Analysis Of Glycopeptides Research Articles

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea.

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

High-throughput rat immunoglobulin G N-glycosylation profiling revealed subclass-specific changes associated with chronic stress

Immunoglobulin G (IgG) glycosylation corresponds well with immune system changes, so it can potentially be used as a biomarker for the consequences of chronic stress such as low-grade inflammation and enhanced immunosenescence in older animals. Here we present a high-throughput glycoproteomic workflow, including IgG enrichment, HILIC glycopeptide purification, and nano-LC-MS analysis of tryptic glycopeptides applied for the analysis of rat IgG. A cohort of 80 animals was exposed to seven stressors in a customized chronic stress protocol with blood and tissue sampling in three timepoints. Young female rats experienced an increase in agalactosylated glycoforms on IgG2a and IgG2c accompanied by a decrease in monogalactosylation. Among old females, increased galactosylation was observed in the IgG2b subclass, pointing to an anti-inflammatory activity of IgG. Additionally, IgG Fc N-glycosylation patterns in Sprague Dawley rats were analyzed, quantified, and reported for the first time. Our findings emphasize age-, sex- and subclass-dependent differences in IgG glycosylation related to chronic stress exposure, confirming the relevance of newly developed methods for further research in glycobiology of rodent immune response. SignificanceIn this study, we showed that a high-throughput streamlined methodology based on protein L 96-well monolithic plates for efficient rat IgG immunoaffinity enrichment from blood plasma, paired with appropriate tryptic glycopeptide preparation, HILIC-SPE enrichment, and nano-LC-MS methods was suitable for quick processing of large sample sets. We report a subclass-specific profiling and changes in rat IgG Fc galactosylation and adrenal gland immunohistochemistry of male and female animals exposed to a customized chronic stress protocol.

Dissociation of Biomolecules by an Intense Low-Energy Electron Beam in a High Sensitivity Time-of-Flight Mass Spectrometer

We report the progress on an electron-activated dissociation (EAD) device coupled to a quadrupole TOF mass spectrometer (QqTOF MS) developed in our group. This device features a new electron beam optics design allowing up to 100 times stronger electron currents in the reaction cell. The electron beam current reached the space-charge limit of 0.5 μA at near-zero electron kinetic energies. These advances enable fast and efficient dissociation of various analytes ranging from singly charged small molecules to multiply protonated proteins. Tunable electron energy provides access to different fragmentation regimes: ECD, hot ECD, and electron-impact excitation of ions from organics (EIEIO). The efficiency of the device was tested on a wide range of precursor charge states. The EAD device was installed in a QqTOF MS employing a novel trap-and-release strategy facilitating spatial mass focusing of ions at the center of the TOF accelerator. This technique increased the sensitivity 6-10 times and allows for the first time comprehensive structural lipidomics on an LC time scale. The system was evaluated for other compound classes such as intact proteins and glycopeptides. Application of hot ECD for the analysis of glycopeptides resulted in rich fragmentation with predominantly peptide backbone fragments; however, glycan fragments attributed to the ECD process were also observed. A standard small protein ubiquitin (8.6 kDa) was sequenced with 90% cleavage coverage at spectrum accumulation times of 100 ms and 98% at 800 ms. Comparable cleavage coverage for a medium-size protein (carbonic anhydrase: 29 kDa) could be achieved, albeit with longer accumulation times.

Bowl-like mesoporous polydopamine with size exclusion for highly selective recognition of endogenous glycopeptides

It has been confirmed that endogenous glycopeptide plays an important role in a variety of pathological and physiological processes. However, direct analysis of endogenous glycopeptide is still a great challenge owing to the low abundance of endogenous glycopeptides and the presence of a large number of interfering substances such as large-sized proteins and heteropeptides in complex biological sample. Herein, we reported a novel bowl-like mesoporous polydopamine nanoparticle modified by carrageenan (denoted as MPDA@PEI@CA) with strong hydrophilicity and size-exclusion effect for high specificity enrichment of endogenous glycopeptides. Thanks to the suitable pore channel structure as well as strong hydrophilic surface, the as-prepared MPDA@PEI@CA nanoparticles exhibited prominent performance in enrichment of N-linked glycopeptide with ultrahigh selectivity (1:5000 M ratio of horseradish peroxidase (HRP) digests/bovine serum albumin (BSA) digests), low detection limit (5 fmol μL−1), outstanding size-exclusion ability (1:1000 mass of HRP/BSA), and unique reusability (five times). 125 N-glycosylation sites of 134 glycopeptides from 65 glycoproteins were identified from 2 μL sample of human serum treated with the MPDA@PEI@CA nanoparticles, which manifested the ability to enrich endogenous N-linked glycopeptides from complex biological samples. These results indicated that the bowl-like MPDA@PEI@CA nanoparticles with novel structure prepared in this work had great potential for glycopeptidome analysis.

