WO2013192530A2

WO2013192530A2 - Methods and reagents for glycoproteomics

Info

Publication number: WO2013192530A2
Application number: PCT/US2013/047073
Authority: WO
Inventors: Richard S. LEE; Hui Zhou
Original assignee: Children's Medical Center Corporation
Priority date: 2012-06-21
Filing date: 2013-06-21
Publication date: 2013-12-27
Also published as: WO2013192530A3

Abstract

Some aspects of this disclosure provide a method for separating a glycan and/or a polypeptide fraction from a mixture comprising a glycan and a polypeptide. In some embodiments, the method comprises acidifying the mixture in order to decrease non-covalent glycan/polypeptide interactions prior to separation. Reagents and devices useful for glycan and/or polypeptide separation are also provided. The technology disclosed herein allows for the rapid, cost-efficient separation and isolation of both glycans and polypeptides obtained from glycosylated proteins after release of the glycans, for example, by enzymatic digest. Uses of the disclosed technology include, for example, the separation and isolation of glycans and/or polypeptides from body fluid samples for diagnostics, and quality control of glycosylation in engineered proteins, e.g., in therapeutic antibodies.

Description

METHODS AND REAGENTS FOR GLYCOPROTEOMICS

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application, U.S. S.N. 61/662,893, filed June 21, 2012, and entitled Methods and Reagents for Glycoproteomics, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] The most abundant post-translational modification of proteins, glycosylation, remains practically unexplored to date at the proteome scale because of a dearth of methods for profiling the complex glycoproteome. Glycosylation of proteins plays an important role in many biological processes, including, for example, cell signaling, cell-cell interactions and the immune response. Further, the majority of protein-based biopharmaceuticals approved or in clinical trials bear some form of post-translational modification (PTM), which, in some cases, can profoundly affect protein properties relevant to their therapeutic application. A better understanding of the biological functions of glycosylation will facilitate the

engineering of next-generation protein and peptide therapeutics with glycosylation profiles optimized for the respective therapeutic approach.

[0003] Analyzing the glycoproteome is technically challenging, because, as a post- translational process, glycosylation nontemplated, and, unlike other post-translational modifications (e.g., phosphorylation), glycan structures found on glycosylated proteins are highly complex. A single protein can have tens to hundreds of different glycan attachments, and glycosylated forms of proteins are often found in low abundance in the cell. The very different chemistries of proteins and glycans present additional challenges in applying analytic methods, such as mass spectrometry, in the glycoproteomic context, and current methods for isolating and/or separating glycoproteins for analytical processing lack in performance. See Doerr, Glycoproteomics Nat Meth 2012, 9(1):36, and Walsh et al., Post- translational modifications in the context of therapeutic proteins Nat Biotech 2006 24(10).

SUMMARY

[0004] Protein glycosylation is one of the most frequent post-translational

modifications and is involved in many biological processes such as, quality control of nascent glycoproteins, protein folding and stability, cell-cell signal transduction, and cellular adhesion. Alterations of N-glycosylation have been reported to be associated with the progress of various kinds of cancers. Moreover, glycosylation fidelity is essential for therapeutic glycoproteins' efficacy and safety. Characterization of the glycans typically requires the release of the glycan from the protein. Different approaches for glycan release are described herein that allow for rapid, reproducible, high-throughput, and unbiased glycan and protein separation, and preservation. The released glycans and proteins are suitable for downstream characterization. Some of the approaches described herein allow, for the first time, for analysis of glycan occupancy of glycosylation sites, in addition to identification of glycosylation sites themselves.

[0005] Some aspects of this disclosure are based on the recognition that there is a need for methods that allow one or more of the following: 1) release a glycan from a protein, 2) separate glycan and protein in a manner that maintains the integrity of both glycan and protein for downstream analysis (including, but not limited to, characterization of

glycosylation sites and occupancy), and 3) accomplish in a rapid, high throughput manner, without cumbersome cleanup.

[0006] Some aspects of this disclosure are based on the recognition that efficient methods for isolating and analyzing glycan chains from glycosylated proteins are a prerequisite for the analysis of protein glycosylation on proteomic scale. Existing methods do not allow for the recovery of the glycan and the polypeptide fractions from glycosylated proteins, but only for one of the two fractions. Further, existing methods for separating or isolating a carbohydrate, e.g., a glycan, from a mixture of carbohydrates, e.g., glycans, and polypeptides are also time consuming, labor intensive and costly, which is prohibitive to their broad use in research and diagnostics.

[0007] Some aspects of this disclosure provide a technology that is useful for separating carbohydrates, e.g., glycans, and/or polypeptides from a mixture comprising both. The technology described herein can be used, for example, in glycosylation analysis of glycoproteins, and can be scaled to be used for the analysis of a single glycoprotein, or for glycoproteome-wide analyses. Glycosylation analysis according to aspects of the technology provided herein, are useful for diagnostic purposes, for example, to detect aberrant glycosylation or glycosylation associated with a disease or disorder in a patient, and for quality control procedures, e.g., in the context of engineered proteins, such as therapeutic antibodies and antibody fragments. In some embodiments, glycosylation analysis according to aspects of this invention can be used for quality control of therapeutic glycoproteins (e.g., glycan occupancy of glycosylation sites for therapeutic antibodies or proteins). [0008] The technology described herein overcomes some of the drawbacks of current methodologies and allows, for the first time, to separate and/or isolate the glycan portion and the protein portion of a glycoprotein while maintaining the structural integrity of each component for downstream analysis, opening up new avenues for glycoproteomic analysis in both clinical and research contexts. Separation of carbohydrate (e.g., glycan) and polypeptide fractions comprised in a mixture can be performed in a selective way, which is useful, e.g., if a specific, known polypeptide or carbohydrate or glycan is to be investigated, or can be performed in an unbiased manner, which is particularly useful for glycoproteomics applications.

[0009] Some aspects of this disclosure relate to the surprising discovery that glycans and polypeptides are difficult to separate after release from a glycoprotein because of non- covalent glycan-polypeptide interactions. Some aspects of this disclosure relate to the surprising discovery that non-covalent interactions between glycans and polypeptides can be reduced via acidification, thus minimizing/reducing non-covalent glycan-polypeptide interactions while maintaining the structural integrity of both glycan and polypeptide. Some aspects of this disclosure relate to the surprising discovery that glycans and/or polypeptides can be physically separated or isolated from glycan-polypeptide mixtures after reduction of non-covalent interaction while continuing to maintain the integrity of each component. Some aspects of this disclosure provide methods, reagents, and devices that allow for the reduction of glycan-polypeptide interactions to a level that is not detrimental for separation of the two fractions.

[0010] Some aspects of this disclosure provide a method of separating a polypeptide and/or a carbohydrate glycan from a mixture comprising the polypeptide and the glycan. In some embodiments, the method comprises (a) acidifying the mixture; and (b) separating the polypeptide and/or the carbohydrate from the mixture. In some embodiments, the

carbohydrate is a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide. In some embodiments, the carbohydrate is a glycan. In some embodiments, the glycan and the polypeptide are cleaved from a glycoprotein, and wherein the method comprises cleaving a bond between the glycan and the polypeptide.

[0011] Some aspects of this disclosure provide a method of separating a polypeptide and/or a glycan from a glycoprotein comprising the polypeptide bound to the glycan. In some embodiments, the method comprises (a) cleaving a bond between the polypeptide and the glycan, thus forming a mixture of the polypeptide and the glycan; (b) acidifying the mixture; and (c) separating the polypeptide and/or the glycan. [0012] In some embodiments, the glycan is selected from the group consisting of an

O-glycan and an N-glycan. In some embodiments, the glycan is bound to the polypeptide via an amino group of an Asparagine residue comprised in the polypeptide. In some

embodiments, the glycan is bound to the polypeptide via a hydroxyl group of a serine or threonine residue comprised in the polypeptide. In some embodiments, the bond is a covalent bond. In some embodiments, the mixture has a pH of 4.5-13. In some embodiments, the mixture has a pH of 6.5-13. In some embodiments, the acidifying comprises changing the pH of the mixture of the polypeptide and the glycan to pH 2-4. In some embodiments, the mixture is acidified by contacting the mixture with formic acid, trifluoro-acetic acid, or acetic acid. In some embodiments, the cleaving of the bond comprises contacting the glycoprotein with a glycosidase. In some embodiments, the glycosidase comprises an endoglycosidase. In some embodiments, the glycosidase comprises an exoglycosidase. In some embodiments, the glycosidase comprises one or more endoglycosidases selected from the group comprising peptide N-glycosidase F, endoglycosidase H, endoglycosidase Hf, endoglycosidase F, endoglycosidase S, peptide N-glycosidase A, and O-glycanase. In some embodiments, the cleaving of the bond comprises base elimination. In some embodiments, the cleaving of the bond comprises exposing the glycoprotein to microwaves. In some embodiments, the microwaves are generated by a domestic microwave device.

[0013] In some embodiments, a sub-moiety glycan is released, separated, and/or isolated. The term sub-moiety glycan refers to a carbohydrate that constitutes part of an intact glycan. For example, in an intact glycan comprising a plurality of monosaccharides linked together, each monosaccharide or each moiety comprising some but not all

monosaccharide units comprised in the glycan would be sub-moiety glycans. In some embodiments, a sub-moiety glycan comprises at least 2, at least 3, at least 4, or at least 5 monosaccharide units. In some embodiments, the intact glycan can be determined by analyzing a sub-moiety glycan released from it.

[0014] In some embodiments, the method comprises separating the glycan released from the polypeptide. In some embodiments, the glycan and the polypeptide have a different molecular weight/size and the separating comprises separating the polypeptide and the glycan based on their molecular weight/size difference. In some embodiments, the separating comprises size fractionation, size exclusion, or filtration over a semi-permeable membrane. In some embodiments, the filtration over a semi-permeable membrane comprises dialysis or spin-filter centrifugation. In some embodiments, the method is carried out under non- denaturing conditions. In some embodiments, the method further comprises fragmenting the polypeptide. In some embodiments, the fragmenting comprises digesting the polypeptide with a protease. In some embodiments, the method comprises isolating the glycan separated from the polypeptide. In some embodiments, the method comprises isolating the polypeptide separated from the glycan. In some embodiments, the method further comprises analyzing the glycan and/or the polypeptide after the separating. In some embodiments, analyzing comprises subjecting the glycan and/or the polypeptide to high performance liquid

chromatography, capillary electrophoresis, and/or mass spectrometry. In some embodiments, the method further comprises modifying or labeling the carbohydrate or the glycan. In some embodiments, the labeling comprises permethylation or fluorophore labeling. In some embodiments, the fluorophore labeling comprises 2-AA (2-aminobenzoic acid (anthranilic acid)) or 2-AB (2-aminobenzamide) labeling.

[0015] Some aspects of this disclosure provide a device for separating a glycan and/or a polypeptide from a glycan/polypeptide mixture. In some embodiments, the device comprises a sample compartment, a collection compartment, and a molecular weight/size- selective material separating the sample compartment from the collection compartment. In some embodiments, the molecular weight/size- selective material has a molecular weight/size cutoff that is between the molecular weight/size of the glycan and the molecular weight/size of the polypeptide. In some embodiments, the molecular weight/size- selective material comprises a polyether sulfone. In some embodiments, the molecular weight/size- selective material comprises an acidic moiety. In some embodiments, the molecular weight/size- selective material comprises a carboxyl- and/or an anhyhdride In some embodiments, the molecular weight/size- selective material is a semi-permeable membrane having a molecular weight/size cutoff above the molecular weight/size of the glycan and below the molecular weight/size of the polypeptide. In some embodiments, the molecular weight/size cutoff of the membrane is 10 kDa - 30 kDa. In some embodiments, the molecular weight/size cutoff of the membrane is 15 kDa - 18 kDa. In some embodiments, the molecular weight/size cutoff of the membrane is 20kDa. In some embodiments, the molecular weight/size cutoff is stable over a pH range of pH 2 - pH 12. In some embodiments, the molecular weight/size- selective material does not comprise a glycan. In some embodiments, the molecular weight/size- selective material is a polyethersulfone membrane. In some embodiments, the device is suitable for exposure to heat. In some embodiments, the device is suitable for exposure to microwaves. In some embodiments, the device comprises a centrifugal filter device. In some embodiments, the centrifugal filter device is suitable for use at up to 10000g. In some embodiments, the centrifugal filter device is suitable for use at more than 10000g, e.g., at 20000 g, at 30000g, at 40000g, or at higher g values.

[0016] Some aspects of this disclosure provide a kit for separating a glycan and/or a polypeptide from a polypeptide/glycan mixture. In some embodiments, the kit comprises a device for separating a glycan and/or a polypeptide as described herein. In some

embodiments, the kit also comprises a buffer or reagent suitable for separating a glycan and a polypeptide. In some embodiments, the kit comprises a glycosidase. In some embodiments, the glycosidase comprises an endoglycosidase. In some embodiments, the kit comprises an exoglycosidase. In some embodiments, the glycosidase comprises one or more

endoglycosidases selected from the group comprising peptide N-glycosidase F,

endoglycosidase H, endoglycosidase Hf, endoglycosidase F, endoglycosidase S, peptide N- glycosidase A, and O-glycanase. In some embodiments, the kit comprises O 18 water. In some embodiments, the endoglycosidase is in a solution comprising O 18 water. In some embodiments, the kit comprises an acidifying reagent, for example, an acid or in dry form or in solution. In some embodiments, the acidifying solution comprises formic acid, trifluoro- acetic acid, or acetic acid. In some embodiments, the kit comprises a protease. In some embodiments, the protease is trypsin. In some embodiments, the kit comprises instructions for separating a glycan and a polypeptide.

[0017] Other advantages, features, and uses of the technology provided by this disclosure will be apparent from the detailed description of certain non-limiting

embodiments; the drawings, which are schematic and not intended to be drawn to scale; and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. An exemplary embodiment of the instantly disclosed technology.

In this embodiment, a sample comprising glycosylated proteins is transferred into the sample compartment of a spin filter device and treated with a glycosidase in order to release glycans from glycosylated proteins. The deglycosylation reaction is performed in a domestic microwave and the sample is subsequently acidified to reduce glycan/polypeptide interactions. Released glycans are separated by spin filtration, and the remainder of the sample, comprising the de-glycosylated protein fraction, is treated with a peptidase (e.g. trypsin) to fragment the remaining proteins. Fragmented peptides are then separated from any large molecular weight molecules left in the sample by spin filtration. The glycans and the peptides obtained from the sample are further processed in glycomics and proteomics applications, respectively.

[0019] Figure 2. An application of the instantly disclosed technology on standard glycoprotein bovine ribonuclease B (Sigma- Aldrich). Released N-glycans from various amounts of ribonuclease B: a) 1 μg; b) 5 μg; and c) 10 μg, were captured by the instantly disclosed technology. Released N-glycans were permethylated and analyzed by MALDI-MS (matrix assisted laser desorption ionization - mass spectrometry). All known five high- mannose N-glycans (Man 5, Man 6, Man7, Man 8, and Man 9) were observed in all spectra, irrespective of the starting amount, demonstrating the high efficiency to capture N- glycans by the instantly disclosed technology.

