Johanna M. Smeekens

A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by Mass Spectrometry (MS)

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.

Comprehensive Analysis of Protein N-Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS

Glycosylation is one of the most important protein modifications in biological systems. It plays a critical role in protein folding, trafficking, and stability as well as cellular events such as immune response and cell-to-cell communication. Aberrant protein glycosylation is correlated with several diseases including diabetes, cancer, and infectious diseases. The heterogeneity of glycans makes comprehensive identification of protein glycosylation sites very difficult by MS because it is challenging to match mass spectra to peptides that contain different types of unknown glycans. We combined a chemical deglycosylation method with LC-MS-based proteomics techniques to comprehensively identify protein N-glycosylation sites in yeast. On the basis of the differences in chemical properties between the amide bond of the N-linkage and the glycosidic bond of the O-linkage of sugars, O-linked sugars were removed and only the innermost N-linked GlcNAc remained, which served as a mass tag for MS analysis. This chemical deglycosylation method allowed for the identification of 555 protein N-glycosylation sites in yeast by LC-MS, which is 46% more than those obtained from the parallel experiments using the Endo H cleavage method. A total of 250 glycoproteins were identified, including 184 membrane proteins. This method can be extensively used for other biological samples.

Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

AbstractCell surface N-glycoproteins play extraordinarily important roles in cell–cell communication, cell–matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide-N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies. Graphical Abstractᅟ

Systematic investigation of cellular response and pleiotropic effects in atorvastatin-treated liver cells by MS-based proteomics.

For decades, statins have been widely used to lower cholesterol levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is well-known that statins have pleiotropic effects including improving endothelial function and inhibiting vascular inflammation and oxidation. However, the cellular responses to statins and corresponding pleiotropic effects are largely unknown at the proteome level. Emerging mass spectrometry-based proteomics provides a unique opportunity to systemically investigate protein and phosphoprotein abundance changes as a result of statin treatment. Many lipid-related protein abundances were increased in HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE, and LDLR, while the abundances of proteins involved in cellular response to stress and apoptosis were decreased. Comprehensive analysis of protein phosphorylation demonstrated that several basic motifs were enriched among down-regulated phosphorylation sites, which indicates that kinases with preference for these motifs, such as protein kinase A and protein kinase C, have attenuated activities. Phosphopeptides on a group of G-protein modulators were up-regulated, which strongly suggests that cell signal rewiring was a result of the effect of protein lipidation by the statin. This work provides a global view of liver cell responses to atorvastatin at the proteome and phosphoproteome levels, which provides insight into the pleiotropic effects of statins.

Yeast rRNA Expansion Segments: Folding and Function

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome. Here, we characterize the binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7), which is the largest and most variable ES of the eukaryotic large ribosomal subunit and is located at the surface of the ribosome. In vitro RNA-protein pull-down experiments using ES7 as a bait indicate that ES7 is a binding hub for a variety of non-ribosomal proteins essential to ribosomal function in eukaryotes. ES7-associated proteins observed here cluster into four groups based on biological process, (i) response to abiotic stimulus (e.g., response to external changes in temperature, pH, oxygen level, etc.), (ii) ribosomal large subunit biogenesis, (iii) protein transport and localization, and (iv) transcription elongation. Seven synthetases, Ala-, Arg-, Asp-, Asn-, Leu-, Lys- and TyrRS, appear to associate with ES7. Affinities of AspRS, TyrRS and LysRS for ES7 were confirmed by in vitro binding assays. The results suggest that ES7 in S. cerevisiae could play a role analogous to the multi-synthetase complex present in higher order organisms and could be important for the appropriate function of the ribosome. Thermal denaturation studies and footprinting experiments confirm that isolated ES7 is stable and maintains a near-native secondary and tertiary structure.

Global Analysis of Secreted Proteins and Glycoproteins in Saccharomyces cerevisiae

Protein secretion is essential for numerous cellular activities, and secreted proteins in bodily fluids are a promising and noninvasive source of biomarkers for disease detection. Systematic analysis of secreted proteins and glycoproteins will provide insight into protein function and cellular activities. Yeast (Saccharomyces cerevisiae) is an excellent model system for eukaryotic cells, but global analysis of secreted proteins and glycoproteins in yeast is challenging due to the low abundances of secreted proteins and contamination from high-abundance intracellular proteins. Here, by using mild separation of secreted proteins from cells, we comprehensively identified and quantified secreted proteins and glycoproteins through inhibition of glycosylation and mass spectrometry-based proteomics. In biological triplicate experiments, 245 secreted proteins were identified, and comparison with previous experimental and computational results demonstrated that many identified proteins were located in the extracellular space. Most quantified secreted proteins were down-regulated from cells treated with an N-glycosylation inhibitor (tunicamycin). The quantitative results strongly suggest that the secretion of these down-regulated proteins was regulated by glycosylation, while the secretion of proteins with minimal abundance changes was contrarily irrelevant to protein glycosylation, likely being secreted through nonclassical pathways. Glycoproteins in the yeast secretome were globally analyzed for the first time. A total of 27 proteins were quantified in at least two protein and glycosylation triplicate experiments, and all except one were down-regulated under N-glycosylation inhibition, which is solid experimental evidence to further demonstrate that the secretion of these proteins is regulated by their glycosylation. These results provide valuable insight into protein secretion, which will further advance protein secretion and disease studies.

Enhancing the mass spectrometric identification of membrane proteins by combining chemical and enzymatic digestion methods

Membrane proteins are critical for many cellular events, including cell signaling, molecular transport, and extracellular interactions. One third of the genome is estimated to encode membrane proteins, which are correlated with disease progression and can serve as promising biomarkers and drug targets. Modern mass spectrometry (MS)-based proteomics techniques facilitate the global analysis of proteins in complex biological samples; however, the hydrophobicity of membrane proteins inhibits their comprehensive analysis. Since membrane proteins are not easily accessible by proteases in aqueous solutions, a combinatorial method incorporating chemical and enzymatic digestion is presented here to improve the digestion efficiency of membrane proteins for MS analysis. Chemical digestion with 2-nitro-5-thiocyanatobenzoic acid (NTCB) was supplemented with enzymatic digestion (Glu-C, or Lys-C and trypsin) to determine the optimal combination of digestion methods. Three parallel experiments were performed with membrane protein extracts from HEK293T cells, and the results demonstrated that combining NTCB with Lys-C and trypsin resulted in the greatest number of identified peptides and proteins while the least number of peptides and proteins were identified by using sequential digestion with NTCB and Glu-C. By integrating chemical digestion before enzymatic digestion, NTCB could more easily access cleavage sites within membrane proteins, and the resulting peptide fragments were thus more accessible by proteases. The combination of chemical and enzymatic digestion presented here proved to be effective for membrane protein analysis.

An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole–glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures.Understanding the functions of protein glycosylation critically depends on methods to efficiently enrich glycoproteins from complex samples. Here, the authors develop a strategy using dendrimer-conjugated benzoboroxole to enhance glycopeptide enrichment, providing the basis for more comprehensive glycoprotein analyses.