Intact Glycoprotein Research Articles

Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans. The most comprehensive characterization of intact glycoproteins requires integration of a suitable separation method and high performance tandem mass spectrometry to provide both protein sequence information and glycosite localization. Here, we couple ultraviolet photodissociation and hydrophilic interaction chromatography with high resolution mass spectrometry to advance the characterization of intact glycoproteins ranging from 15 to 34 kDa, offering site localization of glycans, providing sequence coverages up to 93%, and affording relative quantitation of individual glycoforms.

Comprehensive characterization of bacterial glycoconjugate vaccines by liquid chromatography - mass spectrometry

Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines.

Direct Glycan Analysis of Biological Samples and Intact Glycoproteins by Integrating Machine Learning-Driven Surface-Enhanced Raman Scattering and Boronic Acid Arrays.

Frequent monitoring of glycan patterns is a critical step in studying glycan-mediated cellular processes. However, the current glycan analysis tools are resource-intensive and less suitable for routine use in standard laboratories. We developed a novel glycan detection platform by integrating surface-enhanced Raman spectroscopy (SERS), boronic acid (BA) receptors, and machine learning tools. This sensor monitors the molecular fingerprint spectra of BA binding to cis-diol-containing glycans. Different types of BA receptors could yield different stereoselective reactions toward different glycans and exhibit unique vibrational spectra. By integration of the Raman spectra collected from different BA receptors, the structural information can be enriched, eventually improving the accuracy of glycan classification and quantification. Here, we established a SERS-based sensor incorporating multiple different BA receptors. This sensing platform could directly analyze the biological samples, including whole milk and intact glycoproteins (fetuin and asialofetuin), without tedious glycan release and purification steps. The results demonstrate the platform's ability to classify milk oligosaccharides with remarkable classification accuracy, despite the presence of other non-glycan constituents in the background. This sensor could also directly quantify sialylation levels of a fetuin/asialofetuin mixture without glycan release procedures. Moreover, by selecting appropriate BA receptors, the sensor exhibits an excellent performance of differentiating between α2,3 and α2,6 linkages of sialic acids. This low-cost, rapid, and highly accessible sensor will provide the scientific community with an invaluable tool for routine glycan screening in standard laboratories.

“Small is beautiful” – Examining reliable determination of low-abundant therapeutic antibody glycovariants

Glycans associated with biopharmaceutical drugs play crucial roles in drug safety and efficacy, and therefore, their reliable detection and quantification is essential. Our study introduces a multi-level quantification approach for glycosylation analysis in monoclonal antibodies (mAbs), focusing on minor abundant glycovariants. Mass spectrometric data is evaluated mainly employing open-source software tools. Released N-glycan and glycopeptide data form the basis for integrating information across different structural levels up to intact glycoproteins. Comprehensive comparison showed that indeed, variations across structural levels were observed especially for minor abundant species. Utilizing modification finder (MoFi), a tool for annotating mass spectra of intact proteins, we quantify isobaric glycosylation variants at the intact protein level. Our workflow's utility is demonstrated on NISTmAb, rituximab and adalimumab, profiling their minor abundant variants for the first time across diverse structural levels. This study enhances understanding and accessibility in glycosylation analysis, spotlighting minor abundant glycovariants in therapeutic antibodies.

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Simultaneous Adsorption of Intact Glycoproteins and Phosphoproteins Using Hydrophilic Titanium (IV) Ion-Immobilized Cotton Fiber Functionalized by Phytic Acid Assembly

Protein glycosylation and phosphorylation are two important post-translational modifications and play significant roles in various biological processes. However, both glycoproteins and phosphoproteins are in low abundance in complex samples, which hinders their characterization. In this work, we developed a novel method for the simultaneous adsorption of glycoproteins and phosphoproteins based on solid phase extraction. By virtue of the unique structure of hydrophilic titanium (IV) ion immobilized cotton fiber functionalized with phytic acid (denoted as Ti4+-PA-cotton), glycoproteins, and phosphoproteins could be efficiently enriched in one-step incubation and eluted in sequence. The newly prepared Ti4+-PA-cotton was well characterized, and the adsorption isotherms and kinetics were studied. The maximum adsorption capacities of Ti4+-PA-cotton for β-casein and HRP were 833.3 mg/g and 384.6 mg/g with the adsorption rate of 259.1 and 63.1 mg/g·min. Standard protein mixture of BSA, HRP, and β-casein was used to test the enrichment ability as a proof of concept. The SDS-PAGE results demonstrated that Ti4+-PA-cotton had an excellent enrichment performance and possessed an outstanding application potential in the analysis of glycoproteins and phosphoproteins.