[0020] Figure 3. An application of the instantly disclosed technology on standard glycoprotein bovine fetuin. Even large and complex sialylated released N-glycans from bovine fetuin were efficiently separated and captured by the technology provided herein. The effectiveness of deglycosylation was demonstrated by the protein bands shift in the gel: native fetuin is around 56K Da mass, after the removal of three units of N-glycans per protein, the remaining mass is around 49K Da. Released N-glycans were permethylated and analyzed by MALDI-MS. The peak pattern of four major glycan peaks m/z 3963.8, m/z 3602.6, m/z 3241.5, and m/z 2792.3 are the same as previously reported. Two previously unreported, low abundance glycans with 4 and 5 sialic acid residues (arrows) were identified in this well-characterized model glycoprotein.

[0021] Figure 4. Application of the instantly disclosed technology on standard glycoprotein bovine fetuin.

[0022] Figure 5. Application of the instantly disclosed technology on human IgG from serum (Sigma- Aldrich). Sequences correspond, from top to bottom, to SEQ ID NOs 1- 15, respectively.

[0023] Figure 6. Application of the instantly disclosed technology on human urine.

Figure 7. Application of the instantly disclosed technology on complex human body fluids: urine and plasma from a healthy donor.

[0024] Figure 8. Application of the instantly disclosed technology on three different human urine samples.

[0025] Figure 9. Application of the instantly disclosed technology on three different human plasma samples.

[0026] Figure 10. Application of the instantly disclosed technology on three different pairs of human plasma and urine samples. [0027] Figure 11. Application of the instantly disclosed technology on HeLa cell lysate.

[0028] Figure 12. Comparison of reproducibility and resolution of the instantly disclosed technology with upfront de-N-glycosylation and without upfront de-N- glycosylation.

[0029] Figure 13. Binned density of peptide counts per protein.

[0030] Figure 14. Partial occupancy of a glycosylation site. Schematic of an exemplary method for detection of glycosylation site occupancy. The sequence

KYNSQNQSNNQ corresponds to SEQ ID NO: 16.

[0031] Figure 15. Identification of a partial occupied glycosylation site, a unique outcome provided by the instantly disclosed technology. The sequence DLDMFINASK corresponds to SEQ ID NO: 17.

[0032] Figure 16. An example of the instantly disclosed technology identifying a partial occupied glycosylation site: Apolipoprotein M.

[0033] Figure 17. An example of the instantly disclosed technology identifying a partial occupied glycosylation site: Kininogen-14.

[0034] Figure 18. Identifying a complex glycoprotein (Attractin) with many glycosylation sites.

[0035] Figure 19. Demonstration of de-N-glycosylation by domestic microwave oven.

[0036] Figure 20. De-N-glycosylation of depleted human urine proteins.

[0037] Figure 21. SDS-PAGE of bovine RNase B (A); bovine fetuin (B); human IgG

(C); and MARS-7 bound fractions from two human plasma samples (D & E).

[0038] Figure 22. MALDI-MS spectra of permethylated N-glycans from bovine

RNase B (A); bovine fetuin (B); and human IgG (C).

[0039] Figure 23. Bovine fetuin digested by PNGase F in a traditional 37 °C oven at varying incubation times.

[0040] Figure 24. The MALDI-MS of permethylated N-glycans of bovine fetuin.

[0041] Figure 25 Analysis of an additional urine sample.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0042] Glycosylation is the most abundant protein post-translational modification, playing an important role in protein folding, stability, and function. Current methods for glycosylation analysis do not allow for a simultaneous assessment of the protein and the glycan portion of a glycosylated protein, which is a major drawback for the investigation of glycosylation on a proteomic scale, as well as for engineering and quality control of therapeutic proteins. Based on the emerging market of protein therapeutics, most of which rely on correct glycosylation for efficacy and safety, and glycoprotein-based diagnostics, there is a significant need today for improved technologies that can routinely be applied in both a clinical and a research setting for the analysis of protein glycosylation.

[0043] The term glycosylation, as used herein, refers to either (i) an enzymatic process that attaches a carbohydrate, e.g., a glycan, to a protein, lipid, or other organic molecule, or (ii) a presence of one or more glycans attached to a protein, lipid, or other organic molecule, for example, in the case of proteins, as a result of post-translational modification. Without wishing to be bound by any particular theory, it is believed that naturally occurring protein glycosylation can be a form of co-translational and post- translational modification, in that glycan addition occurs after an amino acid residue is added to the amino acid chain of a peptide or protein.

[0044] The term carbohydrate refers to an organic compound consisting of carbon, hydrogen, and oxygen, including, for example, monomeric sugars (monosaccharides), oligomeric sugars (oligosaccharides, e.g., disaccharides, trisaccharides, etc.), and

polysaccharides. In some embodiments, a carbohydrate is a glycan. Carbohydrates generally have the molecular formula C_nH_2nO_n but important exceptions exist, e.g., deoxyribose, a component of DNA, is a sugar of the formula C₅H₁₀O₄. Carbohydrates are also sometimes referred to as polyhydroxy aldehydes and ketones. Carbohydrates can comprise a single sugar moiety, or a plurality of sugar moieties, and can be classified based on the number of sugar moieties comprised into monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Examples of monosaccharides include, without limitation, glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Naturally produced carbohydrates include N-glycans, O-glycans,

monosaccharides, oligosaccharides, polysaccharides (large polymers of sugar chains e.g. cellulose), and glycolipids. N-glycans are a large and heterogeneous post-translational modification that links to the side chain of asparagine in protein backbone via a β-amide bond. Mammalian N-glycans can be enzymatically released using a universal N-glycanase: PNGase F (peptide-N4-(acetyl-β-glucosaminyl) asparagine amidase). Depending on the nature of the glycoproteins it typically takes at least several hours to perform this enzyme reaction using conventional methods. Carbohydrates may contain modified saccharide units such as 2'-deoxyribose wherein a hydroxyl group is removed, 2'-fluororibose wherein a hydroxyl group is replace with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose, (e.g., 2'-fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

[0045] The term glycan, as used herein, refers to a type of carbohydrate, typically an oligosaccharide or polysaccharide. Glycans typically comprise monosaccharide residues linked by O-glycosidic linkages. Glycans can be homo- or heteropolymers of

monosaccharide residues, and can be linear or branched. In the context of protein

glycosylation, the term glycan refers to the carbohydrate portion of a glycoprotein. In some embodiments, the term glycan refers to glycans having a molecular weight of less than about 200kDa, less than about 150kDa, less than about 100kDa, less than about 50kDa, less than about 40kDa, less than about 30kDa, less than about 25kDa, less than about 20kDa, less than about 15 kDa, less than about 10 kDa, less than about 9 kDa, less than about 8 kDa, less than about 7 kDa, less than about 6 kDa, less than about 5 kDa, less than about 4 kDa, less than about 3 kDa, less than about 2 kDa, less than about 1.5 kDa, less than about 1 kDa, or less than about 500 Da. In some embodiments, the term glycan refers to any glycans except glycosaminoglycans.

[0046] Without wishing to be bound to any particular theory, glycans are typically divided into five classes: (i) N-linked glycans are glycans attached to a nitrogen of an asparagine or arginine residue side-chain of proteins or peptides; (ii) O-linked glycans are glycans attached to the hydroxy group oxygen of a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residue side-chain of proteins or peptides, or to oxygens on lipids such as ceramide; (iii) phospho-glycans are glycans attached to the phosphate moiety of a phospho- serine residue of a protein or peptide; (iv) C-linked glycans are glycans attached to a carbon atom of a tryptophan residue side-chain; and (iv) GPI anchors are glycans linking proteins to lipids.

[0047] In contrast to the non-enzymatic, chemical reaction of protein glycation, protein glycosylation is an enzyme-mediated, site-specific process, in which specific glycan chains are added to a specific residue of a specific protein by the respective glycosyl transferase enzyme. Glycosylation can significantly change the physical and functional properties of a protein, and defective or aberrant glycosylation may result in a loss of function of a given protein. Further, differential glycosylation increases protein diversity in the proteome, because almost every aspect of glycosylation can be varied, including the site of glycan linkage, e.g., the amino residue of a protein to which a given glycan chain is attached; glycan composition (e.g., the type of sugars comprised in a given glycan chain); glycan structure (e.g., branched or unbranched); and glycan chain length (e.g., short- or long-chain oligosaccharides).

[0048] The term protein is used herein interchangeably with the term polypeptide, and refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. A protein may be a naturally occurring protein, a fragment of a naturally occurring protein, or an engineered protein, for example, a recombinant protein, or a protein in which one or more amino acid residues are non-naturally occurring residues, e.g., modified amino acid residues, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex.

[0049] Protein glycosylation serves a variety of structural and functional roles, for example, in membrane and secreted proteins, and the majority of proteins synthesized in the rough ER undergo glycosylation. Glycosylation is also present in the cytoplasm and nucleus as the O-GlcNAc modification. Protein glycosylation plays a role in protein folding and stability. Some proteins do not fold correctly unless they are glycosylated first. Glycans have been reported to modulate protein half-life, e.g., some glycans confer stability to secreted glycoproteins which are rapidly degraded in the absence of correct glycosylation. Glycosylation also plays a role in cell-cell adhesion, e.g., via sugar-binding proteins such as lectins, which recognize specific glycan moieties on specific cell surface glycoproteins.

[0050] Protein glycosylation is an important aspect in the development of therapeutic proteins and peptides, as many protein therapeutics that are developed or already approved are glycosylated and correct glycosylation is important or essential for therapeutic efficacy. For example, glycan composition and structure play a critical and key role in therapeutic antibody and antibody fragment efficacy and safety. Human antibodies have a conserved glycosylation site at Asn-297. Correct glycosylation at Asn-297 has been reported to be essential for therapeutic antibody function, and subtle changes in glycosylation structure have been linked to significant safety issues (see, e.g., Walsh, Biopharmaceutical benchmarks 2010 Nature Biotechnology 28, 917-924 (2010); Anthony, Recapitulation of WIG antiinflammatory activity with a recombinant IgG Fc. Science. 2008 Apr 18;320(5874):373-6; and Bosques, Chinese hamster ovary cells can produce galactose-a-1 ,3 '-galactose antigens on proteins Nature Biotechnology 28, 1153-1156 (2010); the entire contents of each of which are incorporated herein by reference).

[0051] It is important for therapeutic protein approval and production that proteins are produced with the correct glycosylation pattern and that precise quality control measures are implemented to assure correct glycosylation, for example, to avoid loss of efficacy and detrimental effects in the case of aberrant glycosylation. In order to obtain correctly glycosylated peptide or protein therapeutics, mammalian cell lines are often required for their production, since bacterial, yeast, or insect cells often cannot produce the complex glycans and/or glycosylation patterns required for proper protein function.

Technology Overview

[0052] Some aspects of this disclosure provide a technology that allows separation and/or isolation of a carbohydrate (e.g., glycan) and/or a polypeptide from a mixture comprising both. In some embodiments, the separation and/or isolation can be achieved in a single device, e.g., a single centrifugal filter device, thus avoiding extensive sample transfer. Some aspects of this disclosure relate to the recognition that one major roadblock to efficient glycosylation analysis, for example, in the context of glycoproteomic research, diagnostic biomarker discovery, and therapeutic protein production and quality control is that current methods do not allow the isolation and analysis of both the purified glycan portion and the protein portion of glycoprotein molecules from a single sample. Current methods for glycoprotein analysis, include, but are not limited to, C18-SPE(C18-based solid phase extraction) , PGC-SPE (porous graphitized carbon-based solid phase extraction), organic solvent precipitation, size-based gel chromatography, NIBRT glycomics, and prozyme glycoprep. The current methods are burdened by a host of drawbacks, which hamper their broad application to glycoproteomics. For example, existing methods do not allow for the capture of both protein and glycan fractions of a given glycoprotein. Isolation of glycans and other carbohydrates is also typically associated with cumbersome, time consuming and complex purification procedures, and current methods are generally limited to specific glycans or other carbohydrates, or specific proteins, making unbiased capture of, e.g., glycans and/or proteins from a glycoprotein sample impractical. Additionally, current methods typically require extensive sample handling, which is associated with significant

protein/glycan loss and/or damage, limiting the application of these methods to scenarios where large amounts of starting material is readily available.

[0053] For example, some affinity chromatography based methods do not allow for complete elution of pure and intact protein/peptide and/or glycan, and are often burdened by poor recovery, making them impractical in many applications. Additionally, contaminating glycan or peptide/protein must be purified out in an additional step and the data must be adjusted to account undesired cross-contamination. Further, for some mixtures, the elution volume can require evaporative techniques that take days before the additional purification steps can be performed. Further, current methods cannot easily be scaled up, for example, from a single glycoprotein to a plurality of glycoproteins, or to complex mixtures of glycoproteins, and are time- and cost-intensive to the point that broad applications in clinical or research settings are impractical. The technology described herein overcomes the drawbacks of existing methods.

[0054] Some aspects of this disclosure provide a technology that overcomes this shortcoming of current technologies. In some embodiments, a technology is provided herein that allows for the separation and isolation of both the glycan portion and the protein portion of glycoproteins. The instantly disclosed technology allows for the efficient separation and isolation of glycans from glycosylated proteins, without losing the protein fraction of the parent glycosylated proteins. The protein fraction can be isolated as well, from the same glycoprotein, glycan-polypeptide mixture, or biological sample, and both glycan and protein fractions can then be subjected to further analysis. One important advantage of the technology described herein over current technologies is that it allows for an unbiased isolation of glycans and polypeptides from glycoproteins, making it particularly useful for applications in which isolation bias is detrimental, such as glycomics and proteomics applications.

[0055] The term separation, as used herein, refers to a physical separation of an entity, e.g., a molecule or a class of molecules, from another entity, e.g., another molecule or class of molecules, or a mixture of entities. For example, a fractionation of a mixture of molecules by molecular weight results in the separation of molecules below the molecular weight cutoff from those above the molecular weight cutoff. Separation does not necessarily require purification, but a purification typically includes a separation of the entity to be purified from any unwanted entities. The term isolation, as used herein, refers to the removal, through human intervention, of a molecule, for example, a carbohydrate or a polypeptide, from a component, e.g., another molecule or a class of molecules with which it is associated in nature, or, in the case of non-naturally occurring molecules, with which it is associated when originally produced. In some embodiments, the removal may be by separation of the molecule from the component. In other embodiments, the removal may be by a destruction of an association between the molecule and the component, or by a destruction or conversion of the component. For example, a glycan enzymatically cleaved from a naturally occurring or engineered glycoprotein, a glycan physically separated from the polypeptide portion of a parent glycoprotein, a glycan left over after protease digest of a glycoprotein, and a purified glycan are non-limiting examples of what may be referred to as an isolated glycan in some embodiments. The term purification, as used herein, refers to an increase in the concentration of a particular molecule, e.g., a glycan or a polypeptide, in a sample. In some embodiments, purification entails an increase of the abundance of the molecule in the sample to more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, or more than 99.9% of the molecule of its class, or of all molecules, or all molecules other than a solvent or excipient in the sample.

