A Novel Mass Spectrometry Platform for Multiplexed N-Glycoprotein Biomarker Discovery from Patient Biofluids by Antibody Panel Based N-Glycan Imaging.

A new platform for N-glycoprotein analysis from serum that combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) workflows with antibody slide arrays is described. Antibody panel based (APB) N-glycan imaging allows for the specific capture of N-glycoproteins by antibodies on glass slides and N-glycan analysis in a protein-specific and multiplexed manner. Development of this technique has focused on characterizing two abundant and well-studied human serum glycoproteins, alpha-1-antitrypsin and immunoglobulin G. Using purified standard solutions and 1 µL samples of human serum, both glycoproteins can be immunocaptured and followed by enzymatic release of N-glycans. N-Glycans are detected with a MALDI FT-ICR mass spectrometer in a concentration-dependent manner while maintaining specificity of capture. Importantly, the N-glycans detected via slide-based antibody capture were identical to that of direct analysis of the spotted standards. As a proof of concept, this workflow was applied to patient serum samples from individuals with liver cirrhosis to accurately detect a characteristic increase in an IgG N-glycan. This novel approach to protein-specific N-glycan analysis from an antibody panel can be further expanded to include any glycoprotein for which a validated antibody exists. Additionally, this platform can be adapted for analysis of any biofluid or biological sample that can be analyzed by antibody arrays.

Glycan Imaging Mass Spectrometry: Progress in Developing Clinical Diagnostic Assays for Tissues, Biofluids, and Cells.

N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide-based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.

Rapid N-Glycan Profiling of Serum and Plasma by a Novel Slide-Based Imaging Mass Spectrometry Workflow.

Changes in the levels and compositions of N-glycans released from serum and plasma glycoproteins have been assessed in many diseases across many large clinical sample cohorts. Assays used for N-glycan profiling in these fluids currently require multiple processing steps and have limited throughput, thus diminishing their potential for use as standard clinical diagnostic assays. A novel slide-based N-glycan profiling method was evaluated for sensitivity and reproducibility using a pooled serum standard. Serum was spotted on to an amine-reactive slide, delipidated and desalted with a series of washes, sprayed with peptide N-glycosidase F and matrix, and analyzed by MALDI-FTICR or MALDI-Q-TOF mass spectrometry. Routinely, over 75 N-glycan species can be detected from one microliter of serum in less than 6.5 h. Additionally, endoglycosidase F3 was applied to this workflow to identify core-fucosylated N-glycans and displayed the adaptability of this method for the determination of structural information. This method was applied to a small pooled serum set from either obese or nonobese patients that had breast cancer or a benign lesion. This study confirms the reproducibility, sensitivity, and adaptability of a novel method for N-glycan profiling of serum and plasma for potential application to clinical diagnostics.

Antibody Panel Based N-Glycan Imaging for N-Glycoprotein Biomarker Discovery.

Antibody panel based N-glycan imaging is a novel platform for N-glycan analysis of immunocaptured proteins. N-glycosylation is a post-translational modification of pathophysiological importance and is often studied in the context of disease biomarkers. Determination of protein-specific N-glycosylation changes in patient samples has traditionally been laborious or limited to study of a single protein per analysis. This novel technique allows for the multiplexed analysis of N-glycoproteins from biofluids. Briefly, this platform consists of antibodies spotted in an array panel to a microscope slide, specific capture of glycoproteins from a biological sample, and then enzymatic release of N-glycans for analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). N-glycans are detected at each individual spot, allowing N-glycan information to easily be linked back to its protein carrier. Using this protocol, multiplexed analysis of N-glycosylation on serum glycoproteins can be performed. Human serum is discussed here, but this method has potential to be applied to other biofluids and to any glycoprotein that can be captured by a validated antibody. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Antibody panel based N-glycan imaging by MALDI MS Support Protocol: Confirmation of antibody capture by IR-labeled proteins.

The search for biomarkers of hepatocellular carcinoma and the impact on patient outcome.

Hepatocellular carcinoma (HCC) is the 5th most common cancer, but the 3rd leading cause of cancer death globally with approximately 700,000 fatalities annually. The severity of this cancer arises from its difficulty to detect and treat. The major etiologies of HCC are liver fibrosis or cirrhosis from chronic viral infections, as well as metabolic conditions. Since most cases arise from prior pathologies, biomarker surveillance in high-risk individuals is an essential approach for early detection and improved patient outcome. While many molecular biomarkers have been associated with HCC, there are few that have made clinical impact for this disease. Here we review some major approaches used for HCC biomarker discovery-proteomics and glycomics-and describe new methodologies being tested for biomarker development.