Alignment of laser‐induced fluorescence and mass spectrometric detection traces using electrophoretic mobility scaling in CE‐LIF‐MS of labeled N‐glycans

Abstract

The combination of optical detection techniques like photometry (UV) or laser-induced fluorescence (LIF) with mass spectrometry for capillary electrophoresis offers advantages, both for later use of stand-alone CE-UV or CE-LIF systems and for combined CE-UV-MS or CE-LIF-MS analysis. Faster method development is enabled, the identification of analytes is facilitated, and it allows christian the optical detection scheme to be used for more precise quantification. However, shortcomings of current methodology and equipment hindered the broader use of such detection combinations mainly due to the long distance between the detection points (at least 20 cm). Large shifts in migration times and changes in resolution are visible between the detection traces hindering their straightforward comparison. We present here novel equipment for a robust coupling of CE-LIF-MS with the shortest possible distance between detection points (12 cm) determined by the length of the electrospray needle. In addition, we encourage the use of a normalization of detection traces using a scale of effective electrophoretic mobility to obtain the same x-scale for both detection traces. As an example, the proposed methodology is applied to a mixture of labeled as well as non-labeled N-glycans.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…28,[39][40][41][42] All other methods applied fluorescent labeling. Four methods used electrophoretic separation, which included conventional high-resolution capillary gel electrophoresis with laser-induced fluorescence [CE-LIF(APTS-HR1)], [43][44][45][46][47][48] DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis after 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling for high-throughput screening [DSA-FACE(APTS)], [49][50][51][52][53][54] high-resolution capillary gel electrophoresis with rapid labeling with APTS via reductive amination [CE-LIF (APTS-HR2)], and cartridge-based capillary gel electrophoresis with rapid 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) labeling, in development specifically for screening [CCGE(ANTS)]. The CE methods differ with regard to the labeling method: APTS labeling for the "normal" method requires 4 to 24 h for labeling, whereas rapid reductive amination has been optimized Three laboratories were involved in performing the experiments, an analytical laboratory in a development department, a quality control laboratory and a laboratory of a vendor of glycoanalytical tools.…”

A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis

“…Specifically, the use of common DNA sequencing equipment to perform glycan structure analysis by capillary electrophoresis is an excellent alternative to the established methods, with advantages in simplicity, throughput, structural resolution, and sensitivity (Callewaert et al, 2001; Huhn et al, 2012; Laroy et al, 2006; Reusch et al, 2014). Previously described CE techniques for antibody glycan analysis have focused on the analysis of whole IgG, as the large majority of monoclonal antibodies lack Fab glycan-sites (Ritamo et al, 2014).…”

CE‐MS for the analysis of intact proteins 2010–2012

“…The coupling of CE with optical detection and mass spectrometric detection could be an interesting way to improve information content. CE‐MS hyphenated with laser‐induced fluorescence or wavelength‐resolved fluorescence could provide a good platform for selective quantitative detection and identification in a single setup.…”