Oriented immobilization of peptide-N-glycosidase F on a monolithic support for glycosylation analysis

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors

Porovnání isokratických retenčních modelů pro separaci nativních a fluorescenčních derivátů oligosacharidů chromatografií hydrofilních interakcí

In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L-1) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention.V této práci byla zkoumána retence maltooligosacharidů a jejich fluorescenčních derivátů v chromatografii hydrofilních interakcí s použitím čtyř různých stacionárních fází. Nederivatizované maltooligosacharidy (maltosa až maltoheptosa) a jejich deriváty s 2-aminobenzoovou kyselinou, 2-aminobenzamidem, 2-aminopyridinem a 8-aminonaftalen-1,3,6-trisulfonovou kyselinou byly analyzovány na silikagelu, aminopropylsilikagelu, amidu (vázaném karbamoylu) a ZIC-HILIC sulfobetainové fázi. Byly popsány a porovnány vlivy rozdělování analytů mezi volnou mobilní fázi a adsorbovanou vodnou vrstvou, polární a iontové interakce. Byly studovány vlivy aditiv mobilní fáze (0.1% (v/v) kyseliny octové a octanu amonného v koncentračním rozsahu 5-30 mmol L-1) na retenci. Byla testována vhodnost použití různých modelů pro predikci retence pro model lineární solvatační síly, kvadratický model, směsný model a empirický Neue-Kuss model. Směsný model byl rozšířen o parametr popisující příspěvek monomerních glukozových jednotek k retenci nederivatizovaných a derivatizovaných maltooligosacharidů, který byl použit pro hodnocení vlivu oligosacharidové části molekuly a koncové skupiny na retenci

High binding capacity surface grafted monolithic columns for cation exchange chromatography of proteins and peptides

Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 μm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix. These polymers were then photografted in a single step with 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylic acid to afford stationary phases for a strong and a weak cation exchange chromatography, respectively. Alternatively, poly(ethylene glycol) methacrylate was used for grafting in the first step in order to enhance hydrophilicity of the support followed by photografting with 2-acrylamido-2-methyl-1-propanesulfonic acid or acrylic acid in the second step. These new columns were used for the separation of proteins and peptides. A mixture of ovalbumin, α-chymotrypsinogen, cytochrome c, ribonuclease A and lysozyme was used to assess the chromatographic performance for large molecules while a cytochrome c digest served as a model mixture of peptides. All tested columns featured excellent mass transfer as demonstrated with very steep breakthrough curves. The highest binding capacities were found for columns prepared using the two step functionalization. Columns with sulfonic acid functionalities adsorbed up to 21.5 mg/mL lysozyme while the capacity of the weak cation exchange column functionalized with acrylic acid was 29.2 mg/mL. © 2009 Elsevier B.V. All rights reserved