[0056] Some aspects of this disclosure relate to the surprising discovery that non- covalent interactions between free carbohydrates (e.g., glycans) and proteins, for example, hydrogen bonding, ionic bonding, van-der-Waals interactions etc., represent a barrier to the separation and/or isolation of carbohydrates (e.g., glycans) and proteins from mixtures containing both. For example, in a mixture of free carbohydrates (e.g., glycans) and polypeptides, or after release of a glycan fraction from a parent glycoprotein, non-covalent interactions remain a barrier to separation. Some aspects of this disclosure relate to the surprising discovery that glycan and protein fractions of glycoproteins are often difficult or impossible to separate even after release of the glycan from the glycoprotein, because of non- covalent interactions between glycans and proteins. Similarly some aspects of this disclosure relate to the surprising discovery that free carbohydrate and protein fractions comprised in a mixture of both are often difficult or impossible to separate because of non-covalent interactions between carbohydrates and proteins. Some aspects of this disclosure relate to the surprising discovery that non-covalent carbohydrate-protein (e.g., glycan-protein) interactions can be efficiently decreased, for example, in a sample comprising carbohydrates (e.g., glycans) and proteins, by acidification, e.g., by acidifying a sample comprising carbohydrates (e.g., glycans) and proteins, to a level which allows physical separation of carbohydrates (e.g., glycans) and proteins, for example, based on their respective molecular weight. Some aspects of this disclosure relate to the surprising finding that, once the non-covalent interactions between carbohydrates (e.g., glycans) and proteins in a carbohydrate-protein (e.g., glycan-protein) mixture are decreased via acidification, the carbohydrate and protein fractions can be effectively separated from each other via physical separation methods, for example, via a spin filter having a molecular weight cutoff between the molecular weight of the protein fraction and the molecular weight of the carbohydrate (e.g., glycan) fraction, or via size fractionation or dialysis methods.

[0057] In some embodiments, methods, devices, and reagents are provided that allow for the separation and/or isolation of either or both the glycan and protein fractions comprised in a glycoprotein or in a mixture of glycans and proteins. This includes methods, devices, and reagents for the release of glycans from parent glycoproteins, for the decrease of non- covalent interactions between glycans and proteins, and for the separation of glycans and proteins from a mixture of both based on their respective molecular weight.

[0058] The technology described herein can be employed for the separation and/or isolation of carbohydrates (e.g., glycans) and proteins from various types of

carbohydrate/protein (e.g., glycan/protein) mixtures, including, but not limited to, mixtures comprising a single glycan and a single protein, for example, originating from a single glycoprotein; more complex glycoprotein mixtures, for example, mixtures comprising a plurality of glycans released from a single parent glycoprotein or from a plurality of parent glycoproteins and the respective protein fraction(s) of the parent glycoprotein(s); as well as biological samples comprising a carbohydrate and a protein, e.g., body fluid samples (e.g., blood, serum, plasma, urine, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, sweat, tears, etc.) obtained from a subject. In some embodiments, any mixture of glycans and proteins, for example, any sample comprising such a mixture, may be subjected to a procedure described herein for the separation and/or isolation of the glycan and/or protein fractions comprised therein.

[0059] In some embodiments, a method is provided that comprises separating a polypeptide and/or a carbohydrate, for example, a glycan, from a mixture comprising the polypeptide and the carbohydrate. In some embodiments, the method comprises (a) acidifying the mixture; and (b) separating the polypeptide and/or the carbohydrate, for example, the glycan, from the mixture. In some embodiments, the separation of the polypeptide and the carbohydrate is based on a difference in the molecular weight of the carbohydrate and the polypeptide. In some embodiments, the method comprises providing a mixture of the polypeptide and the carbohydrate. In some embodiments, the mixture is obtained by cleaving a glycan and a polypeptide from a glycoprotein. In some embodiments, the method comprises cleaving a covalent bond between the glycan and the polypeptide.

[0060] In some embodiments, a method of separating a polypeptide and/or a glycan from a glycoprotein comprising the polypeptide bound to the glycan is provided. In some embodiments, the method comprises (a) cleaving a bond between the polypeptide and the glycan, thus forming a mixture of the polypeptide and the glycan; (b) acidifying the mixture; and (c) separating the polypeptide and/or the glycan. In some embodiments, the separation of the polypeptide and the carbohydrate is based on a difference in the molecular weight of the carbohydrate and the polypeptide.

[0061] In some embodiments, the glycan and the protein to be separated are comprised in a glycoprotein. In some embodiments, the glycan and the protein fraction of the glycoprotein are conjugated via a covalent bond. In some embodiments, the method comprises releasing the glycan from the glycoprotein. In some embodiments, the releasing of the glycan comprises breaking or cleaving a covalent bond between the glycan and the protein fraction of the glycoprotein. In some embodiments, the covalent bond is cleaved by an enzyme. In some embodiments, a glycan covalently bound to a protein fraction of a glycosylated protein is released from the parent protein by contacting the glycosylated protein with an enzyme that cleaves a covalent bond between the glycan and the parent protein. In some embodiments, the method comprises contacting the glycoprotein with an

endoglycosidase. In some embodiments, the endoglycosidase comprises an N-glycanase (e.g., peptide N-glycosidase F, endoglycosidase H, endoglycosidase Hf, endoglycosidase F, endoglycosidase S, and/or peptide N-glycosidase A), an O-glycosidase or O-glycanase, or with an exoglycosidase. In some embodiments, a glycan is released from a parent glycoprotein by cleaving a covalent bond between the glycan and the protein fraction via base elimination. In some embodiments, glycan release via base elimination comprises contacting a glycoprotein with a base, for example, NaOH or KOH, ammonia, dimethylamine, trimethylamine, etc., in an amount sufficient to create a pH equal to or greater than 11. In some embodiments, release of a glycan from a glycoprotein comprises exposing the glycoprotein to a source of energy, for example, to heat or microwave energy, as described in more detail elsewhere herein.

[0062] In some embodiments, a method of separating a polypeptide and a glycan from a glycoprotein, as provided herein, comprises exposing the glycoprotein to reducing and/or alkylating conditions. In some embodiments, the method comprises digesting the polypeptide or the glycoprotein with a protease. In some embodiments, the protease is trypsin. In some embodiments, the method comprises separating the glycan and a protease- digested fragment of the parent polypeptide.

[0063] In some embodiments, the mixture of carbohydrate (e.g., glycan) and polypeptide from which a carbohydrate and/or a polypeptide is separated and/or isolated has a neutral or basic pH, for example, a pH within the range of about 4.5 to about 13, within the range of about 6.5 to about 13, within the range of about 4.5 to about 9, or within the range of about 6.5 to about 9. In some embodiments, a method a method of separating a polypeptide and/or a glycan from a mixture comprising the polypeptide and the glycan comprises acidifying the mixture. In some embodiments, acidifying comprises changing the pH of the mixture of the polypeptide and the glycan to a pH within the range of about 2 to about 4. In some embodiments, the acidifying comprises contacting the mixture with a volatile organic or inorganic acid, for example, formic acid, trifluoro-acetic acid, or acetic acid. Other suitable acids for acidifying the mixture will be apparent to the skilled artisan. The disclosure is not limited in this respect.

[0064] The technology provided herein can be employed to separate and/or isolate any glycan released from a parent glycoprotein and/or the protein that the glycan was released from. In some embodiments, the glycans and parent proteins are separated and/or isolated based on a difference in molecular weight between the glycan and the protein.

However, the instantly disclosed technology is not limited to separation based on molecular weight. Other approaches for separation of glycans and/or proteins from glycan/protein mixtures suitable for use according to aspects of this disclosure are described herein, and additional approaches will be apparent to the skilled artisan based on the instant disclosure.

Optional pre-processing: alkylation and reduction of glycoproteins

[0065] In some embodiments, a method of separating a polypeptide and a glycan from a glycoprotein, as provided herein, comprises a reduction and/or alkylation step. Such steps are particularly useful if separation and/or isolation of glycans and polypeptides from glycoproteins comprising cysteine residues is attempted. Cysteine residues can form disulfide (S-S)-bonds, which stabilize the 3-D structure of proteins, and may hinder chemical or enzymatic release of glycans from the glycoprotein. For example, in some embodiments comprising an enzymatic release of a glycan from a cysteine residue-comprising

glycoproteins is attempted, the method comprises a step of contacting the glycoprotein with a reduction agent and/or an alkylating agent. In some embodiments, the reduction agent is provided in an amount sufficient to dissociate a cysteine bridge (S-S disulfide bonds) in the glycoprotein. In some embodiments, the reduction agent is provided in an amount sufficient to dissociate all cysteine bridges (S-S disulfide bonds) in the glycoprotein. In some embodiments, the glycoprotein is contacted with an alkylating agent. In some embodiments, the alkylating agent is provided in an amount sufficient to alkylate the S-moiety of a reduced cysteine residue in the glycoprotein. In some embodiments, the alkylating agent is provided in an amount sufficient to alkylate all reduced S-moieties of reduced cysteine residues comprised in the glycoprotein.

[0066] Reduction and/or alkylation are useful to achieve efficient enzymatic release of glycans from glycoproteins by reducing steric hindrance of enzyme access to glycans through reduction of secondary and higher structures of glycoproteins, resulting in an "open" or linearized amino acid sequence, and thus facilitating enzyme access, e.g., PNGase F access to target glycan groups. Reduction and/or alkylation can also be used to attach a specific alkyl group to those cysteine residues that form S-S bonds in the native glycoprotein. Such specific alkyl groups can serve as readily identifiable tags in subsequent analytic approaches, such as mass spectroscopy assays.

[0067] Methods and reagents for reduction and alkylation of proteins, including glycoproteins, are well known to those in the art. Some exemplary methods and reagents (e.g., DTT/IAA) are described in more detail elsewhere herein. Additional suitable methods and reagents will be apparent to those of skill in the art based on the instant disclosure. The disclosure is not limited in this respect.

[0068] In some embodiments, a method of separating a polypeptide and a glycan from a glycoprotein, as provided herein, comprises a step of purifying the reduced and/or alkylated glycoprotein after reduction and/or alkylation. In some embodiments, the purification comprises a buffer exchange in a filter column. Suitable reducing and alkylating reagents for such embodiments typically have a lower molecular weight than the reduced and/or alkylated glycoproteins produced as a result of the reduction and/or alkylation. In some embodiments, reduction and/or alkylation are performed on the same filter column that is used subsequently for separating a glycan released from the reduced and/or alkylated glycoprotein from the parent protein fraction of that glycoprotein, allowing for one-column pre-processing and separation. Purification on a filter column may be carried out according to the recommendations of the manufacturer of the column, or according to embodiments described in more detail elsewhere herein. Using the same column for pre-processing and, if required, purification of the pre-processed glycoproteins or glycan-protein mixtures avoids or minimizes sample transfer steps and any associated loss of starting material. In other embodiments, a reduced and/or alkylated glycoprotein is purified in a purification step that is not carried out on a filter column used for separating a glycan released from the reduced and/or alkylated glycoprotein from the parent protein fraction of that glycoprotein. This is useful if a reduction and/or alkylation protocol is used that is incompatible with a filter column used.

Optional pre-processing: release of gly cans from glycosylated proteins

[0069] In some embodiments, a method as described herein comprises releasing a glycan from a glycoprotein. Typically, such a method comprises breaking or cleaving a covalent bond connecting the glycan and the protein fraction of the glycoprotein. In some embodiments, a covalent bond between a glycan and a protein is cleaved or broken via enzymatic digest or chemical release methods. Enzyme or chemical release methods may be employed when glycans are covalently attached to a polypeptide backbone. The process of separating the glycan and polypeptide fractions starts, in some embodiments, by contacting the glycoprotein with an enzyme or a chemical that can cleave or break the respective covalent glycan-polypeptide bond. In some embodiments, cleaving or breaking a covalent glycan-polypeptide bond further comprises an input of energy, for example, in the form of heat or microwaves. The glycoprotein is contacted with the enzyme or chemical in an amount sufficient and under conditions suitable for the glycan-polypeptide bond to be broken or cleaved.

[0070] Enzymes suitable for catalyzing the cleavage of a covalently bound carbohydrate, or carbohydrate portions, e.g., from a glycoprotein, according to aspects of this disclosure are known to those of skill in the art. Suitable enzymes include, but are not limited to N-glycanases (e.g., PNGase F, PNGase A, endo H, endo Hf, endo F, endo S); O- glycanases (e.g., endo-a-N-acetylgalactosaminidase); Exoglycosidases (e.g., sialidase, fucosidase, mannosidase, neuraminidase, galactosidase, N-acetylgalactosaminidase, N- acetylglucosaminidase; Glycosaminoglycan-degrading enzymes (e.g., chondroitinase, heparinase, hyaluronidase, keratanase); Glycolipid degrading enzymes (e.g.,

endoglycoceramidases); polysaccharide degrading enzymes (e.g., cellulase, carbohydrase, glycogen phosphorylase, etc.). Additional suitable enzymes for use according to aspects of this disclosure will be apparent to those of skill in the art. The disclosure is not limited in this respect.

[0071] In some embodiments, a chemical method is used to break a covalent glycan- polypeptide bond in order to release a glycan from a glycoprotein. One exemplary method of chemical bond disruption is base elimination, also referred to as alkaline elimination (e.g., for N-glycans, O-glycans, phosphorylation, phosphopantetheinylation). In base elimination, the glycoprotein is contacted with a base, for example, an organic or inorganic base, in an amount efficient to disrupt the respective glycan-polypeptide bond. An input of energy, for example, in the form of heat or microwaves, is used in some embodiments, to shorten exposure times required until bond cleavage is achieved. In some embodiments, the base is NaOH or KOH. In some embodiments, base elimination is performed at a pH within the range of about 9 to about 12. In some embodiments, when performing base-induced glycan release, such as O-glycan or N-glycan release, a reducing agent (e.g., a strong reducing agent such as sodium borohydride (NaBH4) is added to prevent the released glycan from

degrading. In some embodiments, the reducing agent is used at a concentration of about 0.1 M, 0.2 M, 0.3M, 0.4 M, 0.5 M, 1M, or 2 M. In some embodiments, a reducing agent, such as, e.g., NaBH4, is used regardless of which method is used for glycan release, e.g., enzymatic or base-induced, in order to reduce the released glycans into the corresponding alditol form

[0072] Enzymatic and chemical release of glycans from glycoproteins typically occurs at neutral or basic pH, respectively. Accordingly, some glycan-polypeptide interactions typically remain when either methodology is used to release a glycan from a glycoprotein, similar to the non-covalent interactions that exist in mixtures of free glycans with free polypeptides (e.g., in human milk). These interactions are overcome, in some embodiments, by acidification subsequent to the glycan release, as described in more detail elsewhere herein. After acidification, the sample can then be subjected to a separation procedure for separating glycan and polypeptide fractions of the parent glycoprotein, as described in more detail elsewhere herein.

