Dataset Information

Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods.

ABSTRACT: Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due to the complexity of biological samples, effective enrichment methods are critical prior to MS analysis. Here, we compared two lectin-independent methods to enrich glycopeptides for the global analysis of protein N-glycosylation by MS. The first boronic acid-based enrichment (BA) method benefits from the universal and reversible interactions between boronic acid and sugars; the other method utilizes metabolic labeling and click chemistry (MC) to incorporate a chemical handle into glycoproteins for future affinity enrichment. We comprehensively compared the performance of the two methods in the identification and quantification of glycoproteins in statin-treated liver cells. Based on the current results, the BA method is more universal in enriching glycopeptides, while with the MC method, cell surface glycoproteins were highly enriched, and the quantification results appear to be more dynamic because only the newly-synthesized glycoproteins were analyzed. In addition, we normalized the glycosylation site ratios by the corresponding parent protein ratios to reflect the real modification changes. In combination with MS-based proteomics, effective enrichment methods will vertically advance protein glycosylation research.

SUBMITTER: Xiao H

PROVIDER: S-EPMC6103449 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods.

Xiao Haopeng H Hwang Ju Eun JE Wu Ronghu R

International journal of mass spectrometry 20170527

Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due ...[more]

PMID: 30147434

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Project description: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.

Project description:Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of O-GalNAc attached to tyrosine. Furthermore, we uncovered ≈ 220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes.

Project description:RationaleThe low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins.MethodsSerum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out.ResultsThe gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa.ConclusionsUsing only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.

Dataset Information

Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods.

Publications

Mass spectrometric analysis of the N-glycoproteome in statin-treated liver cells with two lectin-independent chemical enrichment methods.

Similar Datasets

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