Benjamin F. Mann

High-sensitivity Analytical Approaches for the Structural Characterization of Glycoproteins

1.1. General Considerations Structural intricacies of carbohydrate molecules and their propensity to form varied linkages, substitutions, and branching patterns have fascinated many generations of chemists, as have the three-dimensional aspects of carbohydrate interactions with other biomolecules. The steadily increasing biochemical knowledge in this area has further added to the increasing importance of the field now referred to as “glycobiology” or, more generally, “glycoscience”. Yet, most of the emphasis over the last 50 years or so has been on two other classes of important biopolymers, namely nucleic acids and proteins. However, in the “post-genomic era”, complex carbohydrates can no longer be neglected, as it is becoming clear to many scientists that most mammalian proteins are glycosylated, and microbial systems and plants can have their own unique monosaccharide building blocks and special ways they can be interconnected and branched into unusual structures. Throughout evolution and the development of living organisms, glycoconjugates must have played major roles, no doubt due to their unusual biological selectivities, which, in turn, could well be due to the enormous information capacity of the “sugar code”.1,2 Throughout the 1980s, the multilateral importance of glycoconjugates in biology and medicine was recognized,3-6 albeit with an understanding that only new methodological approaches and systematic investigations would further define new vistas and provide intimate knowledge of how complex carbohydrates participate in all life processes. Today’s glycoscience is a multidisciplinary undertaking in which chemistry is expected to have an important role to describe the most complex structural aspects of sugars and their conjugates with other biological molecules. While the biological and biomedical relevance of studying glycosylation and sugar–protein and sugar–sugar interactions will undoubtedly be guided by advances in other respective fields (immunology, cancer research, parasitology, cell biology, and developmental biology, among others), the chemical disciplines’ two major tasks are to (a) isolate and structurally characterize biologically important glycoconjugates and (b) synthesize carbohydrate structures for biochemical investigations, enabling technologies and medical applications and providing new therapeutics. While the goals and directions of carbohydrate synthesis have been summarized elsewhere,7-11 the focus of our review has been on glycoanalytical chemistry. The synthetic and bioanalytical directions are not mutually exclusive, as new structural findings will undoubtedly provide further rationale for synthetic efforts and these, in turn, the availability of standards for structural verification. Since publication of the review on structural investigations of glycoconjugates at high sensitivity12 in these pages a decade ago, the field of analytical glycobiology has seen dramatic changes in its scope and depth. It is widely appreciated within the glycoscience community and increasingly by others that both new techniques and instrumentation and the established (albeit optimized) analytical methodologies have played very important roles in advancing the science of glycoconjugates to its current stage. Due to their different physical and chemical characteristics, the main classes of glycoconjugates, i.e. glycoproteins, glycolipids, polysaccharides, and proteoglycans with their highly charged constituents, glycosaminoglycans, demand somewhat specialized analytical and structural elucidation approaches. Our review will largely be focused on glycoproteins and their associated glycans, hoping that other scientists will describe the analytical aspects of the remaining glycoconjugate biomolecules elsewhere. The early advances in proteomics, the scientific area mostly preoccupied with identification and structural characterization of proteins, have led to diverse activities in protein post-transitional modifications (PTMs), which are often associated with important biological activities. Glycosylation of proteins is arguably the most widely spread and functionally most intriguing PTM in nature. It is already known that certain glycosylation patterns in proteins give rise to functional variance, with far-reaching consequences for health-disease issues, immunological disorders, toxicity effects, microbial invasion processes, etc. To investigate any of these highly important processes in sufficient molecular detail, analytical