[0073] Some aspects of this disclosure are based on the surprising discovery that glycans can be released from glycoproteins by enzymatic deglycosylation using a microwave, for example, a domestic microwave, to enhance the reaction. Standard de-N-glycosylation protocols usually require a long incubation time ranging from a few hours to overnight.

Although pressure cycling-technology or specific microwave reactors can accelerate the protocol, most researchers have limited access to these instruments due to their high acquisition cost. Some embodiments of this disclosure provide an alternative strategy for de- glycosylation, using an inexpensive domestic microwave oven. In some embodiments, the domestic microwave is a household microwave. In some embodiments, the domestic microwave has a maximum output power of 500 watts, 600 watts, 900 watts, 1000 watts, 1100 watts, 1200 watts, 1300 watts, 1400 watts, 1500 watts, or 2000 watts. In some embodiments, the domestic microwave has a maximum output power of more than 100 watts, more than 200 watts, more than 300 watts, more than 400 watts, more than 500 watts, more than 600 watts, more than 700 watts, more than 800 watts, more than 900 watts, or more than 1000 watts. In some embodiments, the domestic microwave operates at a frequency of 2.45 GHz. In some embodiments, the domestic microwave operates only at a frequency of 2.45 GHz. In some embodiments, the domestic microwave emits microwaves at a wavelength of 122 mm. In some embodiments, a domestic microwave does not comprise a pressurized chamber for sample heating. In some embodiments, the microwave is a microwave as described in Zhou H, Briscoe AC, Froehlich JW, Lee RS. PNGase F catalyzes de-N- glycosylation in a domestic microwave. Anal Biochem. 2012 Apr 16;427(l):33-35, the entire contents are incorporated herein by reference.

[0074] As described in more detail elsewhere herein, purified glycoproteins (bovine

RNase B, bovine fetuin, and human IgGs) and complex glycoprotein samples from human plasma were fully deglycosylated in 20 minutes using this method, without any apparent adverse effects on their glycans or protein backbones. This is the first demonstration of the successful use of a standard domestic microwave oven for de-N-glycosylation for glycomics research.

[0075] Current rapid N-glycan release protocols utilize specific, scientific microwave reactors. With these reactors, rapid N-glycan release was first achieved in 10 minutes on monoclonal antibodies (mAbs), and in 1 hour for standard model glycoproteins (e.g. RNase B). Recent application of high-pressure cycling-technology (PCT) shortened this enzyme reaction to several minutes for RNase B. Immobilization of PNGase F onto solid-phase materials such as capillary columns, and microfluidic-chip have also been reported to improve deglycosylation. Although these techniques can increase the speed of glycan release, they are not widely accessible due to the associated acquisition cost of the required specialized equipment, and the high cost of the immobilized enzyme.

[0076] A domestic microwave-assisted protocol can be very effective for proteolysis digestion with trypsin. Some aspects of this disclosure are based on the surprising discovery that a domestic microwave can also be used for efficient glycan release from glycoproteins using a glycosidase enzyme. Some aspects of this disclosure, accordingly, provide methods for releasing a glycan from a glycoprotein using a domestic microwave. In some embodiments, a rapid (20 minutes or less), simple and inexpensive alternative strategy for N- glycan release using a standard domestic microwave is provided.

Separation of carbohydrate and polypeptide fractions

[0077] Some aspects of this disclosure relate to the surprising discovery that even after release of a glycan from a glycan protein, separation and isolation of the released glycan and the protein based on their molecular weight are often impossible or inefficient because of non-covalent glycan-protein interactions that hinder separation. The same holds true for free carbohydrates (e.g., glycans) and proteins in a carbohydrate-protein mixture. It is believed that the non-covalent interactions are generally stronger in acidic carbohydrates than in neutral carbohydrates, since acidic carbohydrates are typically charged and, thus, may interact with charged groups on the protein backbone or on side chains of amino acids, for example via ionic interactions. Some aspects of this disclosure relate to the surprising discovery that non-covalent interactions between glycans and proteins can efficiently be reduced to a level allowing molecular weight-based separation, for example, by acidifying a mixture of glycans and proteins. Some aspects of this disclosure relate to the surprising discovery that acidification enhances the recovery of acidic carbohydrates, but does not appear to have a detrimental effect on the recovery of neutral carbohydrates.

[0078] In some embodiments, non-covalent interactions between glycans and proteins in a mixture of glycans and proteins are reduced by contacting the mixture with an acid in an amount effective to shift the pH of the mixture into the range of about 1 to about 4.

Acidifying effectively minimizes non-covalent interactions between free glycans and proteins, for example, in a mixture in aqueous solution. In some embodiments, the mixture is generated by release of a glycan from a parent glycoprotein, for example, via enzymatic digest or via chemical treatment as described elsewhere herein. Once the non-covalent interactions between glycans and proteins are minimized, both the glycans and proteins are still present in the same solution, but are now amenable to physical separation based on molecular weight.

[0079] Without wishing to be bound to any particular theory, it is believed that when free carbohydrates, for example, free oligosaccharides (e.g., N-glycans and O-glycans released from glycoproteins, and freely existing oligosaccharides) and proteins or

polypeptides co-exist in an aqueous solution at neutral or basic pH (e.g., pH≥ 4.5 or pH ≥6.5), there are numerous non-covalent interactions between the carbohydrates and proteins, e.g., ionic bonds, hydrogen bonds, and van der Waals interactions, which individually or in combination are strong enough to prevent a separation of the oligosaccharides from the proteins or polypeptides. For example, a carboxyl group of a glycan, e.g., of a sialic acid residue, which is a common residue in acidic glycans, may interact with a primary amine group of a protein or polypeptide backbone, preventing separation of the two molecules.

[0080] It has now been surprisingly found that by acidifying a solution comprising a carbohydrate-protein (e.g., a glycan-protein) mixture, for example, to a pH within the range of about 2 to about 4, the non-covalent carbohydrate-protein interactions are disrupted, or minimized to a level allowing separation of the carbohydrate and protein fractions, for example, by methods that are based on molecular weight differences. Once the non-covalent interactions are disrupted or minimized, the carbohydrates (e.g., glycans) can be subjected to a separation methodology based on the difference in molecular weight between the carbohydrate and the protein fraction. The disruption, in some embodiments, does not have to be complete. It is enough to lower the binding forces between carbohydrate and protein fraction to an extent that can be overcome by the separating force applied in the respective separation approach. For example, if a centrifugation filter column comprising a membrane with a molecular weight cutoff is used for separation, the forces applied by the centrifugation (e.g., g-force) may be large enough to offset a weak, remaining carbohydrate-protein interaction.

[0081] In some embodiments, acidification comprises lowering the pH of a mixture comprising a carbohydrate (e.g., glycan) and a protein fraction to a pH value within the range from about 2 to about 4. This range is suitable for disrupting most glycan-protein interactions to an extent that allows efficient separation of the two fractions. In some embodiments, however, lowering the pH below pH 2 may be necessary to disrupt particularly strong non- covalent glycan-protein interactions. If the composition of a carbohydrate or glycan to be separated or isolated from the protein fraction in a mixture is known, the pH required for disrupting non-covalent glycan-protein interactions can be estimated. For example, a sialic acid residue, a common acidic moiety in glycans, carries one carboxylic group (pKa 3-5). At pH 2-4, carboxylic groups are typically present in a protonated (COOH) form, rather than in a deprotonated (COO-) form, which is found at more basic pH. Without wishing to be bound by any particular theory, it is believed that protonation of carboxylic groups in carbohydrates (e.g., glycans) disrupts the majority of ionic bond interactions between glycans and proteins. Further, at acidic pH there is an abundance of protons (H⁺) in solution, which disrupts hydrogen bond formation between carbohydrates (e.g., glycans) and proteins.

[0082] In some embodiments, the optimal pH for efficient separation of a glycan and a protein in a mixture is determined empirically. In some such embodiments, it is preferred to separate glycan and protein fractions at the highest pH allowing for efficient separation, which avoids exposure of glycan and/or protein fractions to harsh acidic environments, thus minimizing denaturation and fragmentation.

[0083] In some embodiments, an acidic pH above 4 is suitable for disrupting non- covalent glycan -protein interactions. The required pH depends on the strength of the non- covalent glycan-protein interactions and the specific application at hand. For example, if the glycan-protein mixture is a complex biological sample, e.g., a blood, plasma, urine, or milk sample, and quantitative isolation of glycans and proteins is desired, a lower pH value may be preferable, which allows quantitative disruption of even strong non-covalent glycan-protein interactions, whereas, if the mixture is of a single protein and a single glycan, and it is known that the non-covalent interactions between the two fractions are weak, a higher pH may be preferable. Other factors also play a role in determining the optimal pH or pH range, for example, the force driving separation (e.g., high g-force centrifugation protocols may be able to overcome stronger non-covalent interactions than low g-force protocols), the kind and composition of the glycans and proteins to be separated, the difference in molecular weight between glycans and proteins, and the molecular weight cutoff used for separation.

[0084] Suitable reagents for acidifying mixtures of glycans and polypeptides, for example, mixtures in aqueous solution, are provided herein. Such reagents include, without limitation, any organic and inorganic acid that can be provided in an amount sufficient to shift the pH of the mixture to the desired acidic pH, for example, a pH within the range of about 2 to about 4. For acidification of glycan-protein mixtures in aqueous solution, any organic acid, inorganic acid, acidic buffer, or molecule that can generate acids in aqueous solution, such as acidic anhydride will work, as long as the acid, buffer, or molecule can generate enough protons to decrease pH of the mixture to a desired, acidic pH, for example, a pH of about 2 to about 4. In some embodiments, an acidic substance is employed that itself is able to generate a pH below a value of 4. In some embodiments, a volatile acid or acidic molecule is use for acidification of a glycan-protein mixture. Volatile acids and acidic molecules can be removed from the mixture by evaporation. Such acids and acidic molecules include, without limitation, formic acid (organic), acetic acid (organic), trifluoroacetic acid (organic), nitric acid (inorganic) and hydrochloric acid (inorganic). Additional suitable volatile acids and acidic molecules will be apparent to the skilled artisan, and it will be appreciated that this disclosure is not limited in this respect. Non-volatile acids, such as hydrosulfuric acid, may, in some embodiments, require a step of removal or neutralization, if a shift to neutral or basic pH is desired after acidification, while volatile acids or acidic molecules may be removed by evaporation.

[0085] Other factors may play a role in choosing a suitable acid for acidification. For example, in embodiments, in which a glycan-protein mixture is acidified in a spin filter column in contact with a filter membrane, the structural integrity of the membrane must be resistant to the acid and the pH conditions chosen. Those of skill in the art will be able to determine whether a given spin filter membrane or other material in contact with the glycan- protein mixture will be stable when contacted with a given acid or acidic molecule based on this disclosure, the knowledge in the respective arts, and no more than routine

experimentation .

[0086] Some aspects of this disclosure relate to the recognition that most glycans comprised in glycosylated proteins exhibit a lower molecular weight than the protein fraction of the glycosylated protein. For example, the most common glycans, N-glycans and O- glycans (see, e.g., Dell et al., Glycoprotein Structure Determination by Mass Spectrometry Science 2001, 291(5512): 2351-2356 , the entire contents of which are incorporated herein by reference), are oligosaccharides comprised of multiple monosaccharides, e.g., glucose, mannose, fucose, etc. Both N-glycans and O-glycans found in glycosylated proteins typically exhibit a significantly lower molecular weight than the parent protein, with N-glycans typically comprising more monosaccharide residues than O-glycans, and, thus, exhibiting more complex structures and relatively higher molecular weight than O-glycans.

[0087] Some aspects of this disclosure relate to the recognition that this difference in molecular weight between the glycan and the protein fraction can be exploited to separate and/or to isolate the glycan and/or the protein fraction from a mixture comprising both fractions. For example, according to some aspects of this disclosure, glycans and proteins comprised in a mixture of glycans and proteins can be separated based on the difference in their respective molecular weight.

[0088] For example, once N-glycans, O-glycans, or other glycan moieties are released from their parent protein, they typically have a lower molecular weight than the remaining parent protein, allowing separation and isolation of both the glycan and the parent protein. An exception to this notion are glycosaminoglycan, also sometimes referred to as

proteoglycans or mucopolysaccharides, which are long unbranched polysaccharides consisting of a repeating disaccharide unit, for example, a hexose or hexuronic acid, linked to a hexosamine. Glycosaminoglycans may have a similar or higher molecular weight than the parent protein. However, glycosaminoglycans can be fragmented into smaller

oligosaccharides, for example, by enzymatic digest, resulting in low-molecular weight fragments which can then be separated from the parent protein and isolated in analogy to lower molecular weight glycans.

[0089] In some embodiments, a glycan and a polypeptide present in a mixture have a different molecular weight/size and separating the glycan from the polypeptide comprises separating the polypeptide and the glycan based on their molecular weight/size difference. In some embodiments, the separating comprises size fractionation, size exclusion, or filtration over a semi-permeable membrane. In some embodiments, the filtration over a semipermeable membrane comprises dialysis, spin-filter centrifugation, or the application of positive or negative pressure.

[0090] In some embodiments, the glycan is separated from the polypeptide via a spin filter comprising a semi-permeable membrane having a molecular weight cutoff between the molecular weight of the glycan and the molecular weight of the polypeptide. In some embodiments, the spin filter functions as both the reaction vessel (e.g., for reduction, alkylation, glycosidase digest, acidification, and/or protease digest), and as the separation tool.

[0091] In some embodiments, a method of separating a polypeptide and a glycan from a glycoprotein, as provided herein, results in the isolation of both the glycan and the protein fraction comprised in the glycoprotein. In some embodiments, both fractions are subjected to further analysis, for example, of sequencing or other analytical approaches allowing an identification of the glycan and/or the polypeptide.

[0092] It will be apparent to the skilled artisan that any method for separating molecules based on their molecular weight that allows a molecular weight cutoff between the molecular weight of the glycan(s) and protein(s) to be separated and can be performed on a mixture of glycans and proteins at an acidic pH can be employed for separating glycan and protein fractions according to aspects of this disclosure. Three exemplary techniques, filtration, dialysis, and size exclusion chromatography, are particularly useful for application in the context of the instantly provided technology. Common reagents and materials useful in molecular weight cutoff separation methods include, without limitation, membranes for filtration and dialysis, and resins for size exclusion chromatography, e.g., agarose, polyacrylamide, and cross-linked polystyrene resins. [0093] In some embodiments, the technology provide herein utilizes the difference in molecular weight of the protein and glycan fraction of a glycosylated protein to separate the two fractions from each other. Such methodology may be applied to any glycosylated macromolecule in which the glycan fraction and the macromolecule exhibit a difference in molecular weight that is sufficiently large to allow for separation based on that difference. For example, the instantly disclosed technology can be applied to isolate and analyze N- glycans, O-glycans, monosaccharides, and oligosaccharides attached to proteins or lipids, allowing isolation of both the glycan fraction and the protein and/or lipid fraction. The instantly disclosed technology can also be applied to glycosaminoglycans and

polysaccharides as these PTMs can be digested into smaller molecular weight

oligosaccharides.

