Analytical and Bioanalytical Chemistry (v.409, #2)

Recent advances in glycomics, glycoproteomics and allied topics by Yehia Mechref; David C. Muddiman (355-357).
is a professor in the Department of Chemistry and Biochemistry at Texas Tech University, Lubbock, TX, USA. He received a B.Sc. in chemistry from the American University of Beirut (Beirut, Lebanon) and a Ph.D. with an honorable mention from Oklahoma State University (Stillwater, OK, USA). His research focus is on the development of sensitive biomolecular mass spectrometry methods enabling qualitative and quantitative assessments of the roles of proteins, glycoproteins, and glycans in biological systems. Thus far, he has published 21 review articles, 14 book chapters, and 154 peer-reviewed research papers. Currently, his Scopus h-index is 46, with 5862 citations. He has received 11 US patents. He has organized and co-organized numerous symposia and conferences. He received the Barnie E. Rushing, Jr. Faculty Distinguished Research Award in 2016 and the Barnie E. Rushing, Jr. Faculty Outstanding Research Award in 2015. is the Jacob and Betty Belin Distinguished Professor of Chemistry, and founder and director of the W.M. Keck FTMS Laboratory for Human Health Research at North Carolina State University in Raleigh, NC, USA. Prior to moving his research group to North Carolina State University, he was a professor of biochemistry and molecular biology, founder, and director of the Mayo Proteomics Research Center at the Mayo Clinic College of Medicine in Rochester, MN, USA. He is Editor of Analytical and Biological Chemistry and Associate Editor of the Encyclopedia of Analytical Chemistry, and he is on the Editorial Advisory Board of Mass Spectrometry Reviews, Molecular and Cellular Proteomics, Rapid Communications in Mass Spectrometry, and the Journal of Chromatography B. He also serves on the Advisory Board of the NIH-funded Complex Carbohydrate Research Center, University of Georgia, and the Yale/NIDA Neuroproteomics Center, Yale University. He is currently a member of the ASMS Board of Directors and the President of the United States Human Proteome Organization (US-HUPO). His group has published over 225 peer-reviewed papers and received four US patents. He is the recipient of the 2015 ACS Award in Chemical Instrumentation, the 2010 Biemann Medal from the American Society for Mass Spectrometry, the 2009 NCSU Alumni Outstanding Research Award, the 2004 ACS Arthur F. Findeis Award, the 1999 American Society for Mass Spectrometry Research Award, and the 1990–91 Safford Award, University of Pittsburgh, for Excellence in Teaching.

Reversed-phase separation methods for glycan analysis by Gerda C. M. Vreeker; Manfred Wuhrer (359-378).
Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical Abstract Glycan isomer separation by reversed phase liquid chromatography
Keywords: Glycan; Reversed phase; Liquid chromatography; Separation

Brain extracellular matrix (ECM) is a highly organized system that consists of collagens, noncollagenous proteins, glycoproteins, hyaluronan, and proteoglycans. Recognized physiological roles of ECM include developmental regulation, tissue homeostasis, cell migration, cell proliferation, cell differentiation, neuronal plasticity, and neurite outgrowth. Aberrant ECM structure is associated with brain neurodegenerative conditions. This review focuses on two neurodegenerative conditions, schizophrenia and Alzheimer’s disease, and summarizes recent findings of altered ECM components, including proteoglycans, glycosaminoglycans, proteins, and glycoproteins, and proteins and genes related to other brain components. The scope includes immunohistochemical, genomics, transcriptomics, proteomics, and glycomics studies, and a critical assessment of current state of proteomic studies for neurodegenerative disorders. The intent is to summarize the ECM molecular alterations associated with neurodegenerative pathophysiology. Graphical Abstract Brain extracellular matrix showing HSPGs, CSPGs, HA, collagens, and other glycoproteins.
Keywords: Brain extracellular matrix; Proteoglycans; Glycosaminoglycans; Perineuronal nets; Schizophrenia; Alzheimer’s disease