Effective Enrichment Strategy Using Boronic Acid-Functionalized Mesoporous Graphene-Silica Composites for Intact N- and O-Linked Glycopeptide Analysis in Human Serum.

The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO2-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.

Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.

Synthesis of zwitterionic dual-functional metal-organic framework nanocomposite with ultra-hydrophilicity for selective enrichment of glycopeptides

蛋白糖基化是生物体中普遍发生且重要的生物学过程,其参与多种分子生物学的功能和途径,是临床诊断重要的生物标志物。但是,糖肽因其丰度低、离子化效率低、糖链异质性等难点,使糖蛋白分析一直面临巨大的挑战。因此,研究合成了一种新型的两性离子双功能化纳米金(AuGC)修饰的超亲水性沸石咪唑骨架(ZIF-8)纳米复合材料(AuGC/ZIF-8),并建立了亲水相互作用色谱(HILIC)和基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)联用选择性富集糖肽的分析方法。谷胱甘肽和半胱氨酸双功能化的协同作用,使MOF具有超亲水性和低空间位阻,为糖肽选择性富集提供了更多的亲和位点。研究以辣根过氧化物酶(HRP)为模式糖蛋白,通过AuGC/ZIF-8富集后,MALDI-TOF MS分析。结果表明,AuGC/ZIF-8对HRP糖肽的富集能力高达250 μg/mg,且在与牛血清白蛋白(BSA)混合溶液中(HRP-BSA (1∶200,质量比))显示出对HRP糖肽的高选择性,以及极低含量下(0.3 ng/μL)的高灵敏度。因此,在复杂生物样品糖蛋白的富集分离中具有很大的应用潜力。

Profiling the proteoforms of urinary prostate-specific antigen by capillary electrophoresis – mass spectrometry

Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SignificanceThe detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

Quantitative clinical glycomics strategies: A guide for selecting the best analysis approach.

Glycans introduce complexity to the proteins to which they are attached. These modifications vary during the progression of many diseases; thus, they serve as potential biomarkers for disease diagnosis and prognosis. The immense structural diversity of glycans makes glycosylation analysis and quantitation difficult. Fortunately, recent advances in analytical techniques provide the opportunity to quantify even low-abundant glycopeptides and glycans derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Understanding the strengths and weaknesses of different quantitative glycomics analysis methods is important for selecting the best strategy to analyze glycosylation changes in any given set of clinical samples. To provide guidance towards selecting the proper approach, we discuss four widely used quantitative glycomics analysis platforms, including fluorescence-based analysis of released N-linked glycans and three different varieties of MS-based analysis: liquid chromatography (LC)-mass spectrometry (MS) analysis of glycopeptides, matrix-assisted laser desorption ionization-time of flight MS, and LC-ESI-MS analysis of released N-linked glycans. These methods' strengths and weaknesses are compared, particularly associated with the figures of merit that are important for clinical biomarker studies, including: the initial sample requirements, the methods' throughput, sample preparation time, the number of species identified, the methods' utility for isomer separation and structural characterization, method-related challenges associated with quantitation, repeatability, the expertise required, and the cost for each analysis. This review, therefore, provides unique guidance to researchers who endeavor to undertake a clinical glycomics analysis by offering insights on the available analysis technologies.