Downstream applications

[0094] The technology provided herein can be used to isolate glycans and/or polypeptides for any suitable downstream application, for example, for further analysis, identification, measuring, and/or quantification of the isolated glycans and/or polypeptides. Because the technology provided herein allows for the separation and isolation of both a glycan and a polypeptide fraction from a glycan/polypeptide mixture, it is particularly suitable for downstream applications that benefit from the availability of both fractions.

However, it will be understood that the instantly provided technology is not limited to such downstream applications.

[0095] In some embodiments, the technology provided herein is applied in the context of a diagnostic method. For example, a glycan and/or polypeptide fraction may be obtained from a subject using the instantly disclosed technology, and subsequently analyzed. In some embodiments a glycan and/or polypeptide is obtained from a sample from the subject, for example, a tissue, or a body fluid sample.

[0096] The term body fluid, as used herein, refers to any body fluid including, with limitation, serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, sweat, urine, cerebrospinal fluid, saliva, semen, sputum, tears, perspiration, mucus, tissue culture medium, tissue extracts, and cellular extracts. It may also apply to fractions and dilutions of body fluids. The source of a body fluid can be a human body, an animal body, an experimental animal, a plant, or other organism. [0097] The technology provided herein can be applied in the context of samples obtained from a subject. The subject, in some embodiments, is a human. In some embodiments, the subject is a mammal, a mouse, a rat, a cat, a dog, a cattle, a goat, a pig, a sheep, a vertebrate, a fish, a reptile, an amphibian, an insect, a fly, an annelid, or a nematode. The technology described herein is, however, not limited to such samples, and can be applied to samples from other sources as well, for example, to samples obtained from bacteria, yeast, plants, or from environmental samples.

[0098] In some embodiments, a glycan and/or a polypeptide isolated via the instantly disclosed technology is analyzed for the presence or absence of a biomarker. In some embodiments, the biomarker is indicative of the presence or absence of a parameter of interest, for example, a biomarker associated with a disease or disorder is indicative of the absence or the presence of disorder in the subject the sample was derived from. For another example, the presence of a biomarker, for example, a specific glycan structure, may be indicative of the presence of an organism, e.g., a pathogenic organism, in a sample.

[0099] Glycan and polypeptide biomarkers useful according to aspects to this disclosure will be apparent to those of skill in the art. The skilled artisan will, for example, be aware of glycan and/or polypeptide biomarkers associated with a disease or disorder in a human subject.

[00100] Another exemplary, non-limiting downstream application for which the technology disclosed herein is suited is the quality control of proper glycosylation of engineered proteins. Engineered proteins frequently require proper glycosylation to exert their function. For example, antibodies, and antibody fragments are most effective if properly glycosylated, and aberrant or lacking glycosylation abrogates proper function. Accordingly, the technology described herein can be used to monitor glycosylation in engineered proteins, for example, in proteins produced for therapeutic purposes, or for biotechnological applications, such as substrate fermentation.

[00101] The term antibody, as used herein, refers to an immnuoglobulin molecule or an immunologically active portion thereof (e.g., antigen-binding portion). The antibody may be naturally produced or wholly or partially synthetically produced. Examples of immunologically active portion of immnuoglobulin molecules include F(ab), Fv, and F(ab') fragments which can be generated by cleaving the antibody with an enzyme such as pepsin. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are generally preferred in the context of the present disclosure.

[00102] The term antibody fragment refers to any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody' s specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')₂, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages or other more stable linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids. In certain embodiments, the antibody fragment has at least two antigen-binding site. In certain preferred embodiments, the antibody fragment has exactly 2, 3, 4, or 5 antigen-binding sites. Fragments with two antigen-binding sites are particularly useful according to aspects of the present disclosure.

[00103] Single-chain Fvs (scFvs) are recombinant antibody fragments consisting of only the variable light chain (V_L) and variable heavy chain (V_H) covalently connected to one another by a polypeptide linker. Either V_L or V_H may be the NH₂-terminal domain. The polypeptide linker may be of variable length and composition so long as the two variable domains are bridged without serious steric interference. Typically, the linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility. Diabodies are dimeric scFvs. The components of diabodies typically have shorter peptide linkers than most scFvs, and they show a preference for associating as dimers. An Fv fragment is an antibody fragment which consists of one V_H and one V_L domain held together by noncovalent interactions. The term dsFv is used herein to refer to an Fv with an engineered intermolecular disulfide bond to stabilize the V_H-V_L pair. A F(ab')₂ fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins (typically IgG) by digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced. A Fab fragment is an antibody fragment essentially equivalent to that obtained by reduction of the disulfide bridge or bridges joining the two heavy chain pieces in the F(ab')₂ fragment. The Fab' fragment may be

recombinantly produced. A Fab fragment is an antibody fragment essentially equivalent to that obtained by digestion of immunoglobulins (typically IgG) with the enzyme papain. The Fab fragment may be recombinantly produced. The heavy chain segment of the Fab fragment is the Fd piece.

[00104] The skilled artisan will be aware of numerous engineered proteins that require proper glycosylation for proper function. Such proteins include, for example, any therapeutic proteins approved for therapeutic use in human subject.

[00105] Additional downstream applications suitable for further processing and/or analysis of a glycan and/or a polypeptide obtained via the instantly disclosed technology will be apparent to those of skill in the art. The disclosure is not limited in this respect.

Devices

[00106] Some aspects of this disclosure provide a device for separating a carbohydrate, for example, a glycan and/or a polypeptide from a carbohydrate/polypeptide mixture. In some embodiments, the device comprises a sample compartment, a collection compartment, and a molecular weight/size- selective material separating the sample compartment from the collection compartment. In some embodiments, the molecular weight/size- selective material has a molecular weight/size cutoff that is between the molecular weight/size of the carbohydrate and the molecular weight/size of the polypeptide.

[00107] The term molecular weight/ size -selective material, as used herein, refers to a material allowing for separation of molecules by molecular weight and/or molecular size. Such materials are known to those of skill in the art and include, for example, semipermeable membranes and molecular weight/size exclusion resins. Known membrane materials suitable according to aspects of this disclosure include, without limitation, dialysis membranes, and any other membranes that are permeable for molecules up to a certain size, but impermeable for molecules above that size. Such membranes are typically made of a polymer, e.g., PES (poly ether sulfones), polycarbonates, cellulose, regenerated cellulose, and cellulose derivatives (e.g., mixed esters of cellulose, nitrocellulose, cellulose triacetate), nylon, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polypyropylene, glass fiber, quartz fiber, polycarbonate, hydrosart (a cross-linked cellulose based polymer that is chemically and physically stable in a broad pH range (pH 2.0 to pH 14), or other polymers that form a porous membrane. Modulation of pore size provides membranes that have a lower molecular weight cutoff (MWCO) if the pores are smaller, and a higher molecular weight cutoff if the pores are larger. Some exemplary molecular weight cutoff materials are described in more detail elsewhere herein. Additional suitable materials will be apparent to the skilled artisan. The disclosure is not limited in this respect.

[00108] In some embodiments, the device is a filter column. The term filter column, as used herein, refers to a column assembly that is suitable for filtration. Filtration can be accomplished by flowing a liquid sample through a filter membrane. The required force for creating the flow can be created by gravity, by centrifugation, for example, in a laboratory centrifuge, or by pressure. In some embodiments, a filter column is operated in line with an HPLC or with an FPLC device. In some embodiments, a filter column comprises an outer centrifugation vessel, for example, a 1.5ml, 10ml, or 50ml centrifuge tube that serves as the collection compartment, and a filter cartridge that can be inserted into the centrifuge tube. The cartridge typically comprises a fluid reservoir, into which a fluid sample can be transferred, and a molecular weight selective membrane. A limited range of filter columns with different size- selective membranes and suitable for processing different volumes of fluid samples are commercially available. Additional filter columns with novel characteristics that are particularly suitable for use according to aspects of this disclosure are provided herein.

[00109] The methodology provided herein can be performed on a small, laboratory scale, or on a large, industrial scale. The devices provided herein can be scaled in size to allow for the processing of the required sample size. For example, in some embodiments, a device is provided that is suitable for processing a glycan/polypeptide mixture of a volume of about Ιμΐ, about 2μ1, about 5μ1, about ΙΟμΙ, about 50μ1, about ΙΟΟμΙ, about 250μ1, about 500μ1, about 1ml, about 2ml, about 5ml, about 10ml, about 15ml, about 50ml, about 100ml, about 250ml, about 500ml, about 11, about 21, about 51, about 101, about 501, about 1001, about 2501,or about 5001. In some embodiments, a device provided herein is suitable for the processing of a single sample, e.g., a device that comprises a single sample compartment, and a single collection compartment. In other embodiments, a device is provided herein that allows for the simultaneous processing of a plurality of samples. In some embodiments, such a multi-sample device comprises a multi-well plate. In some such embodiments, the device comprises a first plate with multiple wells, in which each well functions as a sample compartment, a weight/size- selective material, e.g., a semi-permeable membrane, at the bottom of each well, and a second plate comprising the same number of wells, which function as the collection compartment. The first plate can be assembled to fit into the second plate, and the plate assembly can then be placed into a centrifuge for sample filtration.

[00110] In some embodiments, the collection compartment is detachable from the sample compartment. In some embodiments, the collection compartment is replaceable. For example, in some embodiments, a first collection compartment is used in pre-processing of the sample, e.g., to collect wash buffers and reagents used in the preparation of a

glycan/polypeptide mixture. Prior to the separation of the glycan from the

glycan/polypeptide mixture, the first collection compartment is replaced by a second collection compartment, which is used to collect the glycan fraction. In some embodiments, a detachable collection compartment is a disposable, single-use tube. In some embodiments, the collection compartment is shaped to fit a centrifuge. In some embodiments, the collection compartment fits a vacuum centrifuge. This is of particular benefit if the separated fractions are to be dried after separation. In some embodiments, the collection compartment can be used for storage and/or further processing, without the need for transferring the eluted fraction to another vessel. In some embodiments, the collection compartment comprises a lid.

[00111] The device is made to withstand the forces and materials it is exposed to during sample processing. For example, the device, in some embodiments, is made to withstand centrifugal forces of at least 10,000g, a pH range from 1-13, and would withstand microwave irradiation to the extent used in the technology described herein..

[00112] In some embodiments, a membrane is used as the molecular weight/size selective material. In some embodiments, the membrane is hydrophobic. In some

embodiments, the membrane is hydrophilic. In some embodiments, the membrane is hydrophobic and has a molecular weight cutoff of about 30kDa. In some embodiments, the membrane is hydrophobic and has a molecular weight cutoff of about 18-20kDa. In some embodiments, the hydrophobic membrane is a PES membrane.

[00113] In some embodiments, a membrane is used that comprises an acidic moiety, for example, a carboxy moiety or an anhydride moiety. In some embodiments, the use of a membrane comprising acidic moieties creates an acidic microenvironment in the vicinity of the membrane. If a glycan/polypeptide mixture comprising non-covalent interactions between the glycan and the polypeptide is contacted with such a membrane, these

interactions will be destabilized in the vicinity of the membrane, even if the sample is not acidified as a whole. Accordingly, the use of an acidic membrane allows, in some

embodiments, for efficient separation of a glycan from a mixture of a glycan and a polypeptide without the need to acidify the mixture by adding an acid or an acid-generating compound.

[00114] It will be apparent to the skilled artisan that the selection of a suitable MWCO of the molecular weight/size selective material for a particular embodiment will dependent on the molecular weight of any carbohydrate to be separated and/or the molecular weight of any polypeptides to remain in the sample during separation of the carbohydrate. Typically, a molecular weight/size- selective material is selected that has a molecular weight/size cutoff (MWCO) above the molecular weight/size of the glycan and below the molecular weight/size of the polypeptide to be separated. Molecular weights of many carbohydrates and polypeptides are well known to those of skill in the art, or, where unknown, can be determined based on methods well known in the art without undue experimentation. In some embodiments, the molecular weight of some, non-limiting examples of glycans may be as follows: Polysaccharides, glycosaminoglycan (> >10 kDa); N-glycans (1.5 to 6 kDa);

oligosaccharides (< 5 kDa); O-glycans (< 3 kDa); and monosaccharide (< 500 Da). The MWCO of the molecular size/weight selective material can then be chosen according to which fraction is to be separated from a sample, e.g., MWCO of 500Da would be sufficient to recover monosaccharides, and MWCO of 6 kDa would be suitable to recover N-glycans in this example.

[00115] In some embodiments, the majority of polypeptides in the polypeptide/glycan mixture is of a molecular weight that is higher than 20 kDa, higher than 30 kDa, or higher than 50 kDa. Accordingly, for many applications in which glycans are to be separated from polypeptides, a suitable MWCO is 20 kDa. In some embodiments, the MWCO of a device provided herein is about 1 kDa, about 2 kDa, about 3 kDa, about 5 kDa, about 6 kDa, about 10 kDa, about 15 kDa, about 18 kDa, about 20 kDa, or about 30 kDa.

[00116] The function and advantage of these and other embodiments of the present disclosure will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure.

Accordingly, it will be understood that the Examples are not meant to limit the scope of the disclosure. EXAMPLES

EXAMPLE 1: Exemplary Protocol

[00117] A protocol for separation of N-glycans and peptides (including deglycosylated peptides) from a glycoprotein sample is described below. This protocol is widely applicable to purified glycoproteins, simple glycoprotein mixtures, and body fluid samples, e.g., human urine, blood, serum, and plasma.

[00118] Materials and Reagents: PNGase F - New England Biolab; Trypsin

(sequencing grade) - Promega; Spin Filters - Vivaspin 2, 10 and 30K MWCO PES (Sartorius Stedium Biotech); Microwave- oven - Sanyo domestic microwave - 1000 Watt (EM- S5120W/B); Centrifuge - Eppendorf centrifuge 5804R. Glass Beakers - VWR (2 Liter volume); FLO 18 (98%) - Rotem Inc.; Other used chemicals in protocol - Sigma Aldrich; Prepared buffer and solutions: 8M Urea/0.2M Tris-HCl buffer (pH 8.5) (prepared fresh); 1M DTT (dithiothreitol); 1M IAA (iodoacetamide); 50 mM ammonium bicarbonate buffer

(ABC); 50 mM ammonium bicarbonate buffer in H₂O¹⁸

[00119] Samples: The following samples were subjected to analysis according to the technology provided herein: Standard glycoproteins: Bovine RNase B, bovine fetuin, and IgG from human plasma (Sigma Aldrich) Human plasma: Human plasma depleted of the top-7 abundant proteins using a MARS-7 depletion (Agilent). Human urine: Human urine samples were depleted of albumin by the One-Step protocol described in Vaezzadeh, A.R., et al., One- step sample concentration, purification, and albumin depletion method for urinary proteomics. J Proteome Res. 9(11): p. 6082-9, the entire contents of which are incorporated herein by refernce.

