Anal Bioanal Chem DOI 10.1007/s00216-015-8717-z RESEARCH PAPER Separation of monosaccharides hydrolyzed from glycoproteins without the need for derivatization Mark S. Lowenthal1 & Eric L. Kilpatrick1 & Karen W. Phinney1 Received: 20 January 2015 /Revised: 27 March 2015 /Accepted: 16 April 2015 # Springer-Verlag Berlin Heidelberg (outside the USA) 2015 Abstract Chromatographic separation of monosaccharides Introduction hydrolyzed from glycoconjugates or complex, aggregate bio- materials, can be achieved by classic analytical methods with- Characterization of carbohydrates and glycans in biological out a need for derivatizing the monosaccharide subunits. A systems is of critical interest in several diverse areas including simple and sensitive method is presented for characterizing therapeutics development [1, 2], disease diagnostics (cancer, underivatized monosaccharides following hydrolysis from heart disease, diabetes, and numerous others) [3–5], and elu- N- and O-linked glycoproteins using high-performance liquid cidation of biological processes [6–9] (cellular metabolism, chromatography separation with mass spectrometry detection biosynthesis, and development). Additionally, greater under- (LC-MS). This method is adaptable for characterizing any- standing of protein folding [10, 11], immunity, energy storage, thing from purified glycoproteins to mixtures of glycoforms, and other areas are dependent on knowledge of glycan com- for relative or absolute quantification applications, and even position. Due to the sheer numbers of possible forms, along for the analysis of complex biomaterials. Use of an amide with their chemical similarity and structural complexity, car- stationary phase with HILIC chromatography is demonstrated bohydrates are among the most difficult classes of biomole- to retain the highly polar, underivatized monosaccharides and cules to characterize and/or quantify whether they exist as to resolve stereoisomers and potentially interfering contami- purely saccharide forms (mono-, oligo-, or poly-saccharide), nants. This work illustrates an original approach for character- or bound to other types of biomolecules forming ization of N- and O-linked glycoprotein standards, mixtures, glycoconjugates (glycoproteins, glycolipids, or proteogly- and for complex biological materials such as a total yeast cans). While complex in the aggregate, glycoconjugates are extract. principally composed of a small class of similar, yet measur- able, subunits of monosaccharides. Mammalian glycosyla- tions are made up of a limited number of monosaccharide Keywords Bioanalytical methods . HPLC . Mass forms – principally galactose, glucose, mannose, fructose, fu- spectrometry / ICP-MS . Separations/Instrumentation cose, N-acetyl glucosamine (GlcNAc), N-acetyl galactos- amine (GalNAc), N-acetyl neuraminic acid (NANA), and rarely, N-glycolyl neuraminic acid (NGNA). Other minor components are seldom included such as xylose, iduronic ac- id, others. Each of these monomeric forms can be uniquely Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8717-z) contains supplementary material, detected in a mass spectrometer as an underivatized com- which is available to authorized users. pound based on a fragmentation pattern alone, or in cases of stereoisomers, with additional chromatographic retention * Mark S. Lowenthal information. [email protected] Monosaccharide characterization offers valuable compli- mentary data to the analysis of intact glycans or glycoprotein 1 Biomolecular Measurement Division, National Institute of Standards characterization. Total monosaccharide characterization is and Technology, Gaithersburg, MD 20899, USA possible for simple or complex mixtures, and can take account M.S. Lowenthal et al. of monosaccharides originating from non-targeted proteins or an approach for monosaccharide characterization that isn’t unexpected impurities. The characterization of biosimilars – currently available. protein therapeutics sharing similar function but with intrinsic structural variability – relies on data obtained at the glycan level, a technically difficult task often hampered by the lack Experimental of commercially available homogenous glycoprotein stan- dards and by the complexity of known and potentially un- Disclaimer Certain commercial equipment, instruments, and known glycoforms. Furthermore, biases of omission are com- materials are identified in this paper to adequately specify the mon in glycan profiling. For instance, contributions from experimental procedure. Such identification does not imply host-cell proteins, excipients, or glycation to the overall car- recommendation or endorsement by NIST nor does it imply bohydrate composition of a biosimilar product are often that the