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Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis Benson C. Selvanesan1,2, Sheelu Varghese1,2, Justyna Andrys5, Ricardo H. Arriaza6, Rahul Prakash6, Purushottam B Tiwari7, Cara Olsen8, Daniel Hupalo2,4, Yuriy Gusev5, Megha N. Patel6, Sara Contente1, Miloslav Sanda9, Aykut Uren7, Matthew D. Wilkerson3,4, Clifton L. Dalgard3,4, Linda S. Shimizu6, Maksymilian Chruszcz6, Tomasz Borowski5, Geeta Upadhyay 1,3,7. Affiliations 1 Department of Pathology, 2 Henry M. Jackson Foundation, 3 Murtha Cancer Center, 4 Department of Anatomy, Physiology, and Genetics 8 Department of Preventive Medicine and Biostatistics Uniformed Services University of the Health Sciences, Bethesda, MD, USA. 5 Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Cracow, Poland. 6 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA. 7 Department of Oncology, Georgetown University Medical Center, Washington, DC, USA. 9 Max Planck Institute for Heart and Lung Research, Ludwigstrasse, 43, 61231 Bad Nauheim, Germany. Correspond Disclaimer The opinions expressed herein are those of the authors and are not necessarily representative of the official policies of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD), the United States Army/Navy/Air Force, the U.S. Government, or any other funding agencies Conflict of Interest None Acknowledgments NIH, NCI, R01 CA227694. NIH, NCI, R21CA256424. DOD, USUHS, VPR-NFP-74-9824. Biomedical Instrumentation Center, USUHS. The American Genome Center, USUHS. Antibody Characterization Program, Clinical Proteomics Tumor Analysis Consortium (CPTAC), National Cancer Institute, National Institute of Health. The Polish Grid Infrastructure, Cracow, Poland. NIH P30CA51008 and 1S10OD019982-01 to Biacore Molecular Interaction Shared Resource (BMISR), Georgetown University. ABSTRACT Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including triple-negative and estrogen receptor-positive breast, ovarian, gastric, head and neck, neuroblastoma, bladder, and lung cancers. Inhibition of LY6K signaling is an ideal therapeutic approach for cancer, since the LY6K protein is not involved in vital organ function. Previously, we identified the small molecule NSC243928 as a binder of LY6K using surface plasmon resonance screening and showed that its activity was dependent on LY6K expression in triple-negative breast cancer cells. Here, we demonstrate the structural basis of the molecular interaction of NSC243928 with LY6K protein and the subsequent inhibition of LY6K function in mitosis and cell division via Aurora B-histone pathway. We observed that LY6K interacts with phosphorylated histones and Aurora B kinases during mitosis and that this interaction was disrupted in the presence of NSC243928. Disruption of LY6K function in mitosis/cytokinesis leads to DNA damage, senescence, and apoptosis of cancer cells. We observed that NSC243928 led to increased binding of LY6K to phosphorylated gammaH2X at S139, which was dependent on NSC243928 interaction with LY6K on phenylalanine 79. Furthermore, we observed increased levels of phosphorylated gammaH2X at S139 and increased caspase-3 activation in the tumor isografts of 4T1 and E0771 mammary tumors treated with NSC243928. These data reveal that LY6K is a novel cell cycle target for therapeutic development in triple-negative breast cancer and other solid cancers with high expression of LY6K, such as bladder cancer, head and neck, and lung cancer. Citation Format: Benson Selvanesan, Sheelu Varghese, Justyna Andrys, Ricardo Arriaza, Rahul Prakash, Purushottam Tiwari, Cara Olsen, Daniel Huplo, yuriy Gusev, Megha Patel, Sara Contente, Miloslav Sanda, Matthew Wilkerson, Clifton Dalgard, Linda S. Shimizu, Maksymilian Chruszcz, Tomasz Borowski, Geeta Upadhyay. Pharmacological inhibition of LY6K induced cell cycle arrest and DNA damage by disrupting the LY6K-Histone-Aurora B signaling axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-04.

Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties, however, specificity of these fragments has never been examined closely. Here, we investigated fragmentation specificity using synthetic stable isotope-labelled glycopeptide standards. These standards were isotopically labelled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false positive structure assignments due to the occurrence of “Ghost” fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent the misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.Graphical abstract

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Supplementary Table S1. Patient Characteristics for patients from the University of California at San Diego; Supplementary Table S2. Fitness of algorithms; Supplementary Table S3. Cross validations of potential models; Supplementary Table S4. Statistical inference of comparing models; Supplementary Figure S1. Scheme of study design;Supplementary Figure S2. Scatter plot; Supplementary Figure S3. AUROC for the individual components analyzed; Supplementary Figure S4. MALDI-TOF analysis of low molecular weight kininogen; Supplementary Figure S5. Glycopeptide analysis of tryptic glycopeptide 44-58; Supplementary Figure S6. Glycopeptide analysis of tryptic glycopeptide 197-208 showing identification of fucosylated glycopeptides; Supplementary Figure S7. Glycopeptide analysis of tryptic glycopeptide 289-300

Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States related to low rates of early tumor detection. Development of noninvasive biomarkers for the early detection of HCC may reduce HCC-related mortality.Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of >3,000 patients from 5 independent sites improved detection of HCC as compared with the currently used biomarker, alpha-fetoprotein (AFP), by 4% to 20%. However, this algorithm had limited benefit in those with AFP Results: The ability to detect early-stage AFP-negative (AFP Conclusions: An algorithm combining fucosylated kininogen, AFP, and clinical characteristics is highly accurate for early HCC detection.Impact: Our biomarker algorithm could significantly improve early HCC detection and curative treatment eligibility in patients with cirrhosis. Cancer Epidemiol Biomarkers Prev; 26(5); 795–803. ©2017 AACR.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N - and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics., This analysis presents the results of a community-based evaluation of existing software for large-scale glycopeptide data analysis.

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this paper, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft collision induced dissociation (CID) conditions which reduces the CVs of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intra-scan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow as well as the CID workflow with the highest sensitivity compared to traditional workflows., This was exemplified by a rapid quantification (13-minute) of IgG1 Fc glycoforms from COVID-19 patients.Graphic Abstract

Hematopoietic acute radiation syndrome (H-ARS) and delayed effects of acute radiation exposure (DEARE) are detrimental health effects that occur after exposure to high doses of ionizing radiation. BIO 300, a synthetic genistein nanosuspension, was previously proven safe and effective against H-ARS when administered (via the oral (po) or intramuscular (im) route) prior to exposure to lethal doses of total-body radiation. In this study, we evaluated the proteomic changes in serum of nonhuman primates (NHP) after administering BIO 300 by different routes (po and im). We utilized nanoflow-ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (NanoUPLC-MS/MS) methods for comprehensive global profiling and quantification of serum proteins. The results corroborate previous findings that suggest a very similar metabolic profile following both routes of drug administration. Furthermore, we observed minor alterations in protein levels, 2 hours after drug administration, which relates to the Cmax of BIO 300 for both routes of administration. Taken together, this assessment may provide an insight into the mechanism of radioprotection of BIO 300 and a reasonable illustration of the pharmacodynamics of this radiation countermeasure.

