Abstract
We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.
Highlights
We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS)
The novel sample preparation protocol introduced in this paper can be scaled up to support deep N-glycomics analysis of the human serum by CE-LIF and the higher sample concentration requirement of CE-ESI-MS
With APTS labeling, negative ionization mode had to be applied resulting lower signal intensity compared with positive ion mode operation
Summary
An efficient sample preparation workflow was developed to facilitate deep N-glycomics analysis of human serum by capillary electrophoresis accommodating the higher sample concentration requirement of CEESI-MS. A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The presence of various glycoforms at a given site (microheterogeneity) or the occupancy of a potential glycosylation site (macroheterogeneity) represents additional analytical challenges Modern glycoanalytical techniques such as HPLC, capillary electrophoresis and mass spectrometry require very efficient sample preparation methods to achieve high sensitivity for deep glycomics analysis. Most of these techniques start with a denaturing step to unfold the glycoproteins in order to allow access for the endoglycosidase enzymes to reach their substrates and release the attached carbohydrate chains. The sample preparation method was tested on hIgG1 and human serum samples
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