Addressing Acid-Catalyzed Deamidation and the Solubility of Hydrophobic Peptides in Multi-Attribute Method Workflows.

Recently, we introduced an optimized and automated Multi-Attribute Method (MAM) workflow, which (a) significantly reduces the number of missed cleavages using an automated two-step digestion procedure and (b) dramatically reduces chromatographic peak tailing and carryover of hydrophobic peptides by implementing less retentive reversed-phase column chemistries. Here, further insights are provided on the impact of postdigest acidification and the importance of maintaining hydrophobic peptides in solution using strong chaotropic agents after digestion. We demonstrate how oxidation can significantly increase the solubility of hydrophobic peptides, a fact that can have a profound impact on quantitation of oxidation levels if care is not taken in MAM workflows. We conclude that (a) postdigestion acidification can result in significant acid-catalyzed deamidation during storage in an autosampler at 5 °C and (b) a strong chaotropic agent, such as guanidine hydrochloride, is critical for preventing loss of hydrophobic peptides through adsorption, which can result in (sometimes extreme) biases in quantitation of tryptophan oxidation levels. An optimized method is presented, which effectively addressed acid-catalyzed deamidation and solubility of hydrophobic peptides in MAM workflows.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins.

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-ß-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.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

Discovery of Highly Active Recombinant PNGase H+ Variants Through the Rational Exploration of Unstudied Acidobacterial Genomes.

Peptide-N 4-(N-acetyl-ß-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-, epoxy- and 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.

Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS.

Neurotransmitter:sodium symporters (NSS) are integral membrane proteins (IMP), responsible for reuptake of neurotransmitters from the synaptic cleft. Due to challenges in production of mammalian NSS in their active form, the prokaryotic hydrophobic amino acid transporter, LeuT, served here as a steadfast model for elucidation of structure-function relationship. As NSS proteins reside within phospholipid bilayer, they require stabilization by artificial membrane systems upon their extraction. Right choice of artificial membrane system is crucial as suboptimal detergent and/or lipids can lead to destabilization or non-native stabilization. Here we study the effect of related detergents, dodecyl maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG), on the conformational dynamics of LeuT by global HDX-MS, in the presence of functionally relevant ligands. We observed that LeuT is more dynamic when solubilized in DDM compared to LMNG. Moreover, LeuT exhibited increased HDX in the presence of K+ compared to Na+, indicating a more dynamic conformation in the presence of K+. Upon addition of leucine, LeuT underwent additional stabilization relative to the Na+-bound state. Finally, peak broadening was observed, suggesting that LeuT undergoes slow unfolding/refolding dynamics in detergent solution. These slow dynamics were verified by local HDX, also proving that detergents modulate the rate of these dynamics. SIGNIFICANCE: Overall, we show the efficacy of global HDX-MS to evaluate the effect of artificial membrane systems on integral membrane proteins and the importance of carefully selecting compatible detergent (and/or lipid) for the solubilization of this class of proteins.

Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.

Structure and Dynamics of a Promiscuous Xanthan Lyase from Paenibacillus nanensis and the Design of Variants with Increased Stability and Activity.

We have characterized the structure and dynamics of the carbohydrate-modifying enzyme Paenibacillus nanensis xanthan lyase (PXL) involved in the degradation of xanthan by X-ray crystallography, small-angle X-ray scattering, and hydrogen/deuterium exchange mass spectrometry. Unlike other xanthan lyases, PXL is specific for both unmodified mannose and pyruvylated mannose, which we find is correlated with structural differences in the substrate binding groove. The structure of the full-length enzyme reveals two additional C-terminal modules, one of which belongs to a new non-catalytic carbohydrate binding module family. Ca2+ are critical for the activity and conformation of PXL, and we show that their removal by chelating agents results in localized destabilization/unfolding of particularly the C-terminal modules. We use the structure and the revealed impact of Ca2+ coordination on conformational dynamics to guide the engineering of PXL variants with increased activity and stability in a chelating environment, thus expanding the possibilities for industrial applications of PXL.

Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.

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.