[00120] The protocol used in these experiments contained three consecutive steps: 1) sample reduction and alkylation; 2) deglycosylation (release of glycans from glycoproteins), and separation and capture of released N-glycans; 3) protein trypsin digestion and the collection of tryptic peptides. All three steps were carried out in a single spin filter.

[00121] Reduction and alkylation: A spin filter was pre -rinsed with 1 mL of HPLC water by spinning (10000g x 5 min). Both the flow-through and the supernatant were discarded. Glycoprotein samples were dissolved in 8M urea/0.2M Tris-HCl Buffer (400 -500 μL) either in the filter reservoir or separately. 1M DTT (25 μL) was added to the filter reservoir and the sample was held at room temperature for 45 min by constant shaking (Mini Vortex, Fisher Scientific). 1M IAA (30-35 μL) was added, and the sample was held at room temperature for 45 min in a dark environment. Excess reagents and small molecules were washed away with 1ml of 50mM ABC (spin @ 10000g x 20 min). Repeat a total of 3 times. Discard all flow-through. B)

[00122] Deglycosylation, and the Separation and Capture ofN-glycans: The sample buffer was exchanged with H₂O¹⁸ based ABC buffer by washing with 500μί of 50mM H₂O¹⁸ based ABC buffer (spin @ 10000g x 15 min). The washing was repeated a total of 4 times.

The flow-through was discarded. 200 μL of 50mM H₂O¹⁸ based ABC buffer was added to the supernatant in the spin filter reservoir, ensuring that the solvent covered the filter membrane area. 2-3 μL of PNGase F (~ 1-2 μL of PNGase F per 100 μg glycoprotein ) were added and the spin filter was floated in a 2L glass beaker filled with 1.5 liters of room temperature tap water. The filter floating in the beaker was placed into the microwave and heated at 30% power for 20 min. The water temperature in the beaker was measured. The final water bath temperature was approximately 70 °C. The spin filter was cooled in an ice bath. lmL of HPLC water was added, and the filter was spun (10000g x 20 min). The flow- through fraction was collected into a 10 mL glass tube (glycan fraction 1). To complete the glycan capture, 1ml of ice cold 0.1% formic acid was added, and the filter was spun (10,000g x 20 min). This step was repeated 3 times and all flow-through was collected (glycan fraction 2). The glycan fractions were combined, and completely dried in a speed- vac centrifuge.

[00123] Trypsin digestion and the collection oftryptic peptides: The pH of the remaining de-N-glycosylated protein fraction (supernatant in the filter reservoir) was adjusted by washing with 50mM ABC buffer (spun at 10,000g x 20 min). The washing was repeated a total of 3 times and the flow-through was discarded. 200μL of 50mM ABC buffer were added to the sample to ensure coverage of the filter membrane. A first trypsin aliquot (trypsin : protein = 1 : 50 by weight) was added to the sample, and the spin filter was floated in a 2L glass beaker filled with 500ml of room temperature tap water. The filter floating in the water was placed into the microwave, and heated at 30% power for 6 min. The filter was removed from the water and cooled in an ice bath. A second aliquot of trypsin (trypsin : protein = 1 : 50 by weight) was added to the sample, and the spin filter was floated in a 2L glass beaker filled with 500ml of room temperature tap water. The filter floating in the water was placed into the microwave, and heated at 30% power for 6 min for the second trypsin digestion. The spin filter was cooled in an ice bath. The tryptic peptides were collected by washing with 0.5ml of 50mM ABC (spun at 10,000g x 15 min). The collection wash was repeated a total of 3 times, and all flow-through fractions (peptide fractions) were combined. Tryptic peptides were then dried. Dried glycans and peptides are ready for downstream processing (e.g. LC-MS experiment).

[00124] Urine samples usually contain a significant amount of metabolites and interfering salts. These need to be removed prior to the deglycosylation step. An effective strategy is to sequentially spin urine samples 3 times in 1 mL of 8 M urea buffer, followed by 5 washes in 1 mL of 50 mM ABC (10000g x 20 min).

[00125] Proteins or other components of the sample may precipitate upon lowering the pH of the solution. Samples should then be reconstituted with 1 mL of 8 M urea buffer (vortex if necessary) and followed by a spin (10000g x 20 min). This step can be repeated if there is still un-dissolved protein pellets. Once all proteins are in the solution, the solvent then can be exchanged into 50 mM ABC buffer by repeated centrifugation as described above. Each step can be considered a "temporary stopping" point. If the protocol is stopped the sample/filter can be stored in at 4 °C as long as solvent covers the filter membrane area.

EXAMPLE 2: Rapid De-N-glycosylation Using a Domestic Microwave

Materials and Methods:

[00126] Materials and Chemicals. Sodium hydroxide, iodomethane, dithiothreitol

(DTT), dimethyl sulfoxide (DMSO), iodoacetamide (IAA), 2,5-dihyroxylbenzoic acid (DHB), urea, ammonium bicarbonate, trifluoroacetic acid (TFA), bovine pancreases

Ribonuclease B (RNase B), bovine fetuin and IgG from human serum were purchased from Sigma- Aldrich (St. Louis, MO). HPLC-grade methanol, water, and acetonitrile were obtained from Honeywell Burdick & Jackson (Muskegon, MI). Dichloromethane was purchased from Fisher Scientific (Fair Lawn, NJ). PNGase F and associated buffers were obtained from New England Biolab. (Ipswich, MA)

[00127] Human plasma. Human blood samples were obtained from healthy volunteers under an Institutional Review Board-approved protocol. Plasma was obtained by

centrifugation of whole blood for 10 min at 3000x g.

[00128] Acquisition of the top 7 abundant proteins from human plasma: Plasma samples were depleted of its seven most abundant proteins by MARS 7 depletion (Agilent) using the vendor's protocol. The depleted top-7 proteins were captured from the column and subjected to the domestic microwave de-N-glycosylation treatment as described below.

[00129] Protein Concentration. The concentration of the plasma samples pre- and post-depletion was measured by Bradford assay (Bio-Rad) using a NanoDrop ND-2000C spectrophotometer (ThermoScienfitic). The Bradford assay was performed according to manufacturer's instructions. The measurements were performed in triplicate for each sample. The average value of the protein concentration is reported.

[00130] One Dimensional Gel Electrophoresis (SDS-PAGE). Samples from each experiment were resuspended in 18 μL of water and 6 μL of 4x LDS sample buffer

(Invitrogen). Samples were heated at 70 °C for 10 min and then were loaded into a 4-12% Bis-Tris precast gel (Invitrogen). Gels were run at 100 V and stained overnight in colloidal Coomassie blue as per manufacturer protocol (Invitrogen). Pro-Q Emerald 300 glycoprotein gel stain kit (p21855) was obtained from Invitrogen. The glycoprotein stain gel was carried out on Invitrogen Novex Tris-glycine gel and stained by the manufacture recommended protocol. The gel image was obtained from a ChemiDoc system (Bio-Rad) with UV absorption.

[00131] Reduction and Alkylation of Human IgG: human IgG was initially dissolved in

8M urea/0.2M Tris-HCl buffer (pH 8.5). The proteins were reduced by 15 μΐ. of 1M DTT and kept at 45 °C for 45 min. After cooling to room temperature, 25 μL of 1M IAA were added and kept in the dark for 40 min.

[00132] Standard PNGase F digestion in water bath: Standard de-N-glycosylation was performed as per the vendor's protocol ^[24]. In brief, glycoproteins were dissolved in 45 μL of glycoprotein denaturing buffer (0.5% SDS, and 0.04M DTT), and heated at 100 °C for 10 minutes. A final reaction volume of 60 μL was obtained by adding 6 μL of 10% NP-40, and 6 μL of G7 buffer (0.5M Sodium phosphate, pH 7.5) and 1-2 μL PNGase F, and HPLC water. The reaction was incubated at 37 °C water bath overnight. The reaction was stopped by acidifying the solution to pH 4 with formic acid.

[00133] De-N-glycosylation by household microwave oven: An LG household microwave oven (Model #: LRM1230W, China) with a maximum output power of 1200w and a frequency of 2.45 GHz was used for microwave irradiation. The digestion protocol was similar to the standard procedure described as above, except that the reaction mixtures were heated in the domestic microwave oven, rather than a water bath. To avoid overheating of the samples, reaction vials (1.5 mL Eppendorf tubes) were placed in a plastic holder floating in a glass beaker with 1.5 liters of cold tap water. Samples were irradiated for 20 minutes at 20% maximum power. After microwave irradiation, the reaction vials were cooled down on ice. After deglycosylation, the samples were then evaporated in the speedvac (SPD1010, Thermo Scientific). In the experiments to determine if additional microwave irradiation beyond 20 minutes were needed, after each 20-minute irradiation, the water bath was replaced with new cold tap water.

[00134] Glycans purification by C18 solid-phase extraction (SPE): Hydrophobic species (proteins, detergents) were removed via C18 SPE cartridge (Sep-Pak Vac, Waters). Briefly, C18 cartridges were activated by 1 mL 100% ACN, and pre-conditioned by 3X 1 mL 2% ACN with 0.1% TFA. Samples were dissolved in 100 of 2% ACN with 0.1% TFA, and loaded onto the cartridges. Glycans were eluted off the cartridge by 3 mL of 2% ACN with 0.1% TFA. The elution fractions were collected in glass tubes (No. 99447-13, Pyrex). The solvents were removed by the speedvac and stored at -20°C.

[00135] Permethylation of glycans. The dried glycan samples were dissolved in 300 μL of DMSO, followed by the addition of powdered NaOH. After a short vortexing to produce a suspension, 50 μL of iodomethane was added, and the reaction was allowed to proceed for 50 min while being vortexed. The reaction was stopped by addition of ice-cold water, and the permethylated glycans were extracted three times with dichloromethane. The extracts were combined and washed with ice-cold water until the pH was 7. The organic layer was evaporated by speed- vac, and the permethylated glycans were re-dissolved in 50-70% methanol.

[00136] MALDI-MS of Permethylated glycans. The dried permethylated glycan samples were dissolved in 50% methanol. 10 mg of DHB was dissolved in 1 mL of 50% methanol aqueous solution containing 1 mM sodium acetate. The matrix solution was centrifuged prior to use. Samples were spotted directly on the MALDI plate and mixed with an equal volume of the DHB solution. The resulting spots were dried under air prior to MALDI-MS analyses. MALDI-MS was carried out on an MDS SCIEX 4800 (Applied Biosystems) using the interactive mode. The external calibration was performed using the ProteoMass Peptide MALDI-MS calibration kit (Sigma- Aldrich). MS data were processed using Data Explorer 4.9 (Applied Biosystems).

Results and Discussion

Effectiveness of enzymatic de-N-glycosylation using a domestic microwave oven

[00137] Previous work has demonstrated that low power microwave irradiation does not affect the protein backbone. Microwave energy can combined with trypsin for effective and rapid protein digestion ^[23]. We demonstrated the effectiveness of using a domestic microwave oven for PNGase F release of N-glycans on standard glycoproteins and a complex plasma mixture. Unlike special scientific microwave reactors, a domestic microwave oven does not have a cooling/pressure control system. We used a simple strategy to prevent overheating of the samples during the microwave digestion by placing the reaction vials in a large water bath, so that the majority of the irradiated energy would be absorbed by the water bath ^[23]. The exact amount of transferred energy by the microwave could not be easily measured, but the final temperature of the water bath was measured to be approximately 70 °C after a 20 min microwave irradiation.

[00138] The model glycoprotein bovine Ribonuclease B (RNase B) was used to perform the initial experiments. RNase B is a small glycoprotein (150 amino acids) with a single N-glycosylation site at asparagine-60 that contains a series of high mannose glycans from Man-5 to Man-9 that account for ~2K Da mass ^{[25 27]}. in its glycosylated form, RNase B has a MW of 18K Da (Figure 1A lane 1). PNGase F de-glycosylation of RNase B in a domestic microwave (Figure 1A lanes 2-4) was as effective as the traditional overnight standard protocol (Figure 1A lane 5). Irradiation beyond 20 minutes (Figure 1A lanes 3 and 4) did not appear to improve the yield of the microwave digestion. In this particular study, we did not optimize the lower limit of enzyme required.

[00139] To further test the protocol, we examined bovine fetuin (Figure IB) because it is an acidic glycoprotein containing three N-glycosylation sites that are large, complex-type, and multi-antenna with various sialic acid residues ^{[28 29]}. Fetuin has one major band at 56K Da and numerous minor bands, indicating the presence of co-purified proteins (Figure IB lane 1). Using the 20-minute microwave oven protocol, we demonstrated effective digestion (Figure IB lane 2) as compared to the standard protocol (Figure IB lane 3). The major fetuin band shifted to 49-50K Da corresponding to the loss of three N-glycans from the fetuin backbone ^[28]. Interestingly, some of the minor bands (98 and 110 K Da) also shifted after digestion implying that the co-purified proteins were also modified by N-glycosylation.

[00140] We tested IgG from human serum. IgG's glycosylation plays a critical role in its effectiveness as a monoclonal antibody. Effective rapid de-glycosylation techniques for improved characterization of IgG glycosylation may significantly benefit this field. IgG (Figure 1C) contains two identical heavy chains (~ 5 IK Da mass each) and two light chains (~25K Da mass each), which are linked by cysteine bridges ^[30]. Each IgG heavy chain contains a conserved N-glycosylation site Asn-297 in its Fc domain that is essential for receptor binding and function ^[8'^30]. To better observe the effect of deglycosylation, we separated the heavy and light chains by reduction and alkylation (Figure 1C lane 1). Using the 20-minute microwave oven protocol we demonstrated a significant shift in the IgG heavy chain (Figure 1C lane 2), supporting the removal of a single N-glycan unit (~ 2K Da). No significant shift was observed on the light chain.

