Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum and can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study a re-defined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding 5 glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid over the course of restenosis progression to investigate potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization.

Intact N-glycopeptide analysis remains challenging due to the complexity of glycopeptide structures, low abundance of glycopeptides in protein digests, and difficulties in data interpretation/quantitation. Herein, we developed a workflow that involved advanced methodologies, the EThcD- MS/MS fragmentation method and data interpretation software, for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis. Haptoglobin was immunopurified from 20 μL of serum in patients with early HCC, liver cirrhosis, and healthy controls, respectively, followed by trypsin/GluC digestion, glycopeptide enrichment, and LC-EThcD-MS/MS analysis. Identification and differential quantitation of site-specific N-glycopeptides were performed using a combination of Byonic and Byologic software. In total, 93, 87, and 68 site-specific N-glycopeptides were identified in early HCC, liver cirrhosis, and healthy controls, respectively, with high confidence. The increased variety of N-glycopeptides in liver diseases compared to healthy controls was due to increased branching with hyper-fucosylation and sialylation. Differential quantitation analysis showed that 5 site-specific N-glycopeptides on sites N184 and N241 were significantly elevated in early HCC compared to cirrhosis (p < 0.05) and normal controls (p ≤ 0.001). The result demonstrates that the workflow provides a strategy for detailed profiles of N-glycopeptides of patient samples as well as for relative quantitation to determine the level changes in site-specific N-glycopeptides between disease states.

Recently developed carbonyl-reactive aminoxy tandem mass tag (aminoxyTMT) reagents enable multiplexed characterization and quantitative comparison of structurally complex glycans between different biological samples. Compared to some previously reported isotopic labeling strategies for glycans, the use of the aminoxyTMT method features a simple labeling procedure, excellent labeling efficiency, and reduced spectral complexity at the MS(1) level. Presence of the tertiary amine functionality in the reporter region of the aminoxyTMT labels leads to increased ionization efficiency of the labeled glycans thus improving electrospray ionization (ESI)-mass spectrometry (MS) detection sensitivity. The use of the labeling reagent also makes electrophoretic separation of the labeled neutral and acidic glycans feasible. In this work, we characterized the ESI and collision induced dissociation (CID) behavior of the aminoxyTMT-labeled neutral and sialylated glycans. For the high-mannose N-glycans and small sialylated oligosaccharides, CID fragmentation of [M + Na + H](2+) provides the most informative MS(2) spectra for both quantitative and qualitative analysis. For complex N-glycans, MS(3) of the protonated Y1(H) ion can be used for relative quantification without interference from the HexNAc fragments. Online capillary electrophoresis (CE)-ESI-MS/MS analyses of multiplexed aminoxyTMT-labeled human milk oligosaccharides (HMOs) and different types of N-glycans released from glycoprotein standards were demonstrated. Improved resolution and quantification accuracy of the labeled HMO isomers was achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. N-Glycans released from human serum protein digests were labeled with six-plex aminoxyTMT and subjected to CE-ESI-MS/pseudo-MS(3) analysis, which demonstrated the potential utility of this glycan relative quantification platform for more complex biological samples.

Nonalcoholic steatohepatitis (NASH) is rising in prevalence in the United States and is a growing cause of hepatocellular carcinomas (HCCs). Site-specific glycan heterogeneity on glycoproteins has been shown as a potential diagnostic biomarker for HCC. Herein, we have performed a comprehensive screening of site-specific N-glycopeptides in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation in HCC, to characterize glycopeptide markers for NASH-related HCCs. In total, 70 NASH patients (22 early HCC, 15 advanced HCC, and 33 cirrhosis cases) were analyzed, with Hp purified from 20 μL of serum in each patient, and 140 sets of mass spectrometry (MS) data were collected using liquid chromatography coupled with electron-transfer high-energy collisional dissociation tandem MS (LC-EThcD-MS/MS) for quantitative analysis on a novel software platform, Byos. Differential quantitation analysis revealed that five N-glycopeptides at sites N184 and N241 were significantly elevated during the progression from NASH cirrhosis to HCC (p < 0.05). Receiver operating characteristic (ROC) curve analysis demonstrated that the N-glycopeptides at sites N184 and N241 bearing a monofucosylated triantennary glycan A3G3F1S3 had the best diagnostic performance in detection of early NASH HCC, area under the curve (AUC) = 0.733 and 0.775, respectively, whereas α-fetoprotein (AFP) had an AUC of 0.692. When combined with AFP, the two panels improved the sensitivity for early NASH HCC from 59% (AFP alone) to 73% while maintaining a specificity of 70%, based on the optimal cutoff. Two-dimensional (2-D) scatter plots of the AFP value and N-glycopeptides showed that these N-glycopeptide markers detected 58% of AFP-negative HCC patients as distinct from cirrhosis. These site-specific N-glycopeptides could serve as potential markers for early detection of HCC in patients with NASH-related cirrhosis.