Glycans and glycoproteins as specific biomarkers for cancer by Muchena J. Kailemia; Dayoung Park; Carlito B. Lebrilla (395-410).
Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments, as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies such as global glycan release and site-specific glycosylation analysis and quantitation are also discussed. Graphical Abstract Steps involved in the biomarker discovery
Keywords: Mass spectrometry; Cancer; Disease biomarker; Glycomics; Glycoproteomics; Site-specific glycosylation

Analysis of heparin oligosaccharides by capillary electrophoresis–negative-ion electrospray ionization mass spectrometry by Lei Lin; Xinyue Liu; Fuming Zhang; Lianli Chi; I. Jonathan Amster; Franklyn E. Leach III; Qiangwei Xia; Robert J. Linhardt (411-420).
is a Postdoctoral Research Fellow in the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute. Her research mainly focuses on analytical method development using mass spectrometry for heparin analysis. is a graduate student from Shandong University, attending an international program in the Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute. Her research mainly focuses on mass spectrometry method development and glycosaminoglycan structure analysis. (Ph.D.) is a Research Professor in the Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute. His research focus is in glycomics, glycoengineering, and molecular interactions using surface plasmon resonance (SPR). is a Professor at the National Glycoengineering Research Center, Shandong University, China. He received his Ph.D. in analytical chemistry under the supervision of Professor Robert J. Linhardt from Rensselaer Polytechnic Institute. Currently he is studying in the field of proteomics and post-translational modification proteomics, as well as structural characterization of low molecular weight heparins. is Professor of Chemistry at the University of Georgia, and Head of the Department of Chemistry. His research interests include understanding the fundamentals of Fourier transform mass spectrometry and its application to bioanalytical chemistry, the development of methods for the structural analysis of glycosaminoglycan oligosaccharides, and the application of ion mobility for examining the three-dimensional structure of glycan–protein complexes. is currently the lead scientist for research and development at Photochem Technologies in Athens, GA. His primary interests are the development of mass spectrometry based instrumentation and methodologies for the structural characterization of biomolecules. (Ph.D.) is the CEO and Principal Scientist of CMP Scientific, Corp. His current research interest is mainly the electrophoretic separation and mass spectrometric detection of analytes that are relevant to life science and the biopharmaceutical industry. More specifically, he is devoted to making capillary electrophoresis–mass spectrometry (CE–MS) coupling technology simpler, faster, and more robust. is the Senior Constellation Professor of Biocatalysis and Metabolic Engineering at Rensselaer, holding appointments in Chemistry, Biology, Chemical Engineering & Biomedical Engineering. He holds an Adjunct Professor of Orthopaedics at the Icahn School of Medicine at Mount Sinai and an Adjunct Professor of Pharmacy at Albany College of Pharmacy. His research focuses on glycoscience and glycotechnology, with specific emphasis on heparin. Most hyphenated analytical approaches that rely on liquid chromatography–MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE–MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE–MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography–MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE–MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE–MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins
Keywords: Hyphenated techniques; CE–MS; Heparin; Low molecular weight heparin; Oligosaccharides; Glycosaminoglycan