[00141] To determine the use of the domestic microwave protocol on complex samples, we tested the top-7 abundant proteins from 2 different human plasma samples. We did not test crude plasma because of the over- abundance of proteins would make it difficult to interpret the effectiveness of the protocol by standard means. We obtained the top-7 proteins from plasma using a well-established depletion protocol (MARS-7) ^[31]. Of the top-7 proteins, 6 are known to be glycoproteins (a- 1 -antitrypsin, haptoglobin, serotransferrin, IgG, IgA and fibrinogen-a-chain). Even though the top-7 proteins make up the majority of the sample, a large number of co-eluting proteins made this a complex mixture (Figure 2A lanes 1 and 3) ^[31]. Both control samples showed a similar pattern with three regions of high intensity: 20-30K, 50-60K, and 160K Da. Three of the glycoproteins (a- 1 -antitrypsin, haptoglobin and serotransferrin) have a MW range of 50-60K Da. After microwave-assisted digestion, there was a visible shift at 50-60K Da (Figure 2A lanes 2 and 4). Furthermore, using a specific glycoprotein stain (Pro-Q, Invitrogen) we noted a dramatic decrease in total glycan signal intensity after microwave assisted PNGaseF digestion (Figure 2B, Lanes 1 vs. 2, and 3 vs. 4), particularly at 20-30K Da and 50-60K Da (Figure 2B arrows). Because PNGase F specifically removes N-glycans, the residual absorption after digestion may be from O-glycosylations that were not removed by PNGase F. Overall, these experiments demonstrated that a domestic microwave could be used effectively to release N-glycans from standard glycoproteins, IgG, and also complex samples.

Analysis of Released Glycans

[00142] To determine the stability of N-glycans after microwave irradiation, released glycans were purified and permethylated using conventional methods ^[32]. Permethylated glycans were analyzed by MALDI-MS. We show the N-glycan spectra of the model glycoproteins and human IgG. Five high mannose-type N-glycans were observed from bovine RNase B with Man5 the most abundant, and Man7 and Man9 less abundant (Figure 3A). Their peak patterns are similar to those previously reported ^[27], suggesting that microwave deglycosylation does not bias the analysis.

[00143] Because of the labile nature of the sialic acid bond, we were concerned that microwave irradiation would potentially result in a loss of terminal sialic acid residues. Bovine fetuin has multiple acidic glycans (Figure 3B). In the analysis of the released fetuin glycans, the three most abundant peaks (m/z 3602.7, 3963.8, and 2792.2) were all fully terminated by sialic acids. The two minor peaks observed (m/z 2431.1 and 3227.4) are partially sialyated and have been previously reported to occur naturally ^[33]. The analysis of the released fetuin N-glycans indicates that even fragile sialic acid linkages are compatible to the new domestic microwave protocol.

[00144] As for IgG glycan analysis, we observed all the major peaks corresponding to complex-type biantennary N-glycans (Figure 3C). The three most abundant peaks (m/z: 1836.02, 2040.13, and 2244.23) are the well-known GOF, GIF, and G2F molecules with core fucose modifications. The remaining glycans were further modified by one or two sialic acids, or bisecting GlcNAc, which is in agreement with previous reports ^[34]. Overall, it appears that the domestic microwave protocol had no adverse effect on the released N- glycans.

Conclusion:

[00145] High-throughput, reproducible, and rapid sample preparation is critical to advance glycomics research, especially for glycan-based biomarker discovery and quality control of therapeutics. We describe a simple protocol to accelerate N-glycan release from either purified or complex samples using a domestic microwave oven. The benefits of this protocol include: a significantly shorter reaction time, easy manipulation, decreased capital expense, no negative effects on glycans (e.g. desialylation), and its ability to be readily couple to other high-throughput protocols. We expect that this protocol can be easily adopted in both academic and industrial laboratories.

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2. P. Perego, L. Gatti, G.L. Beretta. The ABC of Glycosylation. Nat. Rev. Cancer. 2010, 10, 523.

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R. Jefferis, Glycosylation as a strategy to improve antibody-based therapeutics. Nat. Rev. Drug. Discov. 2009, 8, 226.

G. Walsh, R. Jefferis, Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 2006, 24, 1241.

M. Schiestl, T. Stangler, C. Torella, T. Cepeljnik, H. Toll, R. Grau, Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat. Biotechnol. 2011, 29, 310. D. Ghaderi, R.E. Taylor, V. Padler-Karavani, S. Diaz, A. Varki, Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat. Biotechnol. 2010, 28, 863.

P. M. Rudd, I. Rudan, A.F. Wright, High-throughput glycome analysis is set join high- throughput genomics. J. Proteome. Res. 2009, 8, 1105.

M. S. Bereman, D. D. Yong, A. Deiters, D. C. Development of a robust and high throughput method for profiling N-linked glycans derived from plasma glycoproteins by NanoLC-FTICR mass spectrometry, J. Proteome. Res. 2009, 8, 3764.

S. R. Kronewitter, M. L. de Leoz, K. S. Peacock, etc. Human serum processing and analysis methods for rapid and reproducible N-glycan mass profiling. J. Proteome. Res. 2010, 9, 4952.

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W.N. Sandoval, F. Arellano, D. Arnott, H. Rabb, R. Vandlen, J.R. LiU, Rapid removal of N-linked oligosaccharides using microwave assisted enzyme catalyzed deglycosylation. Int. J. Mass Spectrom. 2007, 259, 117.

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http ://www .neb . com/nebecomm/products/productP0705. asp

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[00146] All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EXAMPLE 3:

Analysis of milk oligosaccharides

[00147] Milk is an example of a complex mixture in which there are free

oligosaccharides (more than 300) that are not covalently bound to any protein or lipid. There are, however, enough "interactions" that the oligosaccharides are not "free", and thus not readily amenable to spin filtration. These oligosaccharides are typically referred to as Human Milk Oligosaccharides (HMO) (Bode 2006). In addition, there are plenty of proteins in milk (whey proteins) that carry both N-glycans and O-glycans. A recent publication compared the N-glycans of human milk with those from bovine milk (Lebrilla 2012).

[00148] In this circumstance, free oligosaccharides (HMO) can be separated from other components (proteins, lipids, etc.) by using the technology provided herein. After the HMOs are separated from milk proteins, the technology could again be utilized for the separation of carbohydrates (e.g., glycans) from proteins, e.g., after release of glycans from parent glycoproteins . This second procedure would require the performance of a PNGase F digestion to release the N-glycans. The technology provided herein could again be employed to separate the N-glycans from the proteins. Although N-glycans and HMO are similar compounds, N-glycans are usually larger and more complex than HMO. Thus, leveraging the instantly disclosed technology twice to exploit the covalent and non-covalent interactions of different carbohydrates allows one to obtain both a covalent-link and free-oligosaccharide profile of the mixture via downstream analysis. Additionally, because all fractions are preserved, a protein profile can be obtained by downstream analysis. PTM

occupancy/characterization could also be clarified downstream via O 18 water.

[00149] Further, O-glycans could also be released on the same sample, e.g., after release of N-glycans, or on a sample processed in parallel. There is no universal enzyme that, like PNGase F for N-glycans, can release all O-glycans. In some embodiments, release is carried out using base-induced elimination (β-elimination). It will be apparent to the skilled artisan, that the use of a strong base for β-elimination may result in protein damage. Any base (organic or inorganic) could be employed for β-elimination, for example, any base that can maintain the solution at pH > 11. In some embodiments, conditions are chosen that minimize protein damage. Such conditions (base, pH, temperature, etc.) are known to those of skill in the art.

EXAMPLE 4:

Analysis of N-glycans in urine

[00150] N-glycans were released from glycoproteins in a human urine sample as described elsewhere herein. The released N-glycans were separated from the parent proteins and analyzed. Table 1 shows a list of N-glycans released and captured from a urine sample by the disclosed technology. N-glycans were analyzed by a high-mass accuracy ESI-Orbitrap mass spectrometry (electrospray ionization). More than 9 compositions were detected larger than 4000 Da, indicating a high degree of separation efficiency of N-glycans provided by the disclosed technology. (H: Hex; N: HexNAc; F: Fuc; and A: Neu5Ac).

EXAMPLE 5:

Analysis of N-glycoproteins in urine

[00151] Polypeptides were released from glycoproteins in a human urine sample as described elsewhere herein. The released polypeptides were separated from the glycans and analyzed. Table 2 shows a list of identified glycoproteins from a human urine sample (urine - 01). Captured deglycosylated proteins were analyzed by LC-MS/MS after protease digestion.

EXAMPLE 6:

Glycoprotein analysis in various samples.

[00152] Figure 4 illustrates an application of the instantly disclosed technology on standard glycoprotein bovine fetuin. After releasing the N-glycans, deglycosylated fetuin was subjected to trypsin catalyzed digestion. The tryptic peptides were captured by technology provided herein. The tryptic peptides were analyzed by MALDI-MS. The majority of fetuin sequence was detected including two de-N-glycosylated peptides (amino acids 145-159, and 160-187, representing the former N-glycosylation sites, Asn-156 and Asn-177, underlined in the table).

[00153] Figure 5 illustrates an application of the instantly disclosed technology on human IgG from serum (Sigma- Aldrich). IgG sample was processed by the disclosed technology. Released and captured N-glycans were permethylated and analyzed by MALDI- MS. As expected, N-glycans from human IgG were dominated by biantennary complex-type molecules (FG0, FG1, and FG2) with a few being further modified by sialic acid or bisecting GlcNAc. The detection of biantennary type N-glycans indicated that these glycans were derived from Fc -portion (Asn-297) in the heavy chain of IgG. This is in agreement with the observation of the heavy chain band shift in SDS-PAGE gel (insert - Lane 1: native IgG; Lane 2: deglycosylated IgG).

[00154] Figure 6 illustrates an application of the instantly disclosed technology on human urine. Two samples of urine were obtained from a single donor and were processed by the disclosed technology in parallel as biological replicates. After being processed by the disclosed technology, deglycosylated proteins were recovered from the sample compartment of a filter device and compared to native urine by SDS - PAGE. It was evident that multiple protein bands have shifted dramatically after deglycosylation demonstrating that urine is a complex sample that contains many glycosylated proteins. Lane M: molecular marker standard; Urine-01 (native); Urine-02 (native); Urine- 01+PNGase F (deglycosylated proteins from Urine-01); and Urine- 02+PNGase F (deglycosylated proteins from Urine-02).

[00155] Figure 7 illustrates an application of the instantly disclosed technology on complex human body fluids: urine and plasma from a healthy donor. Both urine and plasma samples from the same donor were processed by the disclosed technology. Captured and released N-glycans were permethylated and analyzed by MALDI-MS. A large number of glycans were detected in human urine, many with extensive acidic residues and branching. N- glycans from plasma were dominated by biantennary complex type.

[00156] Figure 8 illustrates an application of the instantly disclosed technology on three different human urine samples. The urine samples were processed by the disclosed technology. Captured and released N-glycans were permethylated and analyzed by MALDI- MS. A dramatic difference was observed among three N-glycan profiles. N-glycans from Sample A were dominated by complex and sialic acidic glycans, and few neutral glycans with fucose residues. Sample B contained mostly all neutral N-glycans with various fucose residues, and only one sialic acid glycans was found (m/z 2605.4). Sample C contained both neutral glycans with various fucose residues, but several biantennary sialic acid glycans were also detected.

[00157] Figure 9 illustrates an application of the instantly disclosed technology on three different human plasma samples. The plasma samples were processed by the disclosed technology. Released N-glycans were permethylated and analyzed by MALDI-MS. The three spectra were highly similar, demonstrating the reproducibility of disclosed technology.

[00158] Figure 10 illustrates an application of the instantly disclosed technology on three different pairs of human plasma and urine samples. Each pair of urine/plasma samples were obtained from a different individual healthy donor. All samples were processed by the instantly disclosed technology. After capture of their released N-glycans, the remaining de-N- glycosylated proteins were digested by trypsin, and the resultant tryptic peptides were captured by disclosed technology. Tryptic peptides were fractionized into 24 fractions by Off-Gel technique and each fraction was analyzed by LC-MS/MS on a LTQ-Orbitrap. All proteins and peptides were identified by MASCOT searching against human Uniprot database (v2.3) with the following requirements: 1) FDR (false discover rate) of 1% at the peptide level; and 2) at least two unique peptides per protein. The total identified proteins and peptides were consistent among the samples, indicating the reproducibility of the instant disclosed technology. Deglycosylated peptides were determined by two criteria: 1) a peptide sequence containing a N-glycosylation consensus motif Asn-Xaa-Ser/Thr (where Xaa represents any amino acid except proline); and 2) Asn in the N-glycosylation consensus motif was characterized by the incorporation of ¹⁸O into the aspartic acid derivative. A glycoprotein was defined as the detection of its corresponding unique deglycosylated peptide(s). This demonstrates a high degree of coverage of the glycoproteome of both urine and plasma.

[00159] Figure 11 illustrates an application of the instantly disclosed technology on

HeLa cell lysate. HeLa cell lysate was processed by the disclosed technology. Released N- glycans were permethylated and analyzed by MALDI-MS. The profile demonstrates a highly heterogeneous glycan composition and demonstrates the ability to capture and evaluate N- glycans from a cell lysate.

[00160] Figure 12 illustrates a comparison of reproducibility and resolution of the instantly disclosed technology with upfront de-N-glycosylation and without upfront de-N- glycosylation.

[00161] Figure 13 shows binned density of peptide counts per protein. Data from six paired experiments (deglycosylated vs. control) are shown. X-axis: log2 value of

(deglycosylated/ctrl) unique peptide count per protein; Y-axis: Binned density of peptide count. The majority of glycoproteins gain more unique peptide sequences. Whereas there is no bigger difference for other proteins.

[00162] Figure 14 illustrates partial occupancy of glycosylation sites. The instantly disclosed technology allows for detection of a peptide having a glycosylation site in both its occupied (glycosylated) and non-occupied (native, non-glycosylated) form.

[00163] Figure 15 illustrates the identification of a partial occupied glycosylation site, a unique outcome provided by the instantly disclosed technology. This is an example of a partial glycosylated site (Asn- 1198) from the glycoprotein Attractin (075882

ATRN_HUMAN). Two different MS/MS spectra were used to identify the peptide sequence "DLDMFINASK". Both spectra were obtained from sample urine-02 (Figure 6) a) MS/MS spectra demonstrates the native non-glycosylated form of the peptide with the consensus site at the y4/b7 position (asparagine - N) b) MS/MS spectra demonstrates the deglycosylated form with one ¹⁸O-incorporated deamidated Asn (aspartic acid, D at y4/b7, red-highlighted). The mass increase (3 Da) of red-highlighted b- and y- ions in (b) unambiguously assigned the

¹⁸O-atom incorporated aspartic acid position. This assignment confirms that this N- glycosylation consensus site was previously occupied. By identifying both the native, non- glycosylated form and the deglycosylated form (e.g. ¹⁸O-incorporated deamidated Asn) using the disclosed technology, we demonstrate that site Asn-1198 is partially occupied in the glycoprotein Attractin.