Application of magnetic solid phase extraction in separation and enrichment of glycoproteins and glycopeptides

The analysis of endogenous glycoproteins and glycopeptides in human body fluids is of great importance for screening and discovering disease biomarkers with clinical significance. However, the presence of interfering substances makes the direct quantitative detection of low-abundance glycoproteins and glycopeptides in human body fluids one of the great challenges in analytical chemistry. Magnetic solid phase extraction (MSPE) has the advantages of easy preparation, low cost and good magnetic responsiveness. Magnetic adsorbents are the core of MSPE technology, and magnetic adsorbents based on different functional materials are widely used in the quantitative analysis of glycoproteins and glycopeptides in human body fluids, making it possible to analyze glycoproteins and glycopeptides with low abundance as well as multiple types, which provides a technical platform for screening and evaluating glycoproteins and glycopeptides in body fluids as disease biomarkers. In this paper, we focus on the recent advances in the application of MSPE technology and magnetic adsorbents for the separation and enrichment of glycoproteins and glycopeptides in human body fluids, and the future trends and application prospects in this field are also presented.

Towards structure-focused glycoproteomics.

Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While diverse quantitative glycoproteomics strategies capable of mapping monosaccharide compositions of N- and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018–2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle diverse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.

Endolysosomal N-glycan processing is critical to attain the most active form of the enzyme acid alpha-glucosidase

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo, thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

Analysis of Monoclonal Antibody Glycopeptides by Capillary Electrophoresis-Mass Spectrometry Coupling (CE-MS).

Glycosylation is a crucial posttranslational modification (PTM) that might affect the safety and efficacy of monoclonal antibodies (mAbs). Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of mAbs. A bottom-up proteomic workflow is designed to provide detailed information about the glycosylation. In this chapter, we describe the validated experimental protocol applied for the characterization and relative quantification of mAbs N-glycosylation at the glycopeptide level.

Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development

AbstractMany cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Site-specific glycoproteomic analysis revealing increased core-fucosylation on FOLR1 enhances folate uptake capacity of HCC cells to promote EMT.

Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous.Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches.Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells.Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.

Peak Filtering, Peak Annotation, and Wildcard Search for Glycoproteomics

AbstractGlycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, e.g., HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges: finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra. Here, we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, e.g., at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, e.g., ± 0.01 Da. Also, new is glycan “wildcard” search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally, the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Risk Factors for Extensively Drug-Resistant Acinetobacter baumannii in Neonatal Patients

Abstract Introduction Extensively drug-resistant Acinetobacter baumannii infections (XDRABIs) are significant causes of mortality and morbidity in neonatal patients due to the treatment difficulties and limitations. In the present study, we aimed to evaluate the characteristics and risk factors for XDRABIs acquisition during hospitalization. Methods We investigated the risk factors of health care-associated infections (HC-AIs) caused by multidrug-resistant A. baumannii in the four neonatal units between January 2008 and December 2018. The patients and the control group's data and culture results were recorded and analyzed on a daily basis. Results A total of 70 patients infected with XDRABIs and 118 control patients were included in the study. Univariate analysis showed that prematurity, gestation age, birth weight, mechanical ventilation, parenteral nutrition, transfusion, use of glycopeptides, and aminoglycosides were significantly associated with XDRABIs. In the univariate analysis, gestation age, mechanical ventilation, transfusion, parenteral nutrition, use of glycopeptides, carbapenems, and aminoglycosides were significantly associated with mortality. In the multiple logistic regression analysis, gestation age (p = 0.034), transfusion (p < 0.001), and use of glycopeptides (p = 0.019) were determined to be independent predictors of mortality in newborn infants with XDRABIs (R-square = 0.546). Conclusion Determination of the risk factors, avoiding invasive procedures, rational use of antibiotics will lead to a decrease in the mortality and morbidity rates related to XDRABIs.

Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco.

Recent advances in methods for enrichment and mass spectrometric analysis of intact glycopeptides have produced large-scale glycoproteomics datasets, but interpreting this data remains challenging. We present MSFragger-Glyco, a glycoproteomics mode of the MSFragger search engine, for fast and sensitive identification of N- and O-linked glycopeptides and open glycan searches. Reanalysis of recent N-glycoproteomics data resulted in annotation of 80% more glycopeptide-spectrum matches (glycoPSMs) than previously reported. In published O-glycoproteomics data, our method more than doubled the number of glycoPSMs annotated when searching the same glycans as the original search and yielded 4–6-fold increases when expanding searches to include additional glycan compositions and other modifications. Expanded searches also revealed many sulfated and complex glycans that remained hidden to the original search. With greatly improved spectral annotation, coupled with the speed of index-based scoring, MSFragger-Glyco makes it possible to comprehensively interrogate glycoproteomics data and illuminate the many roles of glycosylation.