High throughput plasma N-glycome profiling using multiplexed labelling and UPLC with fluorescence detection

The Analyst ◽  
2011 ◽  
Vol 136 (22) ◽  
pp. 4670 ◽  
Author(s):  
Ana Knežević ◽  
Jonathan Bones ◽  
Stjepan Krešimir Kračun ◽  
Olga Gornik ◽  
Pauline M. Rudd ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
High Throughput ◽  
Glycome Profiling

scholarly journals Understanding The Structure And Composition of Recalcitrant Oligosaccharides In Ammonia Treated Lignocellulosic Biomass Hydrolysate Using High-Throughput Biotin- Based Glycome Profiling And Mass Spectrometry.

2021 ◽  
Author(s):  
Saisi Xue ◽  
Sivakumar Pattathil ◽  
Leonardo Costa Sousa ◽  
Bryan Ubanwa ◽  
Bruce Dale ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lignocellulosic Biomass ◽  
Plant Cell ◽  
High Throughput ◽  
Plant Cell Wall ◽  
Enzyme Hydrolysis ◽  
Size Exclusion ◽  
Negative Ion ◽  
Fungal Enzymes ◽  
Glycome Profiling

Abstract Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to immobilize these molecules for mAbs binding and characterization. To overcome this problem, we have developed a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

scholarly journals High-throughput fluorescence detection using an integrated zone-plate array

Lab on a Chip ◽  
2010 ◽  
Vol 10 (7) ◽  
pp. 852 ◽  
Author(s):  
Ethan Schonbrun ◽  
Adam R. Abate ◽  
Paul E. Steinvurzel ◽  
David. A. Weitz ◽  
Kenneth B. Crozier
Keyword(s):  
Fluorescence Detection ◽  
High Throughput ◽  
Zone Plate ◽  
Plate Array
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