Abstract: Provided herein are stationary phase compositions comprising a chromatographic surface of porous or non-porous core material comprising a surface modifier for use in chromatographic separations.

Abstract: Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent.

Abstract: Provided herein are methodologies where a glycosylated protein or peptide is subjected to peptide bond cleavage to produce a glycan amino acid complex wherein the N-linked or O-linked glycan is attached. A derivatization reagent is then attached to the N terminus of the amino acid to provide a labeled glycan amino acid complex. The labeled glycan amino acid complex is then separated from the matrix via one or more methods including HILIC SPE, and injected directly onto an LC or LC/MS system for analysis, detection and characterization of the glycosylated protein or the peptide.

Abstract: Methods are provided for making rapid labeled dextran ladders and other calibrants useful in liquid chromatography. The methodologies include a two-step process comprising a reductive amination step of providing a reducing glycan and reacting it with a compound having a primary amine to produce an intermediate compound. The intermediate compound is then rapidly tagged with a rapid tagging reagent to produce the rapid labeled dextran ladder.

Abstract: Reagents comprising MS active, fluorescent molecules with an activated functionality for reaction with amines useful in tagging biomolecules such as N-glycans and uses thereof are taught and described.

Abstract: The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.

Abstract: The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.

Abstract: Reagents comprising MS active, fluorescent molecules with an activated functionality for reaction with amines useful in tagging biomolecules such as N-glycans and uses thereof are taught and described.