2-Dimensional ultra-high performance liquid chromatography and DMT-MM derivatization paired with tandem mass spectrometry for comprehensive serum N-glycome characterization.

Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation.

ADP-dependent glucokinase regulates energy metabolism via ER-localized glucose sensing.

Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division.

Salivary N-glycosylation as a biomarker of oral cancer: A pilot study.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers.

Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry.

Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE).

Glycosylation Analysis of Therapeutic Glycoproteins Produced in CHO Cells.

In the last decades, the number of approved therapeutic proteins drugs is increasing exponentially and a large number of new therapeutic entities are progressing through clinical trials, solidifying biologics as the most promising class of pharmaceuticals on the market. Several cell lines are available for biopharmaceutical processes but mammalian cells are preferred since they give fewer problems for immunogenicity as they produce human-like post-translational modifications (PTMs). Glycosylation is the most common and complex (for both bioprocess engineering and quality control) of these modifications.

Quantitative glycomics using liquid phase separations coupled to mass spectrometry.

Post-translational modification of proteins by the attachment of glycans is governed by a variety of highly specific enzymes and is associated with fundamental impacts on the parent protein's physical, chemical and biological properties. The inherent connection between cellular physiology and specific glycosylation patterns has been shown to offer potential for diagnostic and prognostic monitoring of altered glycosylation in the disease state. Conversely, glycoprotein based biopharmaceuticals have emerged as dominant therapeutic strategies in the treatment of intricate diseases.

Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma.

Twoplex C aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing.

Quantitative twoplex glycan analysis using C and C stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS).

Quantitative host cell protein analysis using two dimensional data independent LC-MS(E).

Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed.

Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis.

A CE-based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin-specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled, and profiled by CE.

Unraveling the glyco-puzzle: glycan structure identification by capillary electrophoresis.

State-of-the-art high-resolution separation techniques play an important role in the full structural elucidation of glycans. Capillary electrophoresis (CE) offers a rapid yet simple method for exhaustive carbohydrate profiling. CE is a versatile analytical platform, which can be operated in several separation modes, simply by altering separation conditions during operation.

Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries.

Capillary electrophoresis enables fast, high efficiency separations of oligosaccharides, wherein positional and/or linkage isomers, bearing the same charge-to-mass ratio, can readily be separated based on hydrodynamic radius differences. Fundamental electrophoretic mobility theory was used to investigate the correlation between changes in hydrodynamic volume equivalent radius and corresponding electrophoretic characteristics of oligosaccharides with different molecular properties. Fluorescently derivatized isomeric malto-, cello-, and isomaltooligosaccharide ladders, differing only in their linkage type of α1→4, β1→4, and α1→6, respectively, as well as a sterically larger N-acetylchitooligosaccharide ladder were used as model compounds.

Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation.

N-glycans attached to the C(H)2 domains of the Fc or the antigen binding regions of IgG play an important role in stabilizing and modulating antibody activity. Exhaustive elucidation of 32 IgG N-glycans using a combination of weak anion exchange enrichment and exoglycosidase array digestion with subsequent profiling exceeded 48 h. Pursuing increased throughput and associated structural annotation confidence, we compared the 1.

Identification of N-glycans displaying mannose-6-phosphate and their site of attachment on therapeutic enzymes for lysosomal storage disorder treatment.

Characterization of mono- and bis-mannose-6-phosphate (M6P) bearing oligosaccharides present on acid hydrolase enzymes poses a considerable analytical challenge. In the current paper, we investigated the use of UPLC profiling on a 1.7 μm HILIC phase and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) combined with exoglycosidase digestion and weak anion exchange fractionation for the characterization of M6P bearing glycans on recombinant β-glucuronidase expressed in Chinese Hamster Ovary (CHO) cells.

Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.

Glycosylation is a diverse but critically important post-translational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest.

Two variable semi-empirical and artificial neural-network-based modeling of peptide mobilities in CZE: the effect of temperature and organic modifier concentration.

This work was focused on investigating the effects of two separation influencing parameters in CZE, namely temperature and organic additive concentration upon the electrophoretic migration properties of model tripeptides. Two variable semi-empirical (TVSE) models and back-propagation artificial neural networks (ANN) were applied to predict the electrophoretic mobilities of the tripeptides with non-polar, polar, positively charged, negatively charged and aromatic R group characteristics. Previously published work on the subject did not account for the effect of temperature and buffer organic modifier concentration on peptide mobility, in spite of the fact that both were considered to be influential factors in peptide analysis.