techniques capable of a high degree of structural elucidation and measurement sensitivity are currently needed. Within the plethora of new “-omics fields” (genomics, transcriptomics, lipidomics, metabolomics, etc.), the fields of glycoproteomics and glycomics have started to assume their respectable roles. Analytical glycobiology, representing both glycomics and glycoproteomics, now shares access to new measurement technologies that enable characterization and quantification of molecular processes in living organisms. Extensive glycomic and glycoproteomic data that can nowadays be generated with modern techniques and instrumentation are likely to enrich the “systems biology” approach.13-17 Both fields have started to contribute substantially to a better understanding of multicellular interactions in eukaryotic systems and important issues pertaining to human health and disease.18-23 Additionally, the long-held view that glycosylation is unimportant in prokaryotic systems is no longer defensible.24,25 Since our previous review12 in this journal, much progress has been achieved in terms of methodological developments toward better, more informative, and more sensitive measurements of glycoproteins and their glycan components. In addition, many conceptually important applications of new tools already point to the future needs for dealing with the enormous complexity of glycopeptides and oligosaccharide mixtures extracted from biological tissues and physiological fluids. The relatively recent interest of the pharmaceutical and biotech industries in recombinant glycoproteins, such as monoclonal antibodies, for treatment of cancer and other diseases,26-30 demands the use and further development of glycomic and glycoproteomic analytical procedures as well. Similarly to our previous report,12 the current review has been organized to discuss separately recent advances in glycoproteomics and glycomics, dealing first with the isolation and direct analysis of glycoproteins, followed by the description of advances in glycopeptide analysis and determination of the sites of glycosylation, and moving toward the analysis of complex glycan mixtures. Even more today than 10 years ago, mass spectrometry (MS) is the most prominent methodology in the arsenal of glycoprotein analysis tools. A number of new MS techniques, previously unexplored or insufficiently developed, are now at the center of attention of glycobiologists. At the sensitivity levels required by contemporary glycobiology, MS and tandem MS (MSn) techniques are currently the only means to provide reliable structural information. Carbohydrate derivatization (chemical modification of carbohydrates at microscale) uniquely enables certain MS measurements in terms of enhanced sensitivity and structural information. Due to the enormous “chemical space” for carbohydrate structural complexity,1,2 MS alone, no matter how sophisticated, is unlikely to provide all needed answers. However, in combinations with modern separation methodologies (different forms of chromatography and electrophoresis) that provide unique component resolution in time and space, MS detection and identification capabilities become enormously enriched. The past decade has seen substantial improvements in the chromatographic analysis of complex carbohydrates: (1) transition from the conventional-scale columns to capillary column dimensions, or even microchips, with the resulting gains in mass sensitivity of measurements; and (2) rapidly increasing use of stable and reliable hydrophilic column materials and graphitized carbon adsorbents. Further advances in capillary chromatographic separations pertain to effective resolution of very complex mixtures as well as the frequently needed separation of different isomers. Chromatographic advances of the recent years also relate to simple purifications of samples (analysis steps now often referred to as solid-phase extraction, or SPE) or the more sophisticated microcolumn lectin or affinity materials needed in group separations and preconcentration of certain glycoproteins for analysis. The past decade has also witnessed a rapid development of glycan array technologies, in which the surface-bound glycan structures (either synthesized or isolated from natural mixtures) are presented to glycan-binding proteins in biological samples.31-33 While these enabling technologies are novel and exciting, they will not be covered in this review, which primarily emphasizes techniques leading to structural elucidation of glycoproteins. Likewise, immunologically based measurements will not be discussed.

Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: the ABRF Glycoprotein Research Multi-Institutional Study 2012

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

ProteinQuant Suite: a bundle of automated software tools for label‐free quantitative proteomics

In simplifying the evaluation and quantification of high-throughput label-free quantitative proteomic data, we introduce ProteinQuant Suite. It comprises three standalone complementary computer utilities, namely ProtParser, ProteinQuant, and Turbo RAW2MGF. ProtParser is a filtering utility designed to evaluate database search results. Filtering is performed according to different criteria that are defined by the end-user. ProteinQuant then utilizes this parsed list of peptides and proteins in conjunction with mzXML or mzData files generated from the raw files for quantification. This quantification is based on the automatic detection and integration of chromatographic peaks representative of the liquid chromatography/mass spectrometry (LC/MS) elution profiles of identified peptides. Turbo RAW2MGF was developed to extend the applicability of ProteinQuant Suite to data collected from different types of mass spectrometers. It directly processes raw data files generated by Xcalibur, a ThermoElectron data acquisition software, and generates a MASCOT generic file (MGF). This file format is needed since the protein identification results generated by the database search employing this file format include information required for the precise identification and quantification of chromatographic peaks. The performance of ProteinQuant Suite was initially validated using LC/MS/MS generated for a mixture of standard proteins as well as standard proteins spiked in a complex biological matrix such as blood serum. Automated quantification of the collected data resulted in calibration curves with R(2) values higher than 0.95 with linearity spanning over more than 2 orders of magnitude with peak quantification reproducibility better than 15% (RSD). ProteinQuant Suite was also applied to confirm the binding preference of standard glycoproteins to Con A lectin using a sample consisting of both standard glycoproteins and proteins.

Multimethodological Approach to Identification of Glycoproteins from the Proteome of Francisella tularensis, an Intracellular Microorganism

It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a takeoff platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis.

Sub 2-μm macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

A new, mechanically stable silica microparticle with macrosized internal pores (1.6 μm particles with 100 nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume, as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ∼200 m(2)/g, making them suitable for large biomolecular separations. To demonstrate their potential for bioseparations, they have been functionalized with lectins for affinity enrichment of glycoproteins. The material was derivatized with two lectins, Concanavalin A (Con A) and Aleuria aurantia lectin (AAL), and binding properties were tested with standard glycoproteins. The columns exhibited excellent binding capacities for microaffinity enrichment: Con A was able to bind 75 μg of a standard glycoprotein in a 50 × 1 mm column. Following initial tests, the lectin microcolumns were utilized for enrichment of glycoproteins from 1 μL volumes of blood serum samples, performed in triplicate for each lectin. The enriched serum fractions were subjected to side-by-side glycomic and glycoproteomic profiling analyses with mass spectrometry to show that the new particles offer excellent sensitivity for microscale analyses of precious biological sample materials. The unique combination of the macroporous architecture and small particle diameter suggests the material may have advantages for conventional modes of chromatographic separation of macromolecules in an ultra-high-pressure liquid chromatography (UHPLC) format.

Glycomic and proteomic profiling of pancreatic cyst fluids identifies hyperfucosylated lactosamines on the N-linked glycans of overexpressed glycoproteins

Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patients prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these “hyperfucosylated” glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively, following A. aurantia lectin enrichment.

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica; identification of a novel glycosylated lipoprotein required for virulence

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.

A quantitative investigation of fucosylated serum glycoproteins with application to esophageal adenocarcinoma

Although glycoproteomic studies provide unique opportunities for cancer research, it has been necessary to develop specific methods for analysis of oncologically interesting glycoproteins. We describe a general, multimethodological approach for quantitative glycoproteomic analysis of fucosylated glycoproteins in human blood serum. A total of 136 putative fucosylated glycoproteins were identified with very high confidence in three clinically relevant sample pools (N=5 for each), with a mean CV of 3.1% observed for replicate analyses. Two samples were collected from subjects diagnosed with esophagus disease states, high‐grade dysplasia plus esophageal adenocarcinoma, while the third sample was representative of a disease‐free condition. Some glycoproteins, observed to be significantly upregulated in esophageal adenocarcinoma, i.e. more than twofold higher than in the disease‐free condition, are briefly discussed. Further investigation will be necessary to validate these findings; however, the method itself is demonstrated to be an effective tool for quantitative glycoproteomics of clinical samples.

Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography.

Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.

Examination of glycan profiles from IgG-depleted human immunoglobulins facilitated by microscale affinity chromatography.

Among the most important proteins involved in disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit an understanding of changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e., A, D, E, and M) that contain kappa light chains that requires only a few microliters of biological material. First, we designed a microcolumn containing recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an online depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. Even though only 3 μL of serum was used in these analyses, we were able to recover a significantly enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF MS. As a proof of principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort.