Disulfide bonding of cysteines is one of the most important protein modifications, and it plays a key role in establishing/maintaining protein structures in biologically active forms. Therefore, the determination of disulfide bond arrangement is one important aspect to understanding the chemical structure of a protein and defining its functional domains. Herein, aiming to understand how the HIV-1 envelope protein’s structure influences its immunogenicity, we used an MS-based approach, liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance (LC/ESI-FTICR) mass spectrometry, to determine the disulfide linkages on an oligomeric form of the group M consensus HIV-1 envelope protein (Env), CON-S gp140 ΔCFI. This protein has marked improvement in its immunogenicity compared to monomeric gp120 and wild-type forms of gp140 Envs. Our results demonstrate that the disulfide connectivity in the N-terminal region of CON-S gp140 ΔCFI is different from the disulfide bonding previously reported in the monomeric form of gp120 HIV-1 Env. Additionally, heterogeneity of the disulfide bonding was detected in this region. These data suggest that the V1/V2 region does not have a single, conserved disulfide bonding pattern and that variability could impact immunogenicity of expressed Envs.Keywords: disulfide; envelope protein; HIV-1; LC/ESI-FTICR; mass spectrometry;

The glycopeptide analysis field is tightly constrained by a lack of effective tools that translate mass spectrometry data into meaningful chemical information, and perhaps the most challenging aspect of building effective glycopeptide analysis software is designing an accurate scoring algorithm for MS/MS data. We provide the glycoproteomics community with two tools to address this challenge. The first tool, a curated set of 100 expert-assigned CID spectra of glycopeptides, contains a diverse set of spectra from a variety of glycan types; the second tool, Glycopeptide Decoy Generator, is a new software application that generates glycopeptide decoys de novo. We developed these tools so that emerging methods of assigning glycopeptides’ CID spectra could be rigorously tested. Software developers or those interested in developing skills in expert (manual) analysis can use these tools to facilitate their work. We demonstrate the tools’ utility in assessing the quality of one particular glycopeptide software package, GlycoPep Grader, which assigns glycopeptides to CID spectra. We first acquired the set of 100 expert assigned CID spectra; then, we used the Decoy Generator (described herein) to generate 20 decoys per target glycopeptide. The assigned spectra and decoys were used to test the accuracy of GlycoPep Grader’s scoring algorithm; new strengths and weaknesses were identified in the algorithm using this approach. Both newly developed tools are freely available. The software can be downloaded at http://glycopro.chem.ku.edu/GPJ.jarKeywords: collision-induced dissociation; decoys; false discovery rate; glycopeptide; liquid chromatography−mass spectrometry;

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others.(1) Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env’s glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.Keywords: envelope glycoprotein; glycosylation; HIV; mass spectrometry; vaccine;

The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years. From a structural perspective, mAbs have high disulfide bond content, and the correct disulfide connectivity is required for proper folding and to maintain their biological activity. Therefore, disulfide linkage mapping is an important component of mAb characterization for ensuring drug safety and efficacy. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been well established since the late 1960s. Among these IgG subtypes, disulfide mediated isoforms have been identified for IgG2 and IgG4, and to a lesser extent in IgG1, which is the most studied IgG subclass. However, no studies have been carried out so far to investigate whether different IgG3 isoforms exist due to alternative disulfide connectivity. In an effort to investigate the presence of disulfide-mediated isoforms in IgG3, we employed a bottom-up mass spectrometry approach to accurately determine the disulfide bond linkages in endogenous human IgG3 monoclonal antibody, and our results show that no such alternative disulfide bonds exist. While many antibody-based drugs are developed around IgG1, IgG3 represents a new, and in some cases, more desirable drug candidate. Our data represent the first demonstration that alternative disulfide bond arrangements are not present in endogenous IgG3; and therefore, they should not be present in recombinant forms used as antibody-based therapeutics.