Introduction N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. Methods Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. Results While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. Conclusions Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

Brain-derived neurotrophic factor (BDNF) is generated by proteolytic cleavage of a prodomain from the proBDNF precursor either intracellularly by furin-like proteases or extracellularly by plasmin or matrix metalloproteinases. ProBDNF carries a single N-glycosylation sequon (Asn-127) that remains virtually unstudied despite being located in a highly conserved region proximal to the proteolytic site. To study the proBDNF structure and function, here we expressed the protein and its nonglycosylated N127Q mutant in HEK293F cells. We found that mutation of the Asn-127 prevents intracellular maturation and secretion, an effect reproduced in WT proBDNF by tunicamycin-induced inhibition of N-glycosylation. Absence of the N-glycan did not affect the kinetics of proBDNF cleavage by furin in vitro, indicating that effects other than a direct furin–proBDNF interaction may regulate proBDNF maturation. Using an optimized LC-MS/MS workflow, we demonstrate that secreted proBDNF is fully glycosylated and carries rare N-glycans terminated by GalNAcβ1–4GlcNAcβ1-R (LacdiNAc) extensively modified by terminal sulfation. We and others noted that this type of glycosylation is protein-specific, extends to proBDNF expressed in PC12 cells, and implies the presence of interacting partners that recognize this glycan epitope. The findings of our study reveal that proBDNF carries an unusual type of N-glycans important for its processing and secretion. Our results open new opportunities for functional studies of these protein glycoforms in different cells and tissues.

Quantitative analysis of site specific glycoforms of proteins is technically challenging but highly desirable; resolution of the fucosylated glycoforms is of particular interest due to their biological importance. In this study, we developed a sensitive and specific LC-MS-MRM quantification method that distinguishes the outer arm and core fucosylated configurations of the N-glycopeptides. We take advantage of limited fragmentation of the glycopeptides at low collision energy CID to produce linkage-specific Y-ions. We select these informative ions as MRM transitions for the quantification of the outer arm and total fucosylation of 12 fucosylated glycoforms of 9 glycopeptides in 7 plasma proteins. Our workflow showed improved sensitivity and specificity of quantification of the glycopeptides compared to oxonium ion transitions which allowed us to quantify the glycoforms directly in plasma or serum without fractionation of the samples or glycopeptide enrichment. A pilot study of fucosylation in liver cirrhosis of the HCV and NASH etiologies confirms the quantitative capabilities of the method and shows that liver cirrhosis is consistently associated with increased outer arm fucosylation of majority of the analyzed proteins. The results show that the outer arm fucosylation of the A2G2F1 glycoform of the VDKDLQSLEDILHQVENK peptide of fibrinogen increases greater than 10-fold in the HCV and NASH patients compared to healthy controls.

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project – Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance forN- andO-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved “high coverage” and “high accuracy” glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.

Immune checkpoint inhibitors, including PD-L1/PD-1, are key regulators of immune response and promising targets in cancer immunotherapy. N-glycosylation of PD-L1 affects its interaction with PD-1 but little is known about the distribution of glycoforms at its four NXS/T sequons. We optimized LC-MS/MS methods using collision energy modulation for the site-specific resolution of specific glycan motifs. Using these methods, we demonstrate that PD-L1 expressed on the surface of breast cancer cells carries mostly complex glycans with high proportion of polyLacNAc structures at the N219 sequon. PD-L1 from whole cell lysate contained, in addition, large proportion of high mannose glycans at all sites. Contrary to the full-length protein, the secreted form of PD-L1 expressed in breast cancer or HEK293 cells demonstrated minimum N219 occupancy and low contribution of the polyLacNAc structures. Molecular modeling of PD-L1/PD-1 interaction with N-glycans suggests that glycans at the N219 site of PD-L1 and N74 and N116 of PD-1 are involved in glycan-glycan interactions, but the impact of this potential interaction on the protein function remains at this point unknown. In addition, the interaction of PD-L1 with clinical antibodies is also affected by glycosylation. In conclusion, our study demonstrates that PD-L1 expressed in the MDA-MB-231 breast cancer cells carries polyLacNAc glycans mostly at the N219 sequon which displays the highest variability in occupancy and is most likely to directly influence the interaction with PD-1.

Carbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.

Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage site that differs from the SARS-CoV spike protein of 2002. By analysis of the protein produced in HEK293 cells, we observe that the spike is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and confirmed that the spike protein is heavily N-glycosylated. Our recently developed liquid chromatography–mass spectrometry methodology allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of O-glycosylation on its proteolytic activation.