equipment, instruments, or materials are necessarily overlooked when measured at the glycan level, but are intrin- the best available for the purpose. sically considered by monosaccharide measurements. The qualitative approach presented here using mass spectrometry detection offers the potential to perform quantitative measure- Materials ments using isotope dilution (ID) techniques, which is not currently possible using HPAEC-PAD or other detection tech- All chemicals and solvents were purchased through Sigma niques. Monosaccharide internal standards are available com- (St. Louis, MO) unless otherwise noted below. Isotope- mercially, and may be applied generally to IS-MS measure- labeled monosaccharide standards were purchased through ments. Monosaccharide characterization is therefore sug- Omicron Biochemicals (South Bend, IN). Unlabeled mono- gested to be a valuable complementary tool for assessing the saccharide standards were purchased through Sigma (Aldrich degree of biosimilarity. and Fluka). Casein from bovine milk (C7078), fetuin from Although there are previous literature reports that describe fetal calf serum (F2379), and yeast extract (Y4250) were also acid hydrolysis of glycoproteins into their constituent mono- purchased through Sigma. saccharide units with subsequent analysis using LC-MS [12–17] or capillary electrophoresis [18], each of these studies Sample preparation requires derivitization of monosaccharides for detection, like- ly incorporating measurement bias in proportion to the label- Stable-isotope (13C) labeled analogs of each monosaccharide ing inefficiency. The current industry standard for analysis of were purchased from commercial sources and pre-spiked at non-derivatized, hydrolyzed sugars – anion exchange chroma- the earliest possible step in the sample preparation (se tography with pulsed amperometric detection [19](HPAEC- Electronic Supplementary Material (ESM) Table SI). In the PAD) – is capable of chromatographic resolution of monosac- case for a total yeast extract sample, a glycoprotein extraction charides and can be a very sensitive detection technique, but is step with methanol/chloroform (MeOH/CHCl3) was neces- inherently incompatible with direct mass spectrometry detec- sary to first precipitate the protein sample prior to spiking tion, and therefore incompatible with isotope dilution tech- the labeled analogs. Protein precipitation was performed with- niques. In contrast, the work described here presents a novel in a 400-μL glass autosampler vial insert (Agilent 5181-3377) method focusing on chromatographic resolution and mass that was then used for the acid hydrolysis, reducing protein spectral detection of monosaccharides without modification. loss. In all other cases, pre-spiked samples were dried in a One certain way to improve monosaccharide measurements is speed vac (ThermoSavant SPD1010) and subjected separately through simplifying the sample preparation. This effort reports to each of three unique acid hydrolyses. Hydrolysis was per- a workflow for releasing monosaccharides from intact glyco- formed in a similar manner to previously reported studies by proteins and characterizing them by multiple reaction moni- Mechref’sgroup[16, 17]. In all cases, gas-phase hydrolysis toring (MRM) using liquid chromatography-tandem mass was achieved within a capped glass vial housing the glass spectrometry (LC-MS/MS) techniques. An amide stationary autosampler insert. Figure 1 provides a schematic of the sam- chemistry was used within a normal phase (HILIC) environ- ple preparation procedure for each monosaccharide class. ment to resolve ten underivatized monosaccharides prior to Briefly, glycoproteins were denatured, and neutral sugars negative polarity electrospray ionization, and detection in a (mannose, galactose, glucose, fructose, and fucose) or amine triple quadrupole mass spectrometer. This analytical approach sugars (GlcNAc and GalNAc) were liberated from their gly- is demonstrated in principle for N-linked glycoproteins (im- coprotein precursors using 2 mol/L trifluoroacetic acid (TFA, munoglobulins, RNAse B), O-linked glycoproteins (casein, Fluka 91699) or 4 mol/L hydrochloric acid (HCl, Fluka fetuin), and a complex yeast extract. The combination of sim- 84410), respectively, at 100 °C for 4 h. Sialic acids (NANA, plified sample preparation presented here with chromato- NGNA), being terminal subunits and less chemically stable, graphic separation and mass spectrometric detection provides were hydrolyzed under milder conditions (0.1 mol/L TFA for Monosaccharide characterization without derivatization Fig. 1 Schematic for the hydrolysis and preparation of each monosaccharide class (neutrals, amine sugars, sialic acids) prior to LC-MS analysis 2 h at