab (TZ). However, 11PNGase H + provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while 12