Glycosylation is a major post-translational modification of proteins that regulates many biological processes including protein folding, structure stability, receptor activation, and immune responses. The glycans attached to proteins represent an important determinant of the protein interaction-specificity and maintain the 3D structure of proteins. Mass spectrometry (MS) is one of the most efficient tools used in the current studies of glycoproteins and structure of their glycoforms. Collision energy (CE) is a crucial instrument parameter that can be exploited to improve structural resolution because different linkages of glycan units show different stabilities under CID/HCD fragmentation. Here we report the utility of CE modulation for qualitative and quantitative analysis of site- and structure-specific glycoforms of proteins. Using CE modulation, we were able to break selectively specific glycan linkages on intact glycopeptides and get, to some degree, structure-specific mass spectrometric signals. Structure- and CE-specific oxonium ions provide sufficient information for the resolution of outer arm structure motifs with recognized biological functions. The complementary Y-ions, generated under optimized low CE (soft) conditions, provide additional structural information including features specific to the chitobiose core. This methodology of multiple CE fragmentation without merging spectral information can significantly improve confidence of glycopeptide identification and structural resolution by providing additional information to the established glycopeptide-search algorithms and tools.

N-Linked glycopeptides have highly diverse structures in nature. Herein, we describe the first synthesis of rare multi-antennary N-glycan bearing glycan chains on 6-OH of both α1,6- and α1,3-linked mannose arms. To expedite divergent generation of N-glycan structures, four orthogonal protective groups were installed at the branching points on the core tetrasaccharide, which could be removed individually without affecting one another. In addition, the synthetic route is flexible, allowing a bisecting glucosamine moiety to be introduced at a late stage of the synthesis, further expanding the diversity of sequences that could be achieved. The bisecting glucosamine unit significantly reduced the glycosylation yields of adjacent mannoses, which was attributed to steric hindrance imposed by the glucosamine based on molecular modelling analysis. The N-glycans were then transformed to oxazoline donors and ligated with a glycopeptide acceptor from haptoglobin promoted by the wild type Arthrobacter endo-β-N-acetylglucosaminidase (Endo-A). Endo-A exhibited interesting substrate preferences depending on donor sizes, which was rationalized through molecular dynamics studies. This is the first time that a glycopeptide bearing a bisecting N-acetyl glucosamine (GlcNAc), the rare N-glycan branch, and two LewisX trisaccharide antennae was synthesized, enabling access to this class of complex glycopeptide structures.

Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of liquid chromatography with tandem mass spectrometry is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin and sex hormone binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times (RRT). These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides. This article is protected by copyright. All rights reserved

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bi-sialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer. Significance The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data.

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.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a ∼5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Sex-hormone-binding globulin (SHBG) is a liver-secreted glycoprotein and a major regulator of steroid distribution. It has been reported that the serum concentration of SHBG changes in liver disease. To explore the involvement of SHBG in liver disease of different etiologies in greater detail, we developed a sensitive and selective liquid chromatography–mass spectrometry parallel reaction monitoring workflow to achieve quantitative analysis of SHBG glycosylation micro heterogeneity. The method uses energy-optimized “soft” fragmentation to extract informative Y ions for maximal coverage of glycoforms and their quantitative comparisons. A total of 15 N-glycoforms of two N-glycosites and 3 O-glycoforms of 1 O-glycosite of this low-abundance serum protein were simultaneously analyzed in the complex samples. At the same time, we were able to partially resolve linkage isoforms of the fucosylated glycoforms and to identify and quantify SHBG N-glycoforms that were not previously reported. The results show that both core and outer-arm fucosylation of the N-glycoforms increases with liver cirrhosis but that a further increase of fucosylation is not observed with hepatocellular carcinoma (HCC). In contrast, the α-2–6 sialylated glycoform of the O-glycopeptide of SHBG increases in liver cirrhosis, and a significant 2-fold further increase is observed in HCC. In general, we do not find a significant contribution of different liver disease etiologies to the observed changes in glycosylation; however, elevation of the newly reported HexNAc(4)Hex(6) N-glycoform is associated with alcoholic liver disease.