Rapid and sensitive MALDI MS analysis of oligosaccharides by using 2-hydrazinopyrimidine as a derivative reagent and co-matrix by Kuan Jiang; Arya Aloor; Jiangyao Qu; Cong Xiao; Zhigang Wu; Cheng Ma; Lianwen Zhang; Peng George Wang (421-429).
Sensitive analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is significantly hampered by the low ionization efficiency of oligosaccharides. Derivatization affords a feasible way to enhance the MALDI intensities of oligosaccharides by introducing an easily ionized and/or hydrophobic tag to their reducing ends. However, tagging and subsequent desalting processes are quite time-consuming. Herein, we develop a rapid and sensitive approach for oligosaccharide derivatization by using 2-hydrazinopyrimidine (2-HPM). As a result of the presence of an electron-withdrawing N-heterocycle, 2-HPM can quantitatively derivatize oligosaccharides within 15 min and selectively facilitate their ionization. Additionally, 2-HPM acts as co-matrix to enhance the MALDI signal of oligosaccharides, and therefore the tedious enrichment and purification processes prior to MALDI analysis are avoided. This approach is applied to the analysis of various oligosaccharides released from glycopeptides, glycoprotein, and biological samples. After derivatization, a significant increase of MALDI intensities (greater than 10-fold) was observed for all the tested neutral and sialylated oligosaccharides. Moreover, the enhanced fragmentation of MS/MS brings much convenience to the structural elucidation of oligosaccharides. Graphical abstract Improved MALDI MS analysis of oligosaccharides by using 2-hydrazinopyrimidine as a derivative tag and co-matrix
Keywords: Oligosaccharides; MALDI-TOF MS; 2-Hydrazinopyrimidine; Derivatization

Protein glycosylation plays a key role in many biological processes. In this study, a novel carbon material with nanopores was prepared by carbonization of metal–organic framework (MOF) Mil-101(Cr). The parent MOF assembled from metal ions with bridging organic linkers had many fascinating properties, such as ultrahigh surface area, suitable nanopore structure, and especially a large amount of carbon after being calcined. Due to the strong interactions between carbon and glycans as well as the size-exclusion effect of pore against protein, the N-linked glycans from standard glycoprotein or complex human serum proteins could be identified with high efficiency. The simple synthesis method as well as good enrichment efficiency made this novel carbon material a promising tool for glycosylation research.
Keywords: Protein glycosylation; Carbon material; Nanopores; MOF; Enrichment; N-linked glycans

Isomeric complexity of glycosylation documented by MSn by David J. Ashline; Hailong Zhang; Vernon N. Reinhold (439-451).
Re-analysis of two breast cancer cell lines, MCF-7 and MDA-MB-231, has shown multiple isomeric structures exposed by sequential mass spectrometry, MS n . Several released glycan compositions were re-evaluated, which indicated variations in polylactosamine and fucosylation structures. Probable isomer numbers, when considering both stereo and structural entities, are significant and the varying types are mentioned. The structural isomers of linkage position are most frequently considered, while stereo isomers are usually assumed from biosynthetic data. Evaluation of any new sample should be cautious and merits careful attention to empirical data. While isomers are usually considered a chromatographic problem (e.g., LCMS, IMMS) and most frequently considered a separations problem, such results will always be challenged by identification and documentation. MSn data provide a direct spatial solution that includes spectral data for characterization (mass and abundance) supported by a universal library match feature.
Keywords: Isomer; MSn ; Sequential mass spectrometry; Carbohydrate; Glycan; Permethylation

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization by Shiyue Zhou; Xue Dong; Lucas Veillon; Yifan Huang; Yehia Mechref (453-466).
The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages. Graphical Abstract LC-MS and tandem MS of terminal galactose isomers
Keywords: N-Glycan isomers; Tandem mass spectrometry; Permethylation; Porous graphitic carbon column; LC-MS/MS

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses by Xueyun Zheng; Xing Zhang; Nathaniel S. Schocker; Ryan S. Renslow; Daniel J. Orton; Jamal Khamsi; Roger A. Ashmus; Igor C. Almeida; Keqi Tang; Catherine E. Costello; Richard D. Smith; Katja Michael; Erin S. Baker (467-476).
Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules, such as proteins and lipids, and alter their properties and functions, so glycan characterization is essential for understanding the effects they have on cellular systems. However, the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest that specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS. Graphical abstract Glycan isomers, such as fructose and glucose, show distinct separations in positive and negative ion mode
Keywords: Ion mobility spectrometry; Mass spectrometry; Glycans; O-Glycans; Isomers

