Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry

Glycosylation is one of the most common post-translational modifications in proteins, existing in ∼50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites

Quantitative Serum Glycomics of Esophageal Adenocarcinoma, and Other Esophageal Disease Onsets

Aberrant glycosylation has been implicated in various types of cancers and changes in glycosylation may be associated with signaling pathways during malignant transformation. Glycomic profiling of blood serum, in which cancer cell proteins or their fragments with altered glycosylation patterns are shed, could reveal the altered glycosylation. We performed glycomic profiling of serum from patients with no known disease (N=18), patients with high grade dysplasia (HGD, N=11) and Barrett’s (N=5), and patients with esophageal adenocarcinoma (EAC, N=50) in an attempt to delineate distinct differences in glycosylation between these groups. The relative intensities of 98 features were significantly different among the disease onsets; 26 of these correspond to known glycan structures. The changes in the relative intensities of three of the known glycan structures predicted esophageal adenocarcinoma with 94% sensitivity and better than 60% specificity as determined by receiver operating characteristic (ROC) analysis. We have demonstrated that comparative glycomic profiling of EAC reveals a subset of glycans that can be selected as candidate biomarkers. These markers can differentiate disease-free from HGD, disease-free from EAC, and HGD from EAC. The clinical utility of these glycan biomarkers requires further validation

Glycomics-based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses.Singapore-MIT Alliance for Research and Technolog

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase

Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmissiondeficits in long-term potentiationand specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients

Progression of esophageal dysplasia to cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108637/1/nyas12523.pd

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N ‐linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross‐ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C−2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2‐aminobenzoic acid (2‐AA) enhanced signal abundance in negative‐ion mode. However, few cross‐ring fragments were observed for 2‐AA‐labeled oligosaccharides. The neutral label 2‐aminobenzamide (2‐AB) resulted in more cross‐ring cleavages compared with 2‐AA‐labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89573/1/elps_201100327_sm_SupplInfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/89573/2/3526_ftp.pd

Glycoproteomic markers of hepatocellular carcinoma‐mass spectrometry based approaches

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149557/1/mas21583_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149557/2/mas21583.pd

The use of biofluid markers to evaluate the consequences of sport-related subconcussive head impact exposure : a scoping review

This review was supported by the University of Stirling (no grant number applies). L.W. also received support as part of Framework 7 programme of the European Union (CENTER-TBI, Grant number: 602150–2). S.M. received research support from the Italian Ministry of Health (GR-2013–02354960).Background Amidst growing concern about the safety of sport-related repetitive subconcussive head impacts (RSHI), biofluid markers may provide sensitive, informative, and practical assessment of the effects of RSHI exposure. Objective This scoping review aimed to systematically examine the extent, nature, and quality of available evidence from studies investigating the effects of RSHI on biofluid markers, to identify gaps and to formulate guidelines to inform future research. Methods PRISMA extension for Scoping Reviews guidelines were adhered to. The protocol was pre-registered through publication. MEDLINE, Scopus, SPORTDiscus, CINAHL, PsycINFO, Cochrane Library, OpenGrey, and two clinical trial registries were searched (until March 30, 2022) using descriptors for subconcussive head impacts, biomarkers, and contact sports. Included studies were assessed for risk of bias and quality. Results Seventy-nine research publications were included in the review. Forty-nine studies assessed the acute effects, 23 semi-acute and 26 long-term effects of RSHI exposure. The most studied sports were American football, boxing, and soccer, and the most investigated markers were (in descending order): S100 calcium-binding protein beta (S100B), tau, neurofilament light (NfL), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), phosphorylated tau (p-tau), ubiquitin C-terminal hydrolase L1 (UCH-L1), and hormones. High or moderate bias was found in most studies, and marker-specific conclusions were subject to heterogeneous and limited evidence. Although the evidence is weak, some biofluid markers—such as NfL—appeared to show promise. More markedly, S100B was found to be problematic when evaluating the effects of RSHI in sport. Conclusion Considering the limitations of the evidence base revealed by this first review dedicated to systematically scoping the evidence of biofluid marker levels following RSHI exposure, the field is evidently still in its infancy. As a result, any recommendation and application is premature. Although some markers show promise for the assessment of brain health following RSHI exposure, future large standardized and better-controlled studies are needed to determine biofluid markers’ utility.Publisher PDFPeer reviewe