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

Software-Assisted Data Processing Workflow for Intact Glycoprotein Mass Spectrometry.

Intact protein analysis by mass spectrometry is important for several applications such as assessing post-translational modifications and biotransformation. In particular, intact protein analysis allows the detection of proteoforms that are commonly missed by other approaches such as proteolytic digestion followed by bottom-up analysis. Two quantification methods are mainly used for intact protein data quantification, namely the extracted ion and deconvolution approaches. However, a consensus with regard to a single best practice for intact protein data processing is lacking. Furthermore, many data processing tools are not fit-for-purpose and, as a result, the analysis of intact proteins is laborious and lacks the throughput required to be implemented for the analysis of clinical cohorts. Therefore, in this study, we investigated the application of a software-assisted data analysis and processing workflow in order to streamline intact protein integration, annotation, and quantification via deconvolution. In addition, the assessment of orthogonal data sets generated via middle-up and bottom-up analysis enabled the cross-validation of cleavage proteoform assignments present in seminal prostate-specific antigen (PSA). Furthermore, deconvolution quantification of PSA from patients' urine revealed results that were comparable with manually performed quantification based on extracted ion electropherograms. Overall, the presented workflow allows fast and efficient processing of intact protein data. The raw data is available on MassIVE using the identifier MSV000086699.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

Molecular determinants of the interaction between HSV-1 glycoprotein D and heparan sulfate.

Literature has well-established the importance of 3-O-sulfation of neuronal cell surface glycan heparan sulfate (HS) to its interaction with herpes simplex virus type 1 glycoprotein D (gD). Previous investigations of gD to its viral receptors HVEM and nectin-1 also highlighted the conformational dynamics of gD's N- and C-termini, necessary for viral membrane fusion. However, little is known on the structural interactions of gD with HS. Here, we present our findings on this interface from both the glycan and the protein perspective. We used C-terminal and N-terminal gD variants to probe the role of their respective regions in gD/HS binding. The N-terminal truncation mutants (with Δ1-22) demonstrate equivalent or stronger binding to heparin than their intact glycoproteins, indicating that the first 22 amino acids are disposable for heparin binding. Characterization of the conformational differences between C-terminal truncated mutants by sedimentation velocity analytical ultracentrifugation distinguished between the "open" and "closed" conformations of the glycoprotein D, highlighting the region's modulation of receptor binding. From the glycan perspective, we investigated gD interacting with heparin, heparan sulfate, and other de-sulfated and chemically defined oligosaccharides using surface plasmon resonance and glycan microarray. The results show a strong preference of gD for 6-O-sulfate, with 2-O-sulfation becoming more important in the presence of 6-O-S. Additionally, 3-O-sulfation shifted the chain length preference of gD from longer chain to mid-chain length, reaffirming the sulfation site's importance to the gD/HS interface. Our results shed new light on the molecular details of one of seven known protein-glycan interactions with 3-O-sulfated heparan sulfate.

Direct MALDI Glycotyping of Glycoproteins toward Practical Subtyping of Biological Samples.

The rapid analysis of glycan patterns (glycoforms) of glycoproteins can accelerate their quality control and biomarker discovery. We have focused on the direct analysis of glycoprotein glycoforms using matrix-assisted laser desorption/ionization in-source decay mass spectrometry (MALDI-ISDMS), called MALDI glycotyping. Our results show that the 1,5-diaminonaphthalene (DAN)/2,5-dihydroxybenzoic acid (DHB)/Na matrix can directly analyze the glycoforms in the femtomolar range of intact glycoproteins. The addition of DAN improved the morphology of the solid matrix due to the mixture of DAN and DHB, which significantly contribute to the high sensitivity of this direct analysis. Adding DAN significantly improved the sensitivity of the glycan precursor ions in the TOF/TOF analysis because of its enhanced fragmentation effect as an efficient UV-MALDI matrix. Further, practical glycoform analysis (glycotyping) of diluted biological samples containing glycoproteins, such as egg whites, was also successfully achieved.