LC-MS analysis combined with principal component analysis and soft independent modelling by class analogy for a better detection of changes in N-glycosylation profiles of therapeutic glycoproteins by Ana Planinc; Bieke Dejaegher; Yvan Vander Heyden; Johan Viaene; Serge Van Praet; Florence Rappez; Pierre Van Antwerpen; Cédric Delporte (477-485).
Therapeutic proteins are among the top selling drugs in the pharmaceutical industry. More than 60 % of the approved therapeutic proteins are glycosylated. Nowadays, it is well accepted that changes in glycosylation may affect the safety and the efficacy of the therapeutic proteins. For this reason, it is important to characterize both the protein and the glycan structures. In this study, analytical and data processing methods were developed ensuring an easier characterization of glycoprofiles. N-glycans were (i) enzymatically released using peptide-N-glycosidase F (PNGase F), (ii) reduced, and (iii) analyzed by hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HRMS). Glycosylation changes were analyzed in human plasma immunoglobulin G samples which had previously been artificially modified by adding other glycoproteins (such as ribonuclease B and fetuin) or by digesting with enzyme (neuraminidase). Principal component analysis (PCA) and classification through soft independent modelling by class analogy (SIMCA) were used to detect minor glycosylation changes. Using HILIC-MS-PCA/SIMCA approach, it was possible to detect small changes in N-glycosylation, which had not been detected directly from the extracted-ion chromatograms, which is current technique to detect N-glycosylation changes in batch-to-batch analysis. The HILIC-MS-PCA/SIMCA approach is highly sensitive approach due to the sensitivity of MS and appropriate data processing approaches. It could help in assessing the changes in glycosylation, controlling batch-to-batch consistency, and establishing acceptance limits according to the glycosylation changes, ensuring safety and efficacy. Graphical abstract N-glycosylation characterization using LC-MS-PCA approach
Keywords: Therapeutic glycoproteins; N-Glycans; Principal component analysis (PCA); Soft independent modelling by class analogy (SIMCA); High-resolution mass spectrometry (HRMS); Hydrophilic interaction liquid chromatography (HILIC)

Our greater understanding of the importance of N-linked glycosylation in biological systems has spawned the field of glycomics and development of analytical tools to address the many challenges regarding our ability to characterize and quantify this complex and important modification as it relates to biological function. One of the unmet needs of the field remains a systematic method for characterization of glycans in new biological systems. This study presents a novel workflow for identification of glycans using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT™) strategy developed in our lab. This consists of monoisotopic mass extraction followed by peak pair identification of tagged glycans from a theoretical library using an in-house program. Identification and relative quantification could then be performed using the freely available bioinformatics tool Skyline. These studies were performed in the biological context of studying the N-linked glycome of differentiating xylem of the poplar tree, a widely studied model woody plant, particularly with respect to understanding lignin biosynthesis during wood formation. Through our workflow, we were able to identify 502 glycosylated proteins including 12 monolignol enzymes and 1 peroxidase (PO) through deamidation glycosite analysis. Finally, our novel semi-automated workflow allowed for rapid identification of 27 glycans by intact mass and by NAT/SIL peak pairing from a library containing 1573 potential glycans, eliminating the need for extensive manual analysis. Implementing Skyline for relative glycan quantification allowed for improved accuracy and precision of quantitative measurements over current processing tools which we attribute to superior algorithms correction for baseline variation and MS1 peak filtering. Graphical abstract Workflow for FANGS-INLIGHT glycosite profiling of plant xylem and monolignol proteins followed by INLIGHT tagging with semi-automated identification of glycans by light-heavy peak pairs. Finally, manual validation and relative quantification was performed in Skyline
Keywords: N-linked glycosite; Skyline; Populus trichocarpa ; Monolignol; N-linked glycans

Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry by John B. Mangrum; Akul Y. Mehta; Alhumaidi B. Alabbas; Umesh R. Desai; Adam M. Hawkridge (499-509).
Structural characterization of the microheterogeneity of heparin, heparan sulfate, and other glycosaminoglycans is a major analytical challenge. We present the use of a stable isotope-labeled hydrazide tag (INLIGHT™) with high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS, which was recently introduced for detailed study of N-glycan heterogeneity, to characterize heparinase-digested heparin (digHep) products without the use of semi-volatile ion pairing reagents. Using both full scan LC-MS and data-dependent LC-MS/MS, we identified 116 unique digHep species, a feat possible because of INLIGHT™ labeling. Of these, 83 digHep products were structurally identified, including the 12 standard disaccharides as well as 34 tetra- (DP4), 26 hexa- (DP6), 21 octa- (DP8), and 2 decasaccharides (DP10). Each of the 116 digHep species co-eluted with both light and heavy INLIGHT™ tags (L/Havg = 1.039 ± 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT™-based comparative analyses of different forms of heparin, heparan sulfate, and other GAGs with high quantitative precision using mainstay reverse-phase HRAM LC-MS/MS. Graphical Abstract Reducing end labeling strategy for mapping depolymerized heparin/heparan sulfate products by reverse-phase LC-MS/MS
Keywords: Mass spectrometry/ICP-MS; Glycosaminoglycan; Heparin; Chemical derivatization

Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides by He Zhu; Xu Li; Jingyao Qu; Cong Xiao; Kuan Jiang; Ebtesam Gashash; Ding Liu; Jing Song; Jiansong Cheng; Cheng Ma; Peng George Wang (511-518).
N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H2 18O. Subsequent LC–MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification. Graphical abstract Proposed mechanism of glycopeptides enrichment using DEAE-Sepharose
Keywords: Biological samples; Mass spectrometry/ICP-MS; Amino acids/peptides

Novel boronate material affords efficient enrichment of glycopeptides by synergized hydrophilic and affinity interactions by Jianying Chen; Xiaohu Li; Mengyu Feng; Kun Luo; Juan Yang; Bo Zhang (519-528).
Development of novel materials for enrichment of glycopeptides is the key to a comprehensive analysis of the glycoproteome, which is closely related to several major diseases and biomarker findings. We synthesized phenylboronic acid (PBA) bound to SiO2 microspheres by a thiol–ene click chemistry method (this material was denoted as click PBA) and used it to separate cis-diol-containing molecules and enrich glycopeptides in hydrophilic interaction chromatography mode. Successful preparation of click PBA was confirmed by elemental analysis, X-ray photoelectron spectroscopy, N2 adsorption–desorption isotherms, and high-resolution scanning electron microscopy. Click PBA showed stronger retention towards glycopeptides under alkaline, higher content of organic solution conditions than under acidic, higher content of organic solution or alkaline aqueous solution conditions. Click PBA exhibited high selectivity for both neutral and acidic glycopeptides, which could resist interference from 100 molar fold of bovine serum albumin digests. The high enrichment efficiency can be ascribed to the synergetic effects of affinity interaction and hydrophilic interaction. The application of click PBA to 1 μL human serum resulted in the identification of 101 unique glycosylation sites from 71 glycoproteins. Such material will facilitate comprehensive glycoproteome analysis.
Keywords: Glycopeptide enrichment; Boronate affinity chromatography; Hydrophilic interaction chromatography; Mass spectrometry

It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment by Kathirvel Alagesan; Sana Khan Khilji; Daniel Kolarich (529-538).
Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed “Drop-HILIC”, which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical Abstract The organic solvent plays a crucial role in ZIC-HILIC glycopeptide enrichment
Keywords: Glycoproteomics; Glycopeptide enrichment; Hydrophilic interaction chromatography; HILIC; Glycopeptide synthesis