The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO® penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer arm linked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via α2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.

Treatment of infections caused by pathogenic bacteria is still harder. Due to increasing number of microbial species resistant against so far invented antibiotics. This presents great problem for public health. One of the potential solutions seems to be antimicrobial peptides. Those peptides are synthetized in all organisms as a part of innate immunity with rapid mode of antimicrobial action. Lot of them have been isolated from bacteria, plants, insects and mammals as well. Our project was aimed on finding such peptides in plant extracts, respectively in leaves of Brassica napus (canola), Solanum lycopersicum (tomato) and Tetragonia tetragonioides (New Zealand spinach). We used several separation techniques to obtain fractions containing compounds of peptide nature with hydrophobic character. Antimicrobial activity of these fractions was tested against several gram-positive and gram-negative bacteria. Mass spectrometry analysis of antimicrobial active fractions proved presence of low molecular peptides with molecular masses 1.9 - 4.9 kDa and a partial amino acid sequence in hydrophobic part of Tetragonia extract. In hydrophilic fraction of the Solanum extract with proved antibacterial activity two patogenesis-related proteins with antifungal activity NP24 and TPM-1 were detected.

Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. BIOLOGICAL SIGNIFICANCE: We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease.

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC–MS–MRM assay to quantify glycoforms of IgG subclasses 1–4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. An increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC–MS–MRM method for the quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC. Biological significance We have demonstrated that both quantities and glycoforms of immunoglobulin subclasses change significantly in liver disease progression to HCC through quantitative study of immunoglobulin subclasses and their site specific glycoforms using a sensitive and selective LC–MS–MRM method. Redistribution of the glycoforms of specific immunoglobulin subclasses could have important implications for receptor mediated responses affecting the progression of liver disease.

Here we report the identification of protein targets of chemopreventive phenethyl isothiocyanate (PEITC) via “click” chemistry in the A549 human lung cancer cell line, using a novel alkyne-tagged PEITC which was also found to show potent in vitro anticancer activity.

Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP- LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.

Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC-MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15-20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains.

Quantitative analysis of site-specific glycosylation of proteins is a challenging part of glycoproteomic research. Multiple enrichment steps are typically required in the analytical workflows to achieve adequate characterization of the site-specific glycoforms. In spite of recent advances, quantitative workflows need further development. Here, we report a selective and sensitive MS2 followed by further fragmentation in the linear IT-MS analyzer (MS3) multiple reaction monitoring workflow mass spectrometric method for direct analysis of O-glycopeptides in difficult matrix such as serum. Method optimization was performed using two serum glycoproteins, hemopexin (HPX) and sex hormone binding globulin. With the optimized MS3 workflow, we were able to analyze major glycoforms of HPX directly in human serum. Quantification of the minor glycoforms of HPX and glycoforms of sex hormone binding globulin required enrichment of the protein because these analytes were below the sensitivity of the 4000 quadrupole ion trap hybrid mass spectrometer in the complex serum background. In conclusion, we present a quantitative method for site-specific analysis of O-glycosylation with general applicability to mucin-type glycoproteins. Our results document reliable application of the optimized MS3 multiple reaction monitoring workflow to the relative quantification of O-glycosylation microheterogeneity of HPX in human serum. Introduction of isotopically labeled standards would be desirable to achieve absolute quantification of the analytes. The possibility to analyze serum samples directly represents a significant improvement of the quantitative glycopeptide workflows with the potential for use in clinical applications.

Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States related to low rates of early tumor detection. Development of noninvasive biomarkers for the early detection of HCC may reduce HCC-related mortality. Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of >3,000 patients from 5 independent sites improved detection of HCC as compared with the currently used biomarker, alpha-fetoprotein (AFP), by 4% to 20%. However, this algorithm had limited benefit in those with AFP Results: The ability to detect early-stage AFP-negative (AFP Conclusions: An algorithm combining fucosylated kininogen, AFP, and clinical characteristics is highly accurate for early HCC detection. Impact: Our biomarker algorithm could significantly improve early HCC detection and curative treatment eligibility in patients with cirrhosis. Cancer Epidemiol Biomarkers Prev; 26(5); 795–803. ©2017 AACR.

Glycosylation regulates functional responses mediated by the interaction of IgG with their receptors. Multiple analytical methods have been designed for the determination of the IgG N-glycan microheterogeneity, including MS methods for the analysis of site specific glycoforms of IgG. However, measurement of low abundant glycoforms remains challenging in complex samples like serum without enrichment of the IgG. We present a workflow for quantitative analysis of site specific glycoforms of IgG based on data independent acquisition (DIA) of Y-ions generated under “minimal” fragmentation conditions. The adjusted collision induced dissociation (CID) conditions generate specific Y-ions in the yield of up to 60% precursor ion intensity. These selective fragments, measured in high resolution, improve specificity of detection compared to the typically quantified B-ions which have higher overall intensity but lower signal-to-noise ratios. Under optimized conditions, we achieve label-free quantification of the majority of previously reported glycoforms of IgG (26 glycoforms of IgG1, 22 glycoforms of IgG 2/3, and 19 glycoforms of IgG4) directly in unfractionated samples of human plasma and we detect traces of previously unreported glycoforms of IgG1, including doubly fucosylated glycoforms. The SWATH data independent quantification of IgG glycoforms in pooled plasma samples of patients with liver cirrhosis detects reliably the expected changes in the quantity of major glycoforms compared to healthy controls. Our results show that optimized CID fragmentation enables DIA of IgG glycoforms and suggest that such workflow may enable quantitative analyses of the glycoproteome in complex matrixes.

Five flavin (isoalloxazine) and alloxazine adducts with O-nucleophiles, 5-ethyl-4a-hydroxy-3,7,8,10-tetramethyl-4a,5-dihydroisoalloxazine (1a-OH), 5-ethyl-4a-hydroxy-3,10-dimethyl-4a,5-dihydroisoalloxazine (1b-OH), 5-ethyl-4a-methoxy-3,10-dimethyl-4a,5-dihydroisoalloxazine (1b-OMe), 5-ethyl-4a-hydroxy-1,3-dimethyl-4a,5-dihydroalloxazine (2a-OH) and 5-ethyl-4a-methoxy-1,3-dimethyl-4a,5-dihydroalloxazine (2a-OMe) were prepared from the corresponding salts, 5-ethyl-3,7,8,10-tetramethylisoalloxazinium (1a), 5-ethyl-3,10-dimethylisoalloxazinium (1b) and 5-ethyl-1,3-dimethylalloxazinium (2a) perchlorates by the addition of a nucleophile (water or methanol) and triethylamine as a base. The prepared adducts represent artificial analogs of flavin cofactor derivatives which are essential for the functioning of flavoenzymes. They were characterized by 1H and 13C NMR, HR-MS and UV–VIS spectra. In the cases of 1a-OH, 1b-OH, and 2a-OMe, the crystal structures were determined by X-ray diffraction. Flavinium and alloxazinium salts are in rapid equilibria with their adducts in water or methanolic solutions without the presence of a base. It was found that the equilibrium constants for flavin adduct formation is higher by six orders of magnitude than those for alloxazine derivatives. The presence of the sp3 hybridized C4a atom in the molecule of the adducts causes deviation from planarity. The interplanar angles between benzene and the pyrimidine ring were found to be 31.5°, 23.64° and 15.62° for 1a-OH, 1b-OH and 2a-OMe, respectively, which are much higher than those of previously published adducts of C-nucleophiles. In isoalloxazine adducts, delocalization of π electrons between the N10–C10a and C10a–N1 bonds was detected while the length of the N10–C10a and C10a–N1 bonds in the alloxazine adducts corresponds to a double and single bond, respectively.