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a “what you see is what you get” mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 μL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Characterization of Stability and Small-scale Structural Differences of Intact Glycoprotein Complexes Using Native Mass Spectrometry Travelling-Wave Ion Mobility Spectrometry

Characterization of Stability and Small-scale Structural Differences of Intact Glycoprotein Complexes Using Native Mass Spectrometry Travelling-Wave Ion Mobility Spectrometry

Characterization of a SARS-CoV-2 spike protein reference material

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Cover Feature: Glycan‐Selective MALDI In‐Source Decay Analysis of Intact Glycoproteins (Anal. Sens. 2/2022)

The Cover Feature shows a solid ionic matrix, DHB-aniline-sodium, which enabled top-down glycomics of glycoprotein via a regioselective in-source decay (ISD) and glycan-selective ionization as a sodium adduct ion. Formation of peptide-fragment-derived ions was significantly reduced, while the glycan pattern of the analyte protein was observed. Thus, this matrix selection can switch conventional peptide-selective MALDI-ISD to a glycan-selective MALDI-ISD. More information can be found in the Communication by S. Urakami and H. Hinou.

Glycan‐Selective MALDI In‐Source Decay Analysis of Intact Glycoproteins

AbstractA simple matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometry (MS) approach has been developed to analyze the structural pattern of glycan on glycoproteins without any digestive or modification pretreatment. For a 7 pmol sample of intact glycoprotein, RNase B, a solid ionic matrix DHB‐aniline‐sodium enabled a regioselective in‐source decay (ISD) and glycan‐selective ionization as a sodium adduct ion, [M+Na]+. This matrix significantly reduced the formation of peptide‐fragment‐derived ions, and a high‐mannose‐type glycan pattern was observed. This ISD product ion was formed by selective cleavage of the reducing end of the glycan by 0,2A or 2,4A type cross‐ring cleavage. Pseudo‐MS3‐type post‐source decay (PSD) analysis of an A type ion indicated the regioselectivity of the ISD fragmentation and the glycan sequence. Combining ISD glycomics with conventional ISD proteomics will accelerate quality control for glycoprotein drugs and biomarker discovery.

Quantifying Carbohydrate-Active Enzyme Activity with Glycoprotein Substrates Using Electrospray Ionization Mass Spectrometry and Center-of-Mass Monitoring

Carbohydrate-active enzymes (CAZymes) play critical roles in diverse physiological and pathophysiological processes and are important for a wide range of biotechnology applications. Kinetic measurements offer insight into the activity and substrate specificity of CAZymes, information that is of fundamental interest and supports diverse applications. However, robust and versatile kinetic assays for monitoring the kinetics of intact glycoprotein and glycolipid substrates are lacking. Here, we introduce a simple but quantitative electrospray ionization mass spectrometry (ESI-MS) method for measuring the kinetics of CAZyme reactions involving glycoprotein substrates. The assay, referred to as center-of-mass (CoM) monitoring (CoMMon), relies on continuous (real-time) monitoring of the CoM of an ensemble of glycoprotein substrates and their corresponding CAZyme products. Notably, there is no requirement for calibration curves, internal standards, labeling, or mass spectrum deconvolution. To demonstrate the reliability of CoMMon, we applied the method to the neuraminidase-catalyzed cleavage of N-acetylneuraminic acid (Neu5Ac) residues from a series of glycoproteins of varying molecular weights and degrees of glycosylation. Reaction progress curves and initial rates determined with CoMMon are in good agreement (initial rates within ≤5%) with results obtained, simultaneously, using an isotopically labeled Neu5Ac internal standard, which enabled the time-dependent concentration of released Neu5Ac to be precisely measured. To illustrate the applicability of CoMMon to glycosyltransferase reactions, the assay was used to measure the kinetics of sialylation of a series of asialo-glycoproteins by a human sialyltransferase. Finally, we show how combining CoMMon and the competitive universal proxy receptor assay enables the relative reactivity of glycoprotein substrates to be quantitatively established.

Native Mass Spectrometry Meets Glycomics: Resolving Structural Detail and Occupancy of Glycans on Intact Glycoproteins.

Glycoproteins are inherently heterogeneous and therefore resolving structures in their entirety remains a major challenge in structural biology. Native mass spectrometry has transformed our ability to study glycoproteins, and despite advances in high-resolution instrumentation, there are comparatively a few studies demonstrating its potential with data largely limited to an overall measure of monosaccharide composition for all glycans across glycosylation sites for a given protein. Clearly, these readouts lack glycan topology information, namely, monosaccharide linkage and glycan branching. To address this deficiency, we developed a new approach that joins native mass spectrometry with glycan exoglycosidase sequencing, the combination of which provides remarkable glycoprotein structural details. We show how N-glycan branching, terminal fucosylation, LacNAc extensions, and N- and O-glycan occupancy (i.e., total number of glycans) can be directly characterized on intact glycoproteins with minimal sample preparation. Taken together, native exoglycosidase sequencing mass spectrometry (NES-MS) notably improves our ability to characterize protein glycosylation, addressing a significant need in structural biology that will enable new routes to understand glycoprotein function.

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy.

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.