Glycans and glycoconjugates are involved in regulating a vast array of cellular and molecular processes. Despite the importance of glycans in biology and disease, characterization of glycans remains difficult due to their structural complexity and diversity. Mass spectrometry (MS)-based techniques have emerged as the premier analytical tools for characterizing glycans. However, traditional MS-based strategies struggle to distinguish the large number of coexisting isomeric glycans that are indistinguishable by mass alone. Because of this, ion mobility spectrometry coupled to MS (IM-MS) has received considerable attention as an analytical tool for improving glycan characterization due to the capability of IM to resolve isomeric glycans before MS measurements. In this review, we present recent improvements in IM-MS instrumentation and methods for the structural characterization of isomeric glycans. In addition, we highlight recent applications of IM-MS that illustrate the enormous potential of this technology in a variety of research areas, including glycomics, glycoproteomics, and glycobiology.

The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.

Primary aldosteronism (PA) is a common cause of secondary hypertension and is associated with worse cardiovascular outcomes. The elevated aldosterone in PA leads to left ventricular (LV) remodeling and dysfunction. In recent decades, clinical studies have demonstrated worse LV remodeling including increased LV mass and cardiac fibrosis in patients with PA compared to patients with essential hypertension. Several mechanisms may explain the process of aldosterone-induced LV remodeling, including directly profibrotic and hypertrophic effects of aldosterone on myocardium, increased reactive oxygen species and profibrotic molecules, dysregulation of extracellular matrix metabolism, endothelium dysfunction and circulatory macrophages activation. LV remodeling causes LV diastolic and systolic dysfunction, which may consequently lead to clinical complications such as heart failure, atrial fibrillation, ischemic heart disease, and other vascular events. Adequate treatment with adrenalectomy or medical therapy can improve LV remodeling and dysfunction in PA patients. In this review, we discuss the mechanisms of aldosterone-induced LV remodeling and provide an up-to-date review of clinical research about LV remodeling-related heart structural changes, cardiac dysfunction, and their clinical impacts on patients with PA.

Primary aldosteronism (PA) is characterized by excess production of aldosterone from the adrenal glands and is the most common and treatable cause of secondary hypertension. Aldosterone is a mineralocorticoid hormone that participates in the regulation of electrolyte balance, blood pressure, and tissue remodeling. The excess of aldosterone caused by PA results in an increase in cardiovascular and cerebrovascular complications, including coronary artery disease, myocardial infarction, stroke, transient ischemic attack, and even arrhythmia and heart failure. Endothelial dysfunction is a well-established fundamental cause of cardiovascular diseases and also a predictor of worse clinical outcomes. Accumulating evidence indicates that aldosterone plays an important role in the initiation and progression of endothelial dysfunction. Several mechanisms have been shown to contribute to aldosterone-induced endothelial dysfunction, including aldosterone-mediated vascular tone dysfunction, aldosterone- and endothelium-mediated vascular inflammation, aldosterone-related atherosclerosis, and vascular remodeling. These mechanisms are activated by aldosterone through genomic and nongenomic pathways in mineralocorticoid receptor-dependent and independent manners. In addition, other cells have also been shown to participate in these mechanisms. The complex interactions among endothelium, inflammatory cells, vascular smooth muscle cells and fibroblasts are crucial for aldosterone-mediated endothelial dysregulation. In this review, we discuss the association between aldosterone and endothelial function and the complex mechanisms from a molecular aspect. Furthermore, we also review current clinical research of endothelial dysfunction in patients with PA.