Growing evidence on the diverse biological roles of extracellular glycosylation as well as the need for quality control of protein pharmaceuticals make glycopeptide analysis both exciting and important again after a long hiatus. High-throughput O-glycosylation studies have to tackle the complexity of glycosylation as well as technical difficulties and, up to now, have yielded only limited results mostly from single enrichment experiments. In this study, we address the technical reproducibility of the characterization of the most prevalent O-glycosylation (mucin-type core 1 structures) in human serum, using a two-step lectin affinity-based workflow. Our results are based on automated glycopeptide identifications from higher-energy C-trap dissociation and electron transfer dissociation MS/MS data. Assignments meeting strict acceptance criteria served as the foundation for generating “spectral families” incorporating low-scoring MS/MS identifications, supported by accurate mass measurements and expected chromatographic retention times. We show that this approach helped to evaluate the reproducibility of the glycopeptide enrichment more reliably and also contributed to the expansion of the glycoform repertoire of already identified glycosylated sequences. The roadblocks hindering more in-depth investigations and quantitative analyses will also be discussed.
Keywords: O-Glycosylation; Electron transfer dissociation; LC-MS/MS; Reproducibility; Lectin affinity chromatography; Glycopeptides

Towards an automated analysis of bacterial peptidoglycan structure by Marshall Bern; Richard Beniston; Stéphane Mesnage (551-560).
Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3–3 and 4–3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3–3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical Abstract The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.
Keywords: Peptidoglycan; Cell wall; Muropeptides; Proteomics; Tandem mass spectrometry; Glycoproteomics; Cross-link

GlycoPep MassList: software to generate massive inclusion lists for glycopeptide analyses by Wenting Hu; Xiaomeng Su; Zhikai Zhu; Eden P. Go; Heather Desaire (561-570).
Protein glycosylation drives many biological processes and serves as markers for disease; therefore, the development of tools to study glycosylation is an essential and growing area of research. Mass spectrometry can be used to identify both the glycans of interest and the glycosylation sites to which those glycans are attached, when proteins are proteolytically digested and their glycopeptides are analyzed by a combination of high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) methods. One major challenge in these experiments is collecting the requisite MS/MS data. The digested glycopeptides are often present in complex mixtures and in low abundance, and the most commonly used approach to collect MS/MS data on these species is data-dependent acquisition (DDA), where only the most intense precursor ions trigger MS/MS. DDA results in limited glycopeptide coverage. Semi-targeted data acquisition is an alternative experimental approach that can alleviate this difficulty. However, due to the massive heterogeneity of glycopeptides, it is not obvious how to expediently generate inclusion lists for these types of analyses. To solve this problem, we developed the software tool GlycoPep MassList, which can be used to generate inclusion lists for liquid chromatography tandem-mass spectrometry (LC-MS/MS) experiments. The utility of the software was tested by conducting comparisons between semi-targeted and untargeted data-dependent analysis experiments on a variety of proteins, including IgG, a protein whose glycosylation must be characterized during its production as a biotherapeutic. When the GlycoPep MassList software was used to generate inclusion lists for LC-MS/MS experiments, more unique glycopeptides were selected for fragmentation. Generally, ∼30 % more unique glycopeptides can be analyzed per protein, in the simplest cases, with low background. In cases where background ions from proteins or other interferents are high, usage of an inclusion list is even more advantageous. The software is freely publically accessible. Graphical abstract Software increases the number of glycopeptides that get selected for MS/MS analysis.
Keywords: N-linked glycosylation; Glycopeptide analysis; Software tool; LC-MS/MS; Data-dependent acquisition

New mass spectrometry instrumentation, particularly those with electron transfer dissociation fragmentation, has made the analysis of complex glycopeptide mixtures accessible. However, software tools need to be optimized for interpretation of this type of data. Glycopeptide identification is challenging due to the number of different peptide and sugar moieties that can be combined, leading to a large number of potential compositions to consider. In this manuscript, different strategies for reducing the number of peptides and glycopeptides considered in database searching are compared. Adaptation of the software Protein Prospector to support the use of a reference modification site database doubled the number of glycopeptide IDs. The potential of this as an improved analysis strategy is discussed. Graphical abstract This manuscript compares the use of a restricted protein database based on a list of accession numbers of identified proteins to the use of a modification site database for intact glycopeptide analysis. It was found that the modification database is more effective for glycopeptide identification, particularly for larger glycopeptides
Keywords: Glycopeptides; Mass spectrometry; Glycopeptide data analysis; Protein Prospector; N-glycosylation; O-glycosylation

Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars by Christina M. Woo; Alejandra Felix; Lichao Zhang; Joshua E. Elias; Carolyn R. Bertozzi (579-588).
Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.
Keywords: Glycoproteomics; Chemical proteomics; LC-MS/MS; Metabolic labeling; Sialic acid

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM) by Jincui Huang; Muchena J. Kailemia; Elisha Goonatilleke; Evan A. Parker; Qiuting Hong; Rocchina Sabia; Jennifer T. Smilowitz; J. Bruce German; Carlito B. Lebrilla (589-606).
Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ
Keywords: Human milk; MRM; Glycoproteomics; UPLC; Mass spectrometry

In order to interpret glycopeptide tandem mass spectra, it is necessary to estimate the theoretical glycan compositions and peptide sequences, known as the search space. The simplest way to do this is to build a naïve search space from sets of glycan compositions from public databases and to assume that the target glycoprotein is pure. Often, however, purified glycoproteins contain co-purified glycoprotein contaminants that have the potential to confound assignment of tandem mass spectra based on naïve assumptions. In addition, there is increasing need to characterize glycopeptides from complex biological mixtures. Fortunately, liquid chromatography-mass spectrometry (LC-MS) methods for glycomics and proteomics are now mature and accessible. We demonstrate the value of using an informed search space built from measured glycomes and proteomes to define the search space for interpretation of glycoproteomics data. We show this using α-1-acid glycoprotein (AGP) mixed into a set of increasingly complex matrices. As the mixture complexity increases, the naïve search space balloons and the ability to assign glycopeptides with acceptable confidence diminishes. In addition, it is not possible to identify glycopeptides not foreseen as part of the naïve search space. A search space built from released glycan glycomics and proteomics data is smaller than its naïve counterpart while including the full range of proteins detected in the mixture. This maximizes the ability to assign glycopeptide tandem mass spectra with confidence. As the mixture complexity increases, the number of tandem mass spectra per glycopeptide precursor ion decreases, resulting in lower overall scores and reduced depth of coverage for the target glycoprotein. We suggest use of α-1-acid glycoprotein as a standard to gauge effectiveness of analytical methods and bioinformatics search parameters for glycoproteomics studies. Graphical Abstract Assignment of site specific glycosylation from LC-tandemMS data
Keywords: Glycoproteomics; Integrated-omics; Glycoinformatics; Mass spectrometry; Glycosylation; Glycomics; Alpha-1-acid glycoprotein

Cirrhosis of the liver is associated with increased fucosylation of proteins in the plasma. We describe a data-independent (DIA) strategy for comparative analysis of the site-specific glycoforms of plasma glycoproteins. A library of 161 glycoforms of 25 N-glycopeptides was established by data-dependent LC-MS/MS analysis of a tryptic digest of 14 human protein groups retained on a multiple affinity removal column. The collision-induced dissociation conditions were adjusted to maximize the yield of selective Y-ions which were quantified by a data-independent mass spectrometry workflow using a 10-Da acquisition window. Using this workflow, we quantified 125 glycoforms of 25 glycopeptides, covering 10 of the 14 proteins, without any further glycopeptide enrichment. Comparison of the proteins in the plasma of healthy controls and cirrhotic patients shows an average 1.5-fold increase in the fucosylation of bi-antennary glycoforms and 3-fold increase in the fucosylation of tri- and tetra- antennary glycoforms. These results show that the adjusted glycopeptide DIA workflow using soft collision-induced fragmentation of glycopeptides is suitable for site-specific analysis of protein glycosylation in complex mixtures of analytes without glycopeptide enrichment.
Keywords: Data-independent analysis; N-glycopeptide; Fucosylation; GP-SWATH