Complementary Sample Preparation Strategies for Analysis of Cereal β-Glucan Oxidation Products by UPLC-MS/MS

Abstract

The oxidation of cereal (1→3,1→4)-β-D-glucan can influence the health promoting and technological properties of this linear, soluble homopolysaccharide by introduction of new functional groups or chain scission. Apart from deliberate oxidative modifications, oxidation of β-glucan can already occur during processing and storage, which is mediated by hydroxyl radicals (HO^•) formed by the Fenton reaction. We present four complementary sample preparation strategies to investigate oat and barley β-glucan oxidation products by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), employing selective enzymatic digestion, graphitized carbon solid phase extraction (SPE), and functional group labeling techniques. The combination of these methods allows for detection of both lytic (C1, C3/4, C5) and non-lytic (C2, C4/3, C6) oxidation products resulting from HO^•-attack at different glucose-carbons. By treating oxidized β-glucan with lichenase and β-glucosidase, only oxidized parts of the polymer remained in oligomeric form, which could be separated by SPE from the vast majority of non-oxidized glucose units. This allowed for the detection of oligomers with mid-chain glucuronic acids (C6) and carbonyls, as well as carbonyls at the non-reducing end from lytic C3/C4 oxidation. Neutral reducing ends were detected by reductive amination with anthranilic acid/amide as labeled glucose and cross-ring cleaved units (arabinose, erythrose) after enzyme treatment and SPE. New acidic chain termini were observed by carbodiimide-mediated amidation of carboxylic acids as anilides of gluconic, arabinonic, and erythronic acids. Hence, a full characterization of all types of oxidation products was possible by combining complementary sample preparation strategies. Differences in fine structure depending on source (oat vs. barley) translates to the ratio of observed oxidized oligomers, with in-depth analysis corroborating a random HO^•-attack on glucose units irrespective of glycosidic linkage and neighborhood. The method was demonstrated to be (1) sufficiently sensitive to allow for the analysis of oxidation products also from a mild ascorbate-driven Fenton reaction, and (2) to be specific for cereal β-glucan even in the presence of other co-oxidized polysaccharides. This opens doors to applications in food processing to assess potential oxidations and provides the detailed structural basis to understand the effect oxidized functional groups have on β-glucan's health promoting and technological properties.

Keywords: Fenton-reaction; MS/MS; UPLC; hydrophilic interaction liquid chromatography; labeling; lichenase; β-glucan oxidation; β-glucosidase.

Figure 1

(A) Chemical structure of the glucopyranose (Glc) repeating unit of cereal β-D-glucan (BG) with numbered carbons and indicated glycosidic linkage proportions, together with the symbolic representation of the polysaccharide and its cellulosic β-(1→4) regions. In addition, the fine structure analysis by selective hydrolysis with lichenase is shown, with blue arrows indicating the specific β-(1→4)-linkages susceptible to cleavage by the endo-enzyme and the resulting characteristic ratios of formed oligosaccharides DP3 and DP4. (B) Workflow of the cereal BG oxidation study (from oat and barley) with the two oxidation conditions (a) and (b) and the four complementary sample preparation strategies I–IV. Blue ○ = G, glucose unit; blue ○–OH = G, reducing end glucose unit; oxBG; oxidized β-glucan; AH₂, ascorbic acid; C=O, carbonyl group; CO₂H, carboxylic acid group; SPE, solid phase extraction; UPLC-MS/MS, ultraperformance liquid chromatography tandem mass spectrometry.

Figure 2

Starting from β-(1→4)-linked glucosyl repeating unit (middle), the detectable oxidation products from hydroxyl radical (HO^•)-attack at any of the C1–6 glucose carbons after reacting with oxygen (O₂) are represented as structures, depicted symbols, abbreviations, and full names. Depending on site of attack, the resulting product is formed with or without chain scission, classified as lytic (plane arrows) or non-lytic oxidation (hashed arrows), respectively. Arabinose can by formed both directly under lytic cross-ring cleavage (C1-C2), or as secondary product of gluconic acid (from C1-oxidation) by the indicated, so-called Ruff-degradation (bottom right). ^*Lytic oxidations under cross-ring cleavage and loss of one or two carbons (e.g., formic acid, glycolic acid).

Figure 3

(A) UPLC-MS base peak chromatogram in the negative ionization mode using a BEH amide column and ACN/H₂O gradient (0.1% NH₃) for analysis of the released oligosaccharides from barley β-glucan (BBG) oxidation (harsh conditions) extracted by SPE and concentrated (sample preparation strategy I). Note the 6x zoom for region 6–12 min. Peaks are labeled with their respective base peak m/z and with n = number of monosaccharide units in blue for gluco-oligosaccharides Glc_n and in red for oxo-Glc_n species (with letter qualifier if same n occurs more than once, e.g., 3a, 3b…). (B) MS spectrum of the acidic products obtained from the indicated chromatogram region in (A). Signals are labeled with m/z, with the glucose-carbon location of the carboxylic acid (C1, C6, or C1+C6), and with type of reducing end if cross-ring cleaved to arabinose (Ara). Glc, glucose; oxo-Glc_n, oligomer Glc_n oxidized anywhere along the chain (new C=O); Glc₃Ara/Ery, glucotriose linked to arabinose/erythrose end group; Glc1A, gluconic acid group (C1 oxidized); GlcA, glucuronic acid group (C6 oxidized).

Figure 4

Depiction of mixed-linkage BG and examples of possible oxidation products formed through attack of HO^• at different glucose carbons, followed by sample preparation strategy II. Lichenase selectively hydrolyzed as expected the β-(1→4)-linkage of the β-(1→3)-linked glucose units. Digestion by β-glucosidase, an exo-enzyme, was found to successively cleave off glucose units, leaving oxidized oligomers behind, whereas native regions largely hydrolyzed to glucose. Graphitized carbon SPE removed monosaccharides from the reaction mixture (acidic monomers only partially removed). Downstream (reducing) end of oligomers is on the right side (symbolic differentiation as ○–OH omitted for clarity).

Figure 5

UPLC-MS/MS results in the negative mode using a BEH amide column and ACN/H₂O gradient for analysis of barley β-glucan (BBG) oxidation products (harsh conditions) after enzymatic digestion and SPE (strategy II). (A) Analysis of the neutral products using basic 0.1% NH₃ eluent additive (oxo-product peaks with carbonyl group at the non-reducing end ^oxoGlcGlc_(n−1) in red) and (B) their respective MS/MS spectra. The main oxo-Glc₅ species with C=O at a different position is also included (bottom spectrum). (C) Analysis of acidic products using a buffered 60 mM ammonium formate eluent additive (pH ~ 8) with glucuronic acid bearing oligomers GlcAGlc_(n−1) in red. (D) Respective MS/MS spectra of acidic products. The MS/MS fragments are named according to the nomenclature of Domon and Costello (1988) (cleaved linkage: C_i fragments; cross-ring cleavage: A_i fragments; i = 1, 2, … n). ^*oxo-products with oxidized carbonyl group not at the non-reducing end; ^**Disaccharide signal from catalase material; ^***Buffer salt/solvent peak.

Figure 6

Susceptibility of native and modified β-glucobioses to hydrolysis by the used exo-β-glucosidase (from Aspergillus niger; EC 3.2.1.21; GH family 3). The enzyme was observed to cleave off native β-(1→3)- and β-(1→4)-linked non-reducing end glucosyl units, unless they are β-(1→3)-linked to certain types of modified units (red ○ with ^*), especially if containing CO₂H or labeled functional groups. Blue ○, glucose unit; blue ○–OH, reducing end glucose unit.

