Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow.

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

Release of protein N-glycans by effectors of a Hofmann carboxamide rearrangement.

Background: Chemical methods for glycan release have gained traction because of their cost efficiency, accelerated reaction time and ability to release glycans not amenable to enzymatic cleavage. Oxidative chemical glycan release via hypochlorite treatment has been shown to be a convenient and efficient method that yields N-glycans similar to classical PNGase F digestion. We observed that the initial steps of the suggested mechanism for the oxidative release of glycans from glycoproteins by hypohalites showed similarities to the initiating steps of the classical Hofmann rearrangement of carboxamides. Therefore, we investigated the ability of different stable effectors of a Hofmann-type carboxamide rearrangement to efficiently and selectively release N-glycans from glycoproteins. Methods: Released glycans obtained from different experimental chemical release approaches were analyzed by HILIC-FLD, BHZ-FACE and ESI-MS and evaluated with respect to electrophoretic mobility, retention time and integrated peak area for resolved glycans. Results: We show that the known Hoffmann catalysts 1,3-dichloro-5,5-dimethylhydantoin, the hypervalent organoiodine (III) compound diacetoxy-iodobenzene as well as in-situ hypobromite generation using Oxone® and potassium bromide are all capable of releasing protein-bound N-glycans in good yield. Among the compounds investigated, diacetoxy-iodobenzene was capable of releasing glycans in the absence of alkali. Detailed investigations of the bromide/Oxone® method revealed a dependence of N-glycan release efficiency from the temporal order of bromide addition to the reaction mix as well as from a molar excess of bromide over Oxone®. Conclusions. These findings suggest that the oxidative release of N-glycans occurs via the initiating steps of a Hofmann carboxamide rearrangement. Hypervalent organoiodine compounds hold the promise of releasing glycans in the absence of alkali. The in-situ generation of hypobromite by bromide/Oxone® produces a consistent defined amount of reagent for rapid N-glycan release for both analytical and preparative purposes.

Principles of Protein Labeling Techniques.

Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

Principles of protein labeling techniques.

Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.

Integrated polymer chip for two-dimensional capillary gel electrophoresis.

Two-dimensional (2D) gel electrophoresis (GE) is one of the most powerful methods for nucleic acid and protein separation, but generally suffers from high laboratory efforts connected with high analysis costs. Therefore, we herein present the development of a miniaturized 2D capillary GE (CGE) device which allows for an efficient protein separation in analysis times of about 1.5 h. This integrated 2D-CGE chip comprises a first channel for isoelectric focussing (IEF), a second specially designed transfer channel, 300 parallel micro channels, each having a cross section of 50 microm x 50 microm, and buffer reservoirs. The present work discusses fabrication aspects, in particular the combination of different microfabrication technologies, experimental separation performances of isoelectric focussing (IEF) and CGE, and presents computer simulations and first experimental results of protein transfer from the first to the second dimension.

Towards disposable lab-on-a-chip: poly(methylmethacrylate) microchip electrophoresis device with electrochemical detection.

A fully disposable microanalytical device based on combination of poly(methylmethacrylate) (PMMA) capillary electrophoresis microchips and thick-film electrochemical detector strips is described. Variables influencing the separation efficiency and amperometric response, including separation voltage or detection potential are assessed and optimized. The versatility, simplicity and low-cost advantages of the new design are coupled to an attractive analytical performance, with good precision (relative standard deviation RSD = 1.68% for n = 10). Applicability for assays of mixtures of hydrazine, phenolic compounds, and catecholamines is demonstrated. Such coupling of low-cost PMMA-based microchips with thick-film electrochemical detectors holds great promise for mass production of single-use micrototal analytical systems.