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: 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: In various aspects, the present disclosure pertains to materials (e.g., kits, column assemblies, liquid chromatography systems, etc.) methods for performing liquid chromatography that employ a first column (e.g., a trapping column) and a second column (e.g., an analytical column). The first column comprises a first chromatographic material having a first chromatographic surface that comprises first hydrophobic surface groups and first ionizable surface groups having a first pKa value. The second column comprises a second chromatographic material having a second chromatographic surface that comprises second hydrophobic surface groups and (a) permanently ionized surface groups or (b) second ionizable surface groups having a second pKa value. The first hydrophobic surface groups have a hydrophobicity that is less than a hydrophobicity of the second hydrophobic surface groups.

Abstract: The present invention is directed to a method for performing size exclusion chromatography. Embodiments of the present invention feature devices and methods for improving the speed and separations of size exclusion chromatography using a stationary phase material comprising small particles (<2 micron in diameter).

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: The present disclosure relates to methods, compositions and kits useful for the enhanced pH gradient cation exchange chromatography of a variety of analytes. In various aspects, the present disclosure pertains to chromatographic elution buffer solutions that comprise a first buffer salt, a second buffer salt, a third buffer salt, and fourth buffer salt. The first buffer salt may be, for example, a diprotic acid buffer salt, the second buffer salt may be, for example, a divalent buffer salt with two amine groups, the third buffer salt may be, for example, a monovalent buffer salt comprising a single amine group, and the fourth buffer salt may be, for example, a zwitterionic buffer salt. Moreover, the buffer solution has a pH ranging from 3 to 11.

Abstract: Novel reagents comprising MS active, fluorescent compounds having an activated functionality for reaction with aldehydes and useful in labeling biomolecules such as glycans and methods of making the same are taught and described.

Abstract: Mass spectrometry (MS) active, fluorescent rapid tagging reagent is provided having three substituent groups: (a) a tertiary amino group or other MS active atom; (b) a highly fluorescent moiety; and (c) a reactive group that can react with an amine. The reactive group provides rapid tagging of desired bio-molecules. The fluorescent moiety provides the fluorescent signal. The tertiary amino group provides the MS signal.

Abstract: The present disclosure relates to the determination of analytes in a sample using chromatography. The present disclosure provides methods of separating an analyte from a sample. A mobile phase is flowed through a chromatography column. The mobile phase includes about 0.005% (v/v) to about 0.20% (v/v) difluoroacetic acid and less than about 100 ppb of any individual metal impurity. A sample including the analyte is injected into the mobile phase. The analyte is separated from the sample.

Abstract: Solutions, detection methods and chromatographic systems are provided for electrospray ionization of glycans modified with amphipathic, strongly basic moieties. The solutions for use in electrospray ionization comprise a plurality of glycans having an amphipathic moiety, a basic residue of pKa>5 and a Log P value between 1 and 3, and one or more volatile components selected from the group consisting of an amine, ammonia, ammonia salt, diethylamine, or trimethylamine. The solutions also have a pH between about 3 to about 6, and ionic strength of between about 0 mM to about 500 mM. The solutions are useful in detecting modified glycans in electrospray ionization and in various chromatographic systems.

Abstract: Methods for derivatization of biomolecules including glycans or other biopolymers with one or more fluorescent, MS active compounds by reductive amination or rapid tagging in order to produce derivatized glycan having a pKa>7 and between about 200 ? and about 1000 ? of nonpolar surface area are described.

Abstract: Methods for preparing labeled glycosylamines from a complex matrix are provided. The methodology includes the steps of: denaturing glycoproteins in a complex matrix to form a denatured complex matrix mixture; loading the denatured complex matrix mixture onto a MWCO filtration device; adding a glycosidase enzymatic solution onto the MWCO filtration device to form a deglycosylated complex matrix mixture comprising glycosylamines; collecting glycosylamines released from the MWCO filtration device; and derivatizing glycosylamines with a rapid tagging reagent to form a plurality of labeled glycosylamines suitable for detection in various liquid chromatography systems and detectors.

Abstract: The present disclosure pertains to sample preparation devices useful for affinity capture and purification that include one or more internal structures that comprise a reservoir, a well, a fluid passageway, sorbent particles, and a filter element that blocks passage of the affinity sorbent particles, which sample preparation devices combine the attributes of both dispersive and flow through designs into a single sample preparation device. The present disclosure also pertains to kits that contain and methods that use such sample preparation devices.

Abstract: The present invention provides the use of charged surface reversed phase chromatographic materials along with standard reversed-phase LC and mass spectrometry compatible conditions for the retention, separation, purification, and characterization of acidic, polar molecules, including, but not limited to, organic acids, ?-amino acids, phosphate sugars, nucleotides, other acidic, polar biologically relevant molecules. The chromatographic materials of the invention are high purity chromatographic materials comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifier.

Abstract: A device for separating analytes is disclosed. The device has a sample injector, sample injection needle, sample reservoir container in communication with the sample injector, chromatography column downstream of the sample injector, and fluid conduits connecting the sample injector and the column. The interior surfaces of the fluid conduits, sample injector, sample reservoir container, and column form a flow path having wetted surfaces. A portion of the wetted surfaces of the flow path are coated with an alkylsilyl coating that is inert to at least one of the analytes. The alkylsilyl coating has the Formula I: R1, R2, R3, R4, R5, and R6 are each independently selected from (C1-C6)alkoxy, —NH(C1-C6)alkyl, —N((C1-C6)alkyl)2, OH, ORA, and halo. RA represents a point of attachment to the interior surfaces of the fluidic system. At least one of R1, R2, R3, R4, R5, and R6 is ORA. X is (C1-C20)alkyl, —O[(CH2)2O]1-20—, —(C1-C10)[NH(CO)NH(C1-C10)]1-20-, or —(C1-C10)[alkylphenyl(C1-C10)alkyl]1-20-.

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: 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.