Figure 7

(A) Depiction of carbonyl labeling strategy III by reductive amination with anthranilic acid (2-AA; X = OH) or 2-aminobenzamide (2-AB; X = NH₂) with enzyme digestion and SPE steps analogous to strategy II (see Figure 4). Negative mode UPLC-MS base peak chromatograms (BPI; basic eluent) resulting from oxidation of barley β-glucan (BBG) under a) harsh and b) mild conditions after the C=O sample preparation are shown for (B) SPE fraction 2 containing labeled reducing termini, and (C) SPE fraction 1 containing labeled oxo-products with extracted ion chromatograms (XIC) of 2AB-(oxo-Glc_n). BPI of (C) were obtained after BPI-subtraction of the control sample (0.6% BBG) subjected to the same sample preparation, and the insert under b) shows the 36x zoomed XIC of m/z 281. AH₂, ascorbic acid; Glc, glucose; Ara, arabinose; Ery, erythrose; Ar, aromatic ring; ^5oxoGlc-2AB_o, presumed cyclic product from 5-oxo-reducing ends (see Figure 8); ^oxoTet-2AB_o = “T,” presumed cyclic product of l-threo-tetrodialdose (m/z 221); 2AB-(oxo-Glc_n), labeled oxo-Glc_n at oxidized C=O group; HexGlc_(n−1), oligosaccharide with one sugar unit being an undefined hexose (side products, presumably from direct reduction of oxo-Glc_n; see Figure S9).

Figure 8

Lytic C5-oxidation forming a new terminus ^5oxoGlc under loss of the non-reducing end-portion of the β-glucan polymer, and its reductive amination with 2-aminobenzamide (2-AB) (C=O labeling strategy III). The shown mechanism explains the cyclic end products ^5oxoGlc-2AB_o and Glcβ(1→3)^5oxoGlc-2AB_o detected in SPE fraction 1), which involves two reductive amination steps to form the piperidine derivative (first intermolecular tagging of the reducing end carbonyl (C1), then intramolecular with the C5-ketone). Glc, Glucose; [O], oxidation (Fenton-induced); [H], reduction (hydride from NaBH₃CN).

Figure 9

Depiction of (A) carboxylic acid labeling strategy IV by EDC-mediated amidation of oxidized β-glucan with aniline (PhNH₂) and enzyme digestion/SPE steps analogous to strategy II (see Figure 4). (B) The resulting base peak ion chromatograms (BPI) from oxidation of barley β-glucan (BBG) under (a) harsh and (b) mild conditions after the CO₂H sample preparation (basic eluent). The peaks are labeled with their base peak ion m/z and corresponding structure (chemical structures on the right-hand side). The insert under (b) shows 100x zoom of overlaid extracted ion chromatograms of m/z 270 and 432. Glc, glucose; Glc1A, gluconic acid; GlcA, glucuronic acid; Ara1A, arabinonic acid; Ery1A, erythronic acid; AH₂, ascorbic acid; Ph, phenyl; -NHPh or PhNH-, aniline amide (anilide) of acid species; EDC, ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Figure 10

Comparison of UPLC-MS base-peak ion (BPI) chromatograms of polysaccharide oxidation under harsh conditions (100 mM H₂O₂, 50 μM FeSO₄) after lichenase and β-glucosidase treatment/SPE for (A) only barley β-glucan (BBG) (0.6%; as in Figure 5A), (B) BBG and corn starch (each 0.6%), and (C) only corn starch (0.6%). (D) UPLC-MS of malto-oligosaccharide standards up to maltohexaose (n = 6). Glc, glucose; Glc1A, gluconic acid; GlcA, glucuronic acid; ^oxoGlc, oxo-glucose; oxo-Glc_n, gluco-oligomer with oxidized carbonyl group anywhere along the chain; ^oxoGlcGlc_(n−1), gluco-oligomer with oxidized carbonyl at non-reducing end; L+βG/SPE, after lichenase and β-glucosidase treatment, followed by fractionation by SPE; ^*, oxo-Glc_n with oxidized C=O not at the non-reducing end; α, malto-oligosaccharides [Glc_n with all α-(1→4)].

Figure 11

Influence of fine structure differences (DP3/DP4) of oat and barley β-glucan on the ratios of oligomeric oxidation products. Explanation for the resulting oligomeric oxidation product ratios (n = 3)/(n = 4) from (A) lytic C3/C4-oxidation and (B) non-lytic C6-oxidation, both after lichenase and β-glucosidase treatment/SPE (strategy II). ^oxoGlcGlc_(n−1), gluco-oligomer with oxidized carbonyl at non-reducing end; GlcAGlc_(n−1), oligomer with glucuronic acid (C6-oxidized) unit at non-reducing end (unless for n = 2, where also at reducing end); BBG, barley β-glucan; OBG, oat β-glucan; DP3/DP4, molar ratio of cellotriosyl to cellotetraosyl units in β-glucan (defines fine structure).