Molecular weight determination of high molecular mass (glyco)proteins using CGE‐on‐a‐chip, planar SDS‐PAGE and MALDI‐TOF‐MS

Abstract

The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1 MDa.

Differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins

“…MALDI‐TOF MS is the most useful technique for measuring the MW of PEG‐conjugated proteins . The MWs of the LPEG‐proteins and BrPEG‐proteins, including the native proteins and PEG derivatives, measured by MALDI‐TOF MS are summarized in Table .…”

Protein separation by capillary gel electrophoresis: A review

“…All these chips have been tested for CGE separation of proteins. Performance of microchip-based gel electrophoresis has been compared with that of capillary-based gel electrophoresis [41, 56, 57, 92–94]. In general, the performances are comparable, while microchip CGE provides faster separations.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2009–2010

“…h. Comparisons of methods for N-glycan analysis A comparative study of three techniques, MALDI-TOF, SDS-PAGE and CGE-on-a-chip, for measuring the MWs of large glycoproteins has been reported by Müller et al (2010b). It was found that all three techniques were capable of determining the MW of all high MW (glyco)proteins tested.…”