Synthesis of hydrazide-functionalized hydrophilic polymer hybrid graphene oxide for highly efficient N-glycopeptide enrichment and identification by mass spectrometry.

Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N-glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N-glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N-glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N-glycosylation sites and 480 N-glycopeptides in 232 N-glycoproteins were enriched and identified from 10µL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N-glycosylation sites, 261 N-glycopeptides and 144 N-glycoproteins in three experiments). Among the identified, 95 N-glycosylation sites were not reported in the Uniprot database, and 106 N-glycoproteins were disease related in the Nextprot database, indicating the potential of this new enrichment material in global mapping of protein N-glycosylation.

A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification.

O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine.

[Synthesis of core-shell hydrophilic polymer-silica hybrid material and its application in N-glycan enrichment].

Protein N-glycosylation is one of the most important post-translational modifications closely correlated with many important biological and pathological processes. The structural alterations of N-linked glycans in glycoproteins are always associated with many diseases, such as diabetes, heart failure and malignant tumors. Therefore, it is very important to establish sensitive methods for high-throughput N-glycan profiling. However, the low abundance of the N-glycoproteins and the heterogeneity of the N-glycans make it a challenge to analyse the protein glycosylation sensitively. In this work, we had synthesized core-shell hydrophilic polymer-silica hybrid materials (pGMAG-SiO2) for the efficient enrichment of protein N-glycans. Firstly, pGMAG-SiO2 was prepared by in situ growth of glucose polymer on the surface of silica microparticles using surface-initiated atom transfer radical polymerization (SI-ATRP) technique. The strong hydrophilicity of the material makes it suitable for the enrichment of N-glycans released from complex samples. Secondly, maltoheptaose and the N-glycans from chicken egg albumin were used as standard samples to optimize the enrichment conditions and evaluate the enrichment efficiency of pGMAG-SiO2. Finally, pGMAG-SiO2 was applied to the enrichment of N-linked glycans from human plasma proteins and 47 glycoforms were successfully identified after enrichment. These results demonstrated the high enrichment efficiency and significant application value of pGMAG-SiO2 in the analysis of N-glycans.

Development and control of a lower extremity assistive device (LEAD) for gait rehabilitation.

This research developed a wearable lower extremity assistive device intended to aid stroke patient during rehabilitation. The device specifically aims to assist the patient in sit-to-stand, stand-to-sit, and level-walking tasks in order to promote active gait rehabilitation exercises. The device adopts an anthropomorphic structure with hip and knee joint actuated in sagittal plane. A finite state machine strategy was proposed to control the device. At different states, appropriate assist torque is added to each joint. EMG signals are used to assess the assist performance. Tests on an able-bodied subject show that the device could successfully detect and transit between states. In sit-to-stand tasks, the integrated EMG (iEMG) of the Vastus Medialis for standing up with 11 Nm of assistance torque were found to be significantly less (P = 0.00187) than the iEMG of without assistance for standing up which indicate reduced muscle effort with the device assistance. Results show the device could potentially assist stroke patient in similar tasks.

A novel Ta.AGP.S.1b transcript in Chinese common wheat (Triticum aestivum L.).

ADP-glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis in plants, is composed of two small and two large subunits, and has plastidial and cytosolic isoforms. In kernels of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), transcripts for cytosolic (Ta.AGP.S1a) and plastidial (Ta.AGP.S1b) small subunits of AGPase were encoded by the same gene (Ta.AGP.S.1) by use of the alternative first exons. In this study, a cDNA sequence (1631 bp) [NCBI: EU586278] encoding a novel Ta.AGP.S1b transcript was isolated in kernels of Chinese common wheat cultivars. Compared with another Ta.AGP.S1b transcript [NCBI: FJ643609] isolated in kernels of non-Chinese wheat cultivars, EU586278 lacked a long fragment (117 bp) at its 5'terminal, resulting in a shorten transit peptide. The lacked fragments of Ta.AGP.S1b (EU586278) were universally found in surveyed 22 Chinese common wheat cultivars. Partial genomic DNA sequence [NCBI: FJ907395] of Ta.AGP.S.1 gene, which was corresponded to 5'terminal of EU586278 transcript, was also isolated in Chinese common cultivars and sequencing indicated that FJ907395 contained the corresponding lacked fragment of EU586278 transcript, inferring the lacked fragment in EU586278 transcript was not present in the genome, but possibly occurred at transcription level. Using TargetP software, the predicated transit peptide of putative plastidial SSU encoded by EU586278 contained merely 25 amino acids, considerably shorter than those of other plant AGP. S.1bs (54-70 amino acids). Phylogenetic tree analysis indicated that the amino acid sequence of EU586278 transit peptide was not clustered together with those of other wheat Ta.AGP.S1bs [NCBI: AF536819 and FJ643609] and barley AGP.S1b [NCBI: Z48563]. These implied that EU586278 could be a novel Ta.AGP.S1b transcript. Semi-quantitative PCR analysis indicated that transcripts of EU586278 were abundantly expressed in leaf, moderately in endosperm and stem, and weakly in root.