A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

Glycoproteins are biologically significant large molecules that participate in numerous cellular activities. In order to obtain site-specific protein glycosylation information, intact glycopeptides, with the glycan attached to the peptide sequence, are characterized by tandem mass spectrometry (MS/MS) methods such as collision-induced dissociation (CID) and electron transfer dissociation (ETD). While several emerging automated tools are developed, no consensus is present in the field about the best way to determine the reliability of the tools and/or provide the false discovery rate (FDR). A common approach to calculate FDRs for glycopeptide analysis, adopted from the target-decoy strategy in proteomics, employs a decoy database that is created based on the target protein sequence database. Nonetheless, this approach is not optimal in measuring the confidence of N-linked glycopeptide matches, because the glycopeptide data set is considerably smaller compared to that of peptides, and the requirement of a consensus sequence for N-glycosylation further limits the number of possible decoy glycopeptides tested in a database search. To address the need to accurately determine FDRs for automated glycopeptide assignments, we developed GlycoPep Evaluator (GPE), a tool that helps to measure FDRs in identifying glycopeptides without using a decoy database. GPE generates decoy glycopeptides de novo for every target glycopeptide, in a 1:20 target-to-decoy ratio. The decoys, along with target glycopeptides, are scored against the ETD data, from which FDRs can be calculated accurately based on the number of decoy matches and the ratio of the number of targets to decoys, for small data sets. GPE is freely accessible for download and can work with any search engine that interprets ETD data of N-linked glycopeptides. The software is provided at https://desairegroup.ku.edu/research.

The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env’s PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens.

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Studying protein O-glycosylation remains an analytical challenge. Different from N-linked glycans, the O-glycosylation site is not within a known consensus sequence. Additionally, O-glycans are heterogeneous with numerous potential modification sites. Electron transfer dissociation (ETD) is the method of choice in analyzing these glycopeptides since the glycan side chain remains intact in ETD, and the glycosylation site can be localized on the basis of the c and z fragment ions. Nonetheless, new software is necessary for interpreting O-glycopeptide ETD spectra in order to expedite the analysis workflow. To address the urgent need, we studied the fragmentation of O-glycopeptides in ETD and found useful rules that facilitate their identification. By implementing the rules into an algorithm to score potential assignments against ETD-MS/MS data, we applied the method to glycopeptides generated from various O-glycosylated proteins including mucin, erythropoietin, fetuin, and an HIV envelope protein, 1086.C gp120. The site-specific O-glycopeptide composition was correctly assigned in every case, proving the merits of our method in analyzing glycopeptide ETD data. The algorithm described herein can be easily incorporated into other automated glycomics tools.

Electron transfer dissociation (ETD) is commonly used in fragmenting N-linked glycopeptides in their mass spectral analyses to complement collision-induced dissociation (CID) experiments. The glycan remains intact through ETD, while the peptide backbone is cleaved, providing the sequence of amino acids for a glycopeptide. Nonetheless, data analysis is a major bottleneck to high-throughput glycopeptide identification based on ETD data, due to the complexity and diversity of ETD mass spectra compared to CID counterparts. GlycoPep Detector (GPD) is a web-based tool to address this challenge. It filters out noise peaks that interfere with glycopeptide sequencing, correlates input glycopeptide compositions with the ETD spectra, and assigns a score for each candidate. By considering multiple ion series (c-, z-, and y-ions) and scoring them separately, the software gives more weighting to the ion series that matches peaks of high intensity in the spectra. This feature enables the correct glycopeptide to receive a high score while keeping scores of incorrect compositions low. GPD has been utilized to interpret data collected on six model glycoproteins (RNase B, avidin, fetuin, asialofetuin, transferrin, and AGP) as well as a clade C HIV envelope glycoprotein, C.97ZA012 gp140ΔCFI. In every assignment made by GPD, the correct glycopeptide composition earns a score that is about 2-fold higher than other incorrect glycopeptide candidates (decoys). The software can be accessed at http://glycopro.chem.ku.edu/ZZKHome.php.

Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of electron transfer dissociation (ETD) facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds.

Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study with the inclusion of a fragmentation technique, electron transfer dissociation (ETD), to complement collision-induced dissociation. The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation, two sites (N386 and N392) in the V4 region were populated with high mannose glycans in the CHO cell-derived 1086.C gp120, while these sites had a mixture of high mannose and processed glycans in the 293T cell-derived 1086.C gp120. Compositional analysis of O-linked glycans revealed that 293T cell-derived 1086.C gp120 consisted of core 1, 2, and 4 type O-linked glycans, while CHO cell-derived 1086.C exclusively consisted of core 1 type O-linked glycans. Overall, glycosylation site occupancy of the CHO and 293T cell-derived 1086.C gp120 showed a high degree of similarity except for one site at N88 in the C1 region. This site was partially occupied in 293T-gp120 but fully occupied in CHO-gp120. Site-specific glycopeptide analysis of transmitted/founder 1086.C gp120 expressed in CHO cells revealed the presence of phosphorylated glycans, while 293T cell-produced 1086.C gp120 glycans were not phosphorylated. While the influence of phosphorylated glycans on immunogenicity is unclear, distinguishing host-cell specific variations in glycosylation profiles provide insights into the similarity (or difference) in recombinant vaccine products. While these differences had minimal effect on envelope antigenicity, they may be important in considering immunogenicity and functional capacities of recombinant envelope proteins produced in different expression systems.

The purpose of this review is to provide those interested in glycosylation analysis with the most updated information on the availability of automated tools for MS characterization of N-linked and O-linked glycosylation types. Specifically, this review describes software tools that facilitate elucidation of glycosylation from MS data on the basis of mass alone, as well as software designed to speed the interpretation of glycan and glycopeptide fragmentation from MS/MS data. This review focuses equally on software designed to interpret the composition of released glycans and on tools to characterize N-linked and O-linked glycopeptides. Several websites have been compiled and described that will be helpful to the reader who is interested in further exploring the described tools.

GlycoPep grader (GPG) is a freely available software tool designed to accelerate the process of accurately determining glycopeptide composition from tandem mass spectrometric data. GPG relies on the identification of unique dissociation patterns shown for high mannose, hybrid, and complex N-linked glycoprotein types, including patterns specific to those structures containing fucose or sialic acid residues. The novel GPG scoring algorithm scores potential candidate compositions of the same nominal mass against MS/MS data through evaluation of the Y1 ion and other peptide-containing product ions, across multiple charge states, when applicable. In addition to evaluating the peptide portion of a given glycopeptide, the GPG algorithm predicts and scores product ions that result from unique neutral losses of terminal glycans. GPG has been applied to a variety of glycoproteins, including RNase B, asialofetuin, and transferrin, and the HIV envelope glycoprotein, CON-S gp140ΔCFI. The GPG software is implemented predominantly in PostgreSQL, with PHP as the presentation tier, and is publicly accessible online. Thus far, the algorithm has identified the correct compositional assignment from multiple candidate N-glycopeptides in all tests performed.

Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To ensure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest SF), reducing agents [dithiothreitol (DTT) and tris(2-carboxyethyl)phophine (TCEP)], and various concentrations of alkylating agent [iodoacetamide (IAM)]. After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2. Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.

The work presented herein describes the first comprehensive analysis of a partially deglycosylated HIV vaccine candidate envelope protein (Env). The Env, JRFL gp140 ΔCF, with 27 potential glycosylation sites, was partially deglycosylated with PNGase F as part of a strategy to generate a more immunogenic HIV vaccine, and the resulting protein glycosylation was characterized in a unique workflow using two different glycosidases, Endo H and Endo F3. This unique analysis protocol provided for coverage on 26 of the 27 glycosylation sites, and the data showed that the biochemical treatment with PNGase F resulted in a highly heterogeneous glycoprotein product that had been partially deglycosylated at most of the glycosylation sites. The protocols described in this work could be useful for characterizing the glycosylation site occupancy of other native or biochemically deglycosylated proteins.Graphical abstractResearch highlights▶ A new method for profiling glycosylation site occupancy is introduced. ▶ The method was applied to a partially deglycosylated Env glycoprotein, which is an HIV vaccine candidate. ▶ Partial deglycosylation of Env glycoproteins produces a highly heterogeneous product.

Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not.

The mass defect of a substance can be used in mass spectral analysis to identify peaks as likely belonging to a compound class, such as peptides, if the mass defect is within the known range for that compound class. For peptides, a range of possible mass defects was calculated previously, using a set of theoretical peptides, where all possible amino acid combinations were considered (Mann, M. Abstract from the 43rd Annual Conference on Mass Spectrometry and Allied Topics; Conference Proceedings, 1995). We compare that range of theoretical peptide mass defects to new values obtained from in silico tryptic digests of proteins that are abundant in human serum and human seminal fluid. The range of mass defect values encompassing 95% of peptides for the human protein data sets was found to be up to 50% smaller than the previously reported mass defect range for the theoretical peptides. The smaller range established for human tryptic peptides can be used to improve peptide mass defect filters by excluding more species that are not likely to be peptides, thus improving filter selectivity for peptides during proteomic data analysis.

The extensive glycosylation of HIV-1 envelope proteins (Envs), gp120/gp41, is known to play an important role in evasion of host immune response by masking key neutralization epitopes and presenting the Env glycosylation as “self” to the host immune system. The Env glycosylation is mostly conserved but continues to evolve to modulate viral infectivity. Thus, profiling Env glycosylation and distinguishing interclade and intraclade glycosylation variations are necessary components in unraveling the effects of glycosylation on Env’s immunogenicity. Here, we describe a mass spectrometry-based approach to characterize the glycosylation profiles of two rVV-expressed clade C Envs by identifying the glycan motifs on each glycosylation site and determining the degree of glycosylation site occupancy. One Env is a wild-type Env, while the other is a synthetic “consensus” Env (C.CON). The observed differences in the glycosylation profiles between the two clade C Envs show that C.CON has more unutilized sites and high levels of high mannose glycans; these features mimic the glycosylation profile of a Group M consensus immunogen, CON-S. Our results also reveal a clade-specific glycosylation pattern. Discerning interclade and intraclade glycosylation variations could provide valuable information in understanding the molecular differences among the different HIV-1 clades and in designing new Env-based immunogens.

Glycoproteomics is an emerging science that shows promise in applications such as biomarker discovery and biopharmaceutical development. One central technique in glycoproteomic analysis is analyzing glycopeptides by mass spectrometry. This challenging technique is still under development, and methods to simplify the data analysis are greatly needed. One potentially attractive analysis approach would be to assign a significant portion of the glycopeptide compositions using high-resolution MS data. In the work described herein, we ask the question: Under what circumstances is it possible to assign glycopeptides to MS data, using only high-resolution mass spectra? Variables investigated include the number of glycosylation sites on the protein, the potential diversity of the glycans attached to the protein, and the mass accuracy obtained. This work outlines guidelines for when it is (and is not) appropriate to rely heavily on high-resolution mass measurements to assign glycopeptide compositions; such guidelines are potentially useful for anyone conducting glycopeptide analysis by mass spectrometry.

We demonstrate herein a method for quantifying glycosylation changes on glycoproteins. This novel method uses MS data of characterized glycopeptides to analyze glycosylation profiles, and several quality control tests were done to demonstrate that the method is reproducible, robust, applicable to different types of glycoproteins, and tolerant of instrumental variability during ionization of the analytes. This method is unique in that it is the first label-free quantitative method specifically designed for glycopeptide analysis. It can be used to monitor changes in glycosylation in a glycosylation site-specific manner on a single glycoprotein, or it can be used to quantify glycosylation in a glycoprotein mixture. During mixture analysis, the method can discriminate between changes in glycosylation of a given protein, and changes in the glycoprotein’s concentration in the mixture. This method is useful for quantitative analyses in biochemical studies of glycoproteins, where changes in glycosylation composition can be linked to functional differences; it could also be implemented in the pharmaceutical industry, where glycosylation profiles of glycoprotein-based therapeutics must be quantified. Finally, quantification of glycopeptides is an important aspect of glycopeptide-based biomarker discovery, and our quantitative approach could be a valuable asset to this field as well, provided the compositions of the glycopeptides to be quantified are identifiable using other methods.