[00164] Figure 16 illustrates an example of the instantly disclosed technology identifying a partial occupied glycosylation site: Apolipoprotein M. The application of the instantly disclosed technology on three different pairs of human plasma and urine samples lead to the identification of a partial occupied glycosylation site in Apolipoprotein M as another example of partial occupancy. Apolipoprotein M is a small glycoprotein (188 amino acid) that contains one consensus N-glycosylation site (Asn-135) and has previously been found in its glycosylated form in human plasma using a hydrazide-capture approach. Using the disclosed technology provided herein, analysis demonstrates that this glycoprotein can also exist as a non-glycosylated form (Plasma-02 and plasma-03 samples). This example further highlights that the occupancy of each individual site can vary in a single glycoprotein. Not detected - the peptide comprising N-glycosylation site was not detected in that sample; NG form - the peptide comprising N-glycosylation site was only identified as non- glycosylated (or native) form in that sample; G and NG-form -the peptide comprising N- glycosylation site was identified by both deglycosylated and non-glycosylated (native) forms in that sample; G form - the peptide comprising N-glycosylation site was only identified as the deglycosylated form in that sample.

[00165] Figure 17 illustrates an example of the instantly disclosed technology identifying a partial occupied glycosylation site: Kininogen-14. The application of the instantly disclosed technology on three different pairs of human plasma and urine samples lead to the identification of partial occupied glycosylation sites in Kininogen-14. Kininogen- 14 is known to have four N-glycosylation sites (Asn-48, Asn-169, Asn-205, and Asn-294). All four sites were detected using disclosed technology. It was further discovered that Asn-48 was exclusively partially occupied in all three plasma and urine samples. There was no evidence to support partial occupancy of Asn-294. Two sites Asn-169 and Asn-205 were intermittently partially occupied. This example further highlights that the occupancy of each individual site can vary in a single glycoprotein. The variation in occupancy may be related to the position of each site, but may also be sample specific. This further demonstrates the capability of the instantly disclosed technology to detect this biologic variation, which can't be detected by common hydrazide and lectin techniques. Figure Legend: Not detected - the peptide comprising N-glycosylation site was not detected in that samples; NG form - the peptide comprising N-glycosylation site was only identified as non-glycosylated (or native) form in that sample; G and NG-form -the peptide comprising N-glycosylation site was identified by both deglycosylated and non-glycosylated (native) forms in that sample; G form - the peptide comprising N-glycosylation site was only identified by deglycosylated form in that sample.

[00166] Figure 18 illustrates an example of the instantly disclosed technology identifying a complex glycoprotein (Attractin) with many glycosylation sites. The application of the instantly disclosed technology on three different pairs of human plasma and urine samples lead to the identification of partial occupied glycosylation sites in Attractin. Attractin has 26 potential N-glycosylation sites, and 15 sites were identified using disclosed technology. This technology identified four additional glycosylation sites (Asn-325, Asn- 1054, Asn-1073, and Asn-1082) that were not previously identified to be glycosylated by common hydrazide technique. This further demonstrates the sensitivity of the disclosed technology to characterize a heavily glycosylated glycoprotein even in a complex mixture (e.g. urine and plasma). The non-glycosylated form of the site Asn-1198 was detected in all six human samples, and was found to be partially occupied in 4 of the 6 samples.

[00167] Figure 19 illustrates a demonstration of de-N-glycosylation by domestic microwave oven on standard glycoproteins: bovine RNase B (A), bovine fetuin (B), human IgG (C ), and human a-1 acidic glycoprotein (D). * : carried out in the traditional water bath.

[00168] Figure 20. De-N-glycosylation of depleted human urine proteins (500 μg per aliquot) in domestic microwave with various amounts of PNGase F, Coommassie blue stain (A), and Glycoprotein stain (B ). Lane 1 is normal urine after albumin depletion. Lanes 2-5 are de-N-glycosylated urine with varying amounts of PNGase F.

[00169] Figure 21 illustrates an SDS-PAGE of bovine RNase B (A); bovine fetuin (B); human IgG (C); and MARS-7 bound fractions from two human plasma samples (D & E); before and after PNGase F digestion by domestic microwave irradiation (DMW) or standard water bath method (* indicates overnight incubation in a 37 °C water bath). (A) to (D) were stained by coomasssie blue stains, and (E) was stained by Pro-Q glycoprotein stain. The band of PNGase F (~ 36K Da) was visible in figures A to D). These examples demonstrate that domestic microwave assisted digestion is effective at catalyzing the PNGase F digestion of glycans in 20 minutes and at low amounts of PNGase F in model glycoproteins, IgG and a complex sample.

[00170] Figure 22 illustrates MALDI-MS spectra of permethylated N-glycans from bovine RNase B (A); bovine fetuin (B); and human IgG (C) after the domestic microwave release protocol. Each peak was assigned a representative cartoon based on their m/z value and biological synthetic pathway, representing composition only and not implicating a fully defined structure. This demonstrates that the domestic microwave assisted digestion does not appear to affect the glycan fraction.

[00171] Figure 23 illustrates bovine fetuin digested by PNGase F in a traditional 37 °C oven at varying incubation times (Lane 2 to Lane 5), as compared to the domestic microwave oven protocol (Lane 6). At shorter times using the conventional method, incomplete digestion is achieved. Complete digestion is achieved using overnight (Lane 5) and the domestic microwave irradiation. Lane M: ladder. Lane 1: native fetuin. Lane 2: conventional heating for 20 minute. Lane 3: 1 hour. Lane 4: 3 hours. Lane 5: overnight (14 hours). Lane 6: 20 minute domestic microwave irradiation.

[00172] Figure 24 illustrates an MALDI-MS of permethylated N-glycans of bovine fetuin. (a) The remaining N-glycans in the sample chamber of the filter device without acidification; and (b) the remaining N-glycans in the sample chamber of the filter device after acidification. Highly sialic acidic N-glycans were selectively retained in the sample chamber (a) due to the ionic interactions between the glycans and protein at neutral or basic conditions. After acidifying, these interactions were effectively interrupted, allowing the elution of large glycans with multiple sialic acid residues by filtration (b). All ions were single sodium adducts [M+Na]+ and the monoisotopic peak was annotated.

Figure 25 illustrates an additional urine sample that was obtained from the donor of Ul, and processed according to the methods provided herein. The N-glycans were eluted by additional acidic washes (X8) to determine if there were residual glycans not captured. The initial three elutions and the remaining five elutions were respectively combined.

Furthermore, the deglycosylated proteins were recovered from the sample chamber of the filter and passed through a C18 SPE cartridge to determine if there were residual glycans with the protein sample. The MALDI-MS of permethylated urinary N-glycans were depicted: (a) the combined first three acidic elutions; (b) the remaining five acidic elutions; and (c) the remaining N-glycans from the protein solution. It clearly demonstrates that three acidic elutions effectively separate the vast majority of urinary N-glycans (including the complex and large acidic glycans) from the proteins by filtration (a). N-glycans were not detected in the additional acidic elutions (b), or in the remaining protein sample processed by C18 SPE (c). All ions were single sodium adducts [M+Na]+ and the monoisotopic peak was annotated. EQUIVALENTS AND SCOPE

[00173] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the technology described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[00174] In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[00175] Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a

composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[00176] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the technology, or aspects of the technology, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the technology or aspects of the technology consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment that comprises one or more elements, features, steps, etc., the disclosure also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[00177] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[00178] In addition, it is to be understood that any particular embodiment of the presently disclosed technology may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods disclosed or provided herein can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Claims

CLAIMS What is claimed is:

1. A method of separating a polypeptide and/or a carbohydrate from a mixture comprising the polypeptide and the carbohydrate, the method comprising:

(a) acidifying the mixture; and

(b) separating the polypeptide and/or the carbohydrate from the mixture.

2. The method of claim 1, wherein the carbohydrate is a monosaccharide, a disaccharide, or an oligosaccharide.

3. The method of claim 1 or 2, wherein the carbohydrate is a glycan.

4. The method of claim 3, wherein the glycan and the polypeptide are cleaved from a glycoprotein, and wherein the method comprises cleaving a bond between the glycan and the polypeptide.

5. A method of separating a polypeptide and/or a glycan from a glycoprotein comprising the polypeptide bound to the glycan, the method comprising:

(a) cleaving a bond between the polypeptide and the glycan, thus forming a mixture of the polypeptide and the glycan;

(b) acidifying the mixture; and

(c) separating the polypeptide and/or the glycan.

6. The method of any of claims 3-5, wherein the glycan is selected from the group consisting of an O-glycan and an N-glycan.

7. The method of any of claims 3-5, wherein the glycan is bound to the polypeptide via a β-amide group which links to the side chain of an asparagine residue comprised in the polypeptide.

8. The method of any of claims 3-5, wherein the glycan is bound to the polypeptide via the hydroxyl group of the side chain within a serine or threonine residue comprised in the polypeptide.

9. The method of claim 4 or claim 5, wherein the bond is a covalent bond.

10. The method of any one of claims 1-9, wherein the mixture has a pH of 4.5-13 or of 6.5-13.

11. The method of any one of claims 1-10, wherein the acidifying comprises changing the pH of the mixture of the polypeptide and the glycan to pH 2-4.

12. The method of any one of claims 1-11, wherein the mixture is acidified by contacting the mixture with formic acid, trifluoro-acetic acid, or acetic acid.

13. The method of any one of claims 4-12, wherein the cleaving of the bond comprises contacting the glycoprotein with a glycosidase.

14. The method of claim 13, wherein the glycosidase comprises an endoglycosidase.

15. The method of claim 13, wherein the glycosidase comprises an exoglycosidase.

16. The method of claim 13, wherein the glycosidase comprises one or more

endoglycosidases selected from the group comprising peptide N-glycosidase F,

endoglycosidase H, endoglycosidase Hf, endoglycosidase F, endoglycosidase S, peptide N- glycosidase A, and O-glycanase.

17. The method of any one of claims 4-12, wherein the cleaving of the bond comprises base elimination.

18. The method of any one of claims 4-17, wherein the cleaving of the bond comprises exposing the glycoprotein to microwaves.

19. The method of claim 18, wherein the microwaves are generated by a domestic microwave device.

20. The method of any one of claims 3-19, wherein the glycan and the polypeptide have a different molecular weight/size and the separating comprises separating the polypeptide and the glycan based on their molecular weight/size difference.

21. The method of claim 20, wherein the separating comprises size fractionation, size exclusion, or filtration over a semi-permeable membrane.

22. The method of claim 21, wherein the filtration over a semi-permeable membrane comprises dialysis, spin-filter centrifugation or the application of positive or negative pressure.

23. The method of any one of claims 1-22, wherein the method is carried out under non- denaturing conditions.

24. The method of any one of claims 3-23, wherein the method comprises isolating the glycan separated from the polypeptide.

25. The method of any one of claims 3-24, wherein the method comprises isolating the polypeptide separated from the glycan.

26. The method of any one of claims 1-25, wherein the method further comprises fragmenting the polypeptide.

27. The method of claim 26, wherein the fragmenting comprises digesting the polypeptide with a protease.

28. The method of any one of claims 3-27, further comprising analyzing the glycan and/or the polypeptide after the separating.

29. The method of claim 28, wherein analyzing comprises subjecting the glycan and/or the polypeptide to high performance liquid chromatography, capillary electrophoresis, and/or mass spectrometry.

30. The method of claim 28 or 29, wherein analyzing comprises identifying a glycosylation site and/or a level of occupation of a glycosylation site.

31. The method of any of claims 1-30, further comprising modifying or labeling the carbohydrate or the glycan.

32. The method of claim 31, wherein the labeling comprises permethylation or fluorophore labeling.

33. The method of claim 32, wherein the fluorophore labeling comprises 2-AA (2- aminobenzoic acid (anthranilic acid)) or 2-AB (2-aminobenzamide) labeling.

34. A device for separating a carbohydrate and/or a polypeptide from a

carbohydrate/polypeptide mixture, the device comprising:

a sample compartment,

a collection compartment, and

a molecular weight/size- selective material separating the sample compartment from the collection compartment, wherein the molecular weight/size- selective material has a molecular weight/size cutoff that is between the molecular weight/size of the carbohydrate and the molecular weight/size of the polypeptide.

35. The device of claim 34, wherein the molecular weight/size- selective material comprises a polyether sulfone.

36. The device of claim 34, wherein the molecular weight/size- selective material comprises an acidic moiety.

37. The device of claim 34, wherein the molecular weight/size- selective material comprises a carboxyl- and/or an acidic anhyhdride moiety.

38. The device of any of claims 34-37, wherein the molecular weight/size- selective material is a semi-permeable membrane having a molecular weight/size cutoff above the molecular weight/size of the carbohydrate and below the molecular weight/size of the polypeptide.

39. The device of claim 38, wherein the molecular weight/size cutoff of the membrane is within the range of 10 kDa - 30 kDa.

40. The device of claim 39, wherein the molecular weight/size cutoff of the membrane is within the range of 15 kDa - 18 kDa.

41. The device of claim 39, wherein the molecular weight/size cutoff of the membrane is about 20kDa.

42. The device of any one of claims 34-41, wherein the molecular weight/size cutoff is stable over a pH range of pH 2 - pH 12.

43. The device of any one of claims 34-42, wherein the molecular weight/size- selective material does not comprise cellulose.

44. The device of any one of claims 34-42, wherein the molecular weight/size- selective material does not comprise a carbohydrate and/or does not produce a carbohydrate moiety during the processing.

45. The device of any one of claims 34-44, wherein the molecular weight/size- selective material is a polyethersulfone membrane.

46. The device of any one of claims 34-45, wherein the device is suitable for exposure to heat.

47. The device of any one of claims 34-46, wherein the device is suitable for exposure to microwaves.

48. The device of any one of claims 34-47, wherein the device comprises a centrifugal filter device.

49. The device of claim 48, wherein the centrifugal filter device is suitable for use at up to 10000g.

50. The device of any one of claims 34-49, wherein the device is suitable for

micro waving.

51. A kit, comprising:

the device for separating a carbohydrate and/or a polypeptide of any one of claims 34-

50; and

a buffer or reagent suitable for separating a carbohydrate and a polypeptide.

52. The kit of claim 51, wherein the carbohydrate is a glycan.

53. The kit of claim 51 or 52, wherein the kit comprises a glycosidase.

54. The kit of claim 53, wherein the glycosidase comprises an endoglycosidase.

55. The kit of claim 53, wherein the kit comprises an exoglycosidase.

56. The kit of claim 53, wherein the glycosidase comprises one or more endoglycosidases selected from the group comprising peptide N-glycosidase F, endoglycosidase H,

endoglycosidase Hf, endoglycosidase F, endoglycosidase S, peptide N-glycosidase A, and O- glycanase.

57. The kit of any one of claims 51-56, wherein the kit comprises O¹⁸ water.

58. The kit of any one of claims 51-57, wherein the endoglycosidase is in a solution comprising O¹⁸ water.

59. The kit of any one of claims 51-58, wherein the kit comprises an acidifying solution.

60. The kit of claim 59, wherein the acidifying solution comprises formic acid, trifluoro- acetic acid, or acetic acid.

61. The kit of any one of claims 51-60, wherein the kit comprises a protease.

62. The kit of any one of claims 51-61, wherein the protease is trypsin.

63. The kit of any one of claims 51-62, wherein the kit comprises instructions for separating a carbohydrate and a polypeptide.