A series of 3-substituted pyrazinium tetrafluoroborates was prepared as simple analogues of flavinium salts which are efficient organocatalysts for oxidations with hydrogen peroxide. It was shown that pyrazinium derivatives with an electron-withdrawing substituent catalyze mild oxidations of sulfides to sulfoxides and Baeyer-Villiger oxidations in a similar way to flavinium catalysts. The most reactive catalyst, 3-cyanopyrazinium tetrafluoroborate, was efficiently employed in preparative sulfoxidations of aromatic and aliphatic sulfides as well as in Baeyer-Villiger oxidations of cyclobutanones. A proposed mechanism for the catalysis is based on the formation of pyrazine hydroperoxide which is the agent oxidizing the substrate.

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic moieties on their amino group.

In the present work, we describe in detail an efficient solution synthesis of norleucine-derived phosphonopeptides mimicking the peptide sequences Nle-Gly(Ala) and Nle-Gly(Ala)-Val. The most efficient strategy involved use of the benzyl group. The synthesis was achieved through BOP-catalysed coupling of the monobenzyl ester of the N-Cbz-protected phosphonate derivative of norleucine with the hydroxyl moieties of derivatised l -lactic or glycolic acid. Subsequently, complete deprotection of the products was achieved in good yields by one-step Pd-catalysed hydrogenolysis. We also prepared the Fmoc-Nle-Ψ[PO(OH)O]-CH2-COOH synthon and demonstrated that this precursor is a suitable building block for the solid-phase synthesis of cysteine-containing phosphonopeptides.

In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and also with their ability to stimulate lipogenesis in vitro. We found that [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2 were very potent insulin analogues with respect to their binding affinities (214 and 465%, respectively, compared to that of human insulin), but they were significantly less potent than human insulin in vivo. Their full-length counterparts, [NMeHisB26]-insulin and [NMeAlaB26]-insulin, were less effective than human insulin with respect to binding affinity (10 and 21%, respectively) and in vivo activity, while [HisB26]-insulin exhibited properties similar to those of human insulin in all of the tests we carried out. The ability of selected analogues to stimulate lipogenesis in adipocytes was correlated with their biological potency in vivo. Taken together, our data suggest that the B26 residue and residues B26-B30 have ambiguous roles in binding affinity and in vivo activity. We hypothesize that our shortened analogues, [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2, have different modes of interaction with the insulin receptor compared with natural insulin and that these different modes of interaction result in a less effective metabolic response of the insulin receptor, despite the high binding potency of these analogues.

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide β-alanyl- L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of 125 I-CART(61–102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61–102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74–86) and CART(62–86) containing disulfide bridges kept partial binding potency of the original molecule with K i in 10 −5 and 10 −4 M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

Quantification of proteins by LC-MS/MS-MRM has become a standard method with broad projected clinical applicability. MRM quantification of protein modifications is, however, far less utilized, especially in the case of glycoproteins. This review summarizes current methods for quantitative analysis of protein glycosylation with a focus on MRM methods. We describe advantages of this quantitative approach, analytical parameters that need to be optimized to achieve reliable measurements, and point out the limitations. Differences between major classes of N- and O-glycopeptides are described and class-specific glycopeptide assays are demonstrated.

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of (18)O water. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

We present herein a straightforward synthesis of N -Fmoc-protected synthons derived from a phosphinic analogue of methionine. These precursors were used successfully for the solid-phase synthesis of methionine-mimic phosphinopeptides using BOP-catalyzed coupling without protection of the phosphoryl moiety. We also prepared a new type of pseudopeptide derived from a phosphinic analogue of norleucine with a –PO(OH)–CH 2 –COOR moiety.