Unique Glycans in Synaptic Glycoproteins in Mouse Brain.

The synapse is an essential connection between neuronal cells in which the membrane and secreted glycoproteins regulate neurotransmission. The post-translational modifications of glycoproteins with carbohydrates, although essential for their functions as well as their specific localization, are not well understood. Oddly, whereas galactose addition to glycoproteins is required for neuronal functions, galactosylation is severely restricted for Asn-linked on N-glycans in the brain, and genetic evidence highlights the important roles of galactose in brain functions and development.

Large-Scale and Site-Specific Mapping of the Murine Brain -Glycoproteome with IMPa.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available -glycoprotease IMPa from for comprehensive -glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa.

Systematic analysis of the impact of phosphorylation and O-GlcNAcylation on protein subcellular localization.

The subcellular localization of proteins is critical for their functions in eukaryotic cells and is tightly correlated with protein modifications. Here, we comprehensively investigate the nuclear-cytoplasmic distributions of the phosphorylated, O-GlcNAcylated, and non-modified forms of proteins to dissect the correlation between protein distribution and modifications. Phosphorylated and O-GlcNAcylated proteins have overall higher nuclear distributions than non-modified ones.

Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma.

GD2-targeting immunotherapies have improved survival in children with neuroblastoma, yet on-target, off-tumor toxicities can occur and a subset of patients cease to respond. The majority of neuroblastoma patients who receive immunotherapy have been previously treated with cytotoxic chemotherapy, making it paramount to identify neuroblastoma-specific antigens that remain stable throughout standard treatment. Cell surface glycoproteomics performed on human-derived neuroblastoma tumors in mice following chemotherapy treatment identified protein tyrosine kinase 7 (PTK7) to be abundantly expressed.

Global quantification of newly synthesized proteins reveals cell type- and inhibitor-specific effects on protein synthesis inhibition.

Manipulation of protein synthesis is commonly applied to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms have been widely investigated and employed in bio-related research, but it is extraordinarily challenging to measure and evaluate the synthesis inhibition efficiencies of individual proteins by different inhibitors at the proteome level. Newly synthesized proteins are the immediate and direct products of protein synthesis, and thus their comprehensive quantification provides a unique opportunity to study protein inhibition.

Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer's Disease Brain.

Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS-based glycoproteomics.

Spatial and temporal proteomics reveals the distinct distributions and dynamics of O-GlcNAcylated proteins.

Protein O-GlcNAcylation plays critical roles in many cellular events, and its dysregulation is related to multiple diseases. Integrating bioorthogonal chemistry and multiplexed proteomics, we systematically and site specifically study the distributions and dynamics of protein O-GlcNAcylation in the nucleus and the cytoplasm of human cells. The results demonstrate that O-GlcNAcylated proteins with different functions have distinct distribution patterns.

Time-Resolved and Comprehensive Analysis of Surface Glycoproteins Reveals Distinct Responses of Monocytes and Macrophages to Bacterial Infection.

Glycoproteins on the surface of immune cells play extremely important roles in response to pathogens. Yet, a systematic and time-resolved investigation of surface glycoproteins during the immune response remains to be explored. Integrating selective enrichment of surface glycoproteins with multiplexed proteomics, we globally and site-specifically quantified the dynamics of surface glycoproteins on THP-1 monocytes and macrophages in response to bacterial infection and during the monocyte-to-macrophage differentiation.

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome.

The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome.

Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics.

The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity.

Systematic characterization of extracellular glycoproteins using mass spectrometry.

Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system-wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)-based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically.

Time-Resolved and Comprehensive Analysis of Surface Glycoproteins Reveals Distinct Responses of Monocytes and Macrophages to Bacterial Infection.

Glycoproteins on the surface of immune cells play extremely important roles in response to pathogens. Yet, a systematic and time-resolved investigation of surface glycoproteins during the immune response remains to be explored. Integrating selective enrichment of surface glycoproteins with multiplexed proteomics, we globally and site-specifically quantified the dynamics of surface glycoproteins on THP-1 monocytes and macrophages in response to bacterial infection and during the monocyte-to-macrophage differentiation.

Enhancing Comprehensive Analysis of Secreted Glycoproteins from Cultured Cells without Serum Starvation.

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches.

Effective Method for Accurate and Sensitive Quantitation of Rapid Changes of Newly Synthesized Proteins.

Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis.

Comprehensive Analysis of Protein Glycation Reveals Its Potential Impacts on Protein Degradation and Gene Expression in Human Cells.

Glycation as a type of non-enzymatic protein modification is related to aging and chronic diseases, especially diabetes. Global analysis of protein glycation will aid in a better understanding of its formation mechanism and biological significance. In this work, we comprehensively investigated protein glycation in human cells (HEK293T, Jurkat, and MCF7 cells).

Surface Glycoproteomic Analysis Reveals That Both Unique and Differential Expression of Surface Glycoproteins Determine the Cell Type.

Proteins on the cell surface are frequently glycosylated, and they are essential for cells. Surface glycoproteins regulate nearly every extracellular event, but compared with global analysis of proteins, comprehensive and site-specific analysis of surface glycoproteins is much more challenging and dramatically understudied. Here, combining metabolic labeling, click-chemistry and enzymatic reactions, and mass spectrometry-based proteomics, we globally characterized surface glycoproteins from eight popular types of human cells.

G-Quadruplexes in Human Ribosomal RNA.

rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans.

Global and site-specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry.

Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site-specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures.

Mass Spectrometry-Based Chemical and Enzymatic Methods for Global Analysis of Protein Glycosylation.

Glycosylation is one of the most common protein modifications, and it is essential for mammalian cell survival. It often determines protein folding and trafficking, and regulates nearly every extracellular activity, including cell-cell communication and cell-matrix interactions. Aberrant protein glycosylation events are hallmarks of human diseases such as cancer and infectious diseases.

A genome-inspired, reverse selection approach to aptamer discovery.

Limitations of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and related methods that depend upon combinatorial oligonucleotide libraries have hindered progress in this area. Our laboratory has introduced a new approach to aptamer discovery that uses oligonucleotides with sequences drawn from the human genome to capture proteins from biological samples. Specifically, we have focused on capture of proteins in nuclear extracts from human cell lines using G-quadruplex (G4) forming genomic sequences.

Evaluation and optimization of reduction and alkylation methods to maximize peptide identification with MS-based proteomics.

Mass spectrometry (MS) has become an increasingly important technique to analyze proteins. In popular bottom-up MS-based proteomics, reduction and alkylation are routine steps to facilitate peptide identification. However, incomplete reactions and side reactions may occur, which compromise the experimental results.