The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others.(1) Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometry-based strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces cross-clade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env’s glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140ΔCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.

The STEP method (Statistical Test of Equivalent Pathways), recently developed to determine primary and secondary fragmentation in the MS/MS of peptides and carbohydrates, is applied in the current study to common pharmaceutical antibiotics. The classification of product ions as primary or secondary is then utilized to construct genealogy diagrams that aid in the structural characterization of the product ions. Four compounds were subjected to the MS/MS conditions used for the STEP method, and the method was used to correctly identify primary and secondary ions in three of the four pharmaceuticals. Calculated STEP values for erythromycin did not match previously characterized fragmentation assignments. This provided an opportunity to explore potential limitations of STEP analysis. It was determined that inaccurate STEP assignments could result, if the starting compound is classified as “fragile”, because fragile ions, such as erythromycin can produce abnormally low STEP ratios. While this finding represents a limitation of using the STEP method to determine whether product ions are due to primary or secondary fragmentation for fragile ions, it suggests the possibility of identifying the presence of “fragile ions” by STEP analysis.

Phosphorylation and sulfation are important modifications affecting the biological properties of carbohydrates, proteins, and glycoproteins. Identification of these two functional groups facilitates the understanding of the structure/function relationship in various species. Mass spectrometry is one of the methods used to detect the presence of these two modifications in complex biological mixtures. However, phosphorylated and sulfated structures are isobaric; thus, differentiation between them in routinely used mass spectrometers is problematic. Herein, we demonstrate that these two groups can be discriminated by using ion-pairing in conjunction with MS/MS experiments. The characteristic product ions are used to successfully identify the phosphorylation and sulfation present in mono-, disaccharides, and the highly sulfated glycoprotein, ovine luteinizing hormone. This method is a robust approach to differentiate the two isobaric functional groups.

We demonstrate a method that enhances the mass spectral signal of mono- and disulfated glycopeptides, present in glycoproteins that contain many other nonsulfated glycoforms. This method utilizes the tripeptide Lys-Lys-Lys as an ion-pairing reagent to complex selectively to sulfated species, and enhance their ion signal. The method is applied to the analysis of glycopeptides released from the enzymatic digestion of ovine luteinizing hormone. In this analysis, a disulfated glycopeptide is identified that was previously not detectable by MS assays, and a monosulfated glycoform, present at less than 1% abundance, is identified without any separation or enrichment of these species prior to analysis. In addition to enhancing the ion signal of sulfated glycopeptides, the ion-pairing technique is useful in obtaining structural information about the sulfated species.

Phosphorylation and sulfation are two important biological modifications present in carbohydrates, proteins, and glycoproteins. Typically, sulfation and phosphorylation cause different biological responses, so differentiating these two functional groups is important for understanding structure/function relationships in proteins, carbohydrates, and metabolites. Since phosphorylated and sulfated compounds are isobaric, their discrimination is not possible in routinely utilized mass spectrometers. Thus, a novel mass spectrometric method to distinguish them has been developed. Herein, we utilize basic peptides as ion-pairing reagents to complex to phosphorylated and sulfated carbohydrates via noncovalent interactions. By performing ESI-MS/MS on the ion-pair complexes, the isobaric compounds can be distinguished. This is the first study demonstrating that ion-pairing can be used for the detection of phosphorylated compounds and the first study to use ion-pairing in conjunction with MS/MS to obtain structural information about the analytes.