RESUMO
Fabrication of poly(glycidyl methacrylate-co-ethylene dimethacrylate) [also referred to as poly(GMA-co-EDMA)] monoliths was optimized as supporting material for trypsin digestion nanoreactors. Reaction parameters, such as polymerization time, porogen concentration, and monomer to crosslinker ratios, were evaluated in respect to the permeability of the resulting monolith and their effect on digestion efficiency, estimated by mass spectrometric analysis of a model protein cytochrome C. The structural homogeneity of the resulting monolithic support was checked by scanning electron microscopy. The best nanoreactor performance, measured by the reduction of nanoreactor backpressure and increased sequence coverage of cytochrome C, was achieved with 8% 2-octanol (porogen) 20%/20% glycidyl methacrylate to ethylene glycol dimethacrylate ratio and 5 h of polymerization time. Digestion of as low as 3 microg of cytochrome C with 77% sequence coverage was obtained using the optimized trypsin nanoreactor.
Assuntos
Metilmetacrilatos/química , Nanotecnologia , Tripsina/química , Sequência de Aminoácidos , Citocromos c/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Dados de Sequência MolecularRESUMO
As a continuation of our work on boronic acid lectin affinity chromatography (BLAC), in this paper we introduce an automated affinity micropartitioning approach using combined boronic acid and concanavalin A (BLAC/Con A) resin-filled micropipette tips to isolate and enrich human serum glycoproteins. The N-linked oligosaccharides of the partitioned glycoproteins were removed by PNGase F enzyme digestion, followed by 8-aminopyrene-1,3,6-trisulfonic acid labeling. Capillary gel electrophoresis with blue LED-induced fluorescence detection was applied in a multiplexed format for comparative glycan profiling. The efficiency of BLAC affinity micropartitioning was compared with that of the individual lectin and pseudolectin affinity enrichment. Finally, we report on our findings in glycosylation differences in human serum samples from healthy and prostate cancer patients by applying BLAC/Con A micropipette tip-based enrichment and comparative multicapillary gel electrophoresis analysis of the released and labeled glycans.
Assuntos
Ácidos Borônicos/química , Cromatografia de Afinidade/métodos , Concanavalina A/química , Glicoproteínas/sangue , Glicoproteínas/isolamento & purificação , Eletroforese Capilar/métodos , Glicoproteínas/análise , Glicosilação , Hemofiltração/métodos , Humanos , Masculino , Oligossacarídeos/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Polissacarídeos/análise , Polissacarídeos/química , Neoplasias da Próstata/sangue , Pirenos/química , Aglutininas do Germe de Trigo/químicaRESUMO
We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid-lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pesudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.
