Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

doi:10.21203/rs.3.rs-4721444/v1

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Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

https://doi.org/10.21203/rs.3.rs-4721444/v1

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Free oligosaccharides in human milk have many biological functions for infant health. The reducing end of most human milk oligosaccharides is lactose, and caprine milk was reported to contain oligosaccharides structurally similar to those present in human milk. The structures of oligosaccharides were traditionally determined using nuclear magnetic resonance spectroscopy or enzyme digestion. Mass spectrometry has much higher sensitivity than nuclear magnetic resonance spectroscopy and enzyme digestion. However, conventional mass spectrometry methods only determine part of the structures of oligosaccharides, i.e., compositions and linkage positions. In this study, we used the latest developed mass spectrometry method, namely logically derived sequence tandem mass spectrometry, to determine the complete structures (i.e., composition, linkage positions, anomericities, and stereoisomers) of free neutral trisaccharides in caprine colostrum and mature milk. The high sensitivity of mass spectrometry enables us to discover oligosaccharides of low abundance. Isomers of (Hex)₂HexNAc, (Hex)₃, and (Hex)₂Fuc which have not been reported before were identified. Many of them do not have lactose at the reducing end. Instead, the reducing end is either Glcb-(1–4)-Glc or Glcb-(1–4)-GlcNAc. These unusual oligosaccharides are higher in concentration and more structurally diverse in caprine colostrum than that in caprine mature milk and human milk. The structural diversity indicates more complicated biosynthetic pathways of caprine milk compared to that of human milk.

Physical sciences/Chemistry/Analytical chemistry

Physical sciences/Chemistry/Biochemistry

Caprine milk

colostrum

oligosaccharides

mass spectrometry

structure

Free oligosaccharides are one major class of macromolecules in human milk^1–3. Though most milk oligosaccharides are indigestible, studies suggest that they act as prebiotics, deflect the adhesion of pathogens in the infant gut, and affect the local intestinal immune system of the breast-fed infant^4–16. The concentration of free oligosaccharides in human is high (5–23 g/L)¹⁷, and more than 200 different oligosaccharides have been reported with predominance of neutral fucosylated oligosaccharides (50 − 70%) compared to acidic ones (10 − 25%)^18–28. In contrast, the concentrations of free oligosaccharides in bovine milk (0.03–0.06 g/L) and caprine milk (0.25–0.30 g/L) are low and are less structurally diverse, but oligosaccharides present in bovine and caprine colostrum are higher in concentration and more structural diversity than those in mature milk^29–43. Most of the free oligosaccharides in human milk consist of a lactose (Galβ-(1–4)-Glc) at the reducing end^{27, 44}. Caprine milk was reported to contain oligosaccharides structurally similar to those present in human milk, suggesting that caprine milk oligosaccharides can be used to mimic human milk oligosaccharides for infant health^45–49.

The complete structures of oligosaccharides were conventionally determined using enzyme digestion¹⁸ or nuclear magnetic resonance spectroscopy^29–33, but these methods require a large amount of samples and are not suitable for oligosaccharides of low abundances. Since the sensitivity of mass spectrometry is three to four orders of magnitude higher than that of the aforementioned methods, mass spectrometry has been widely used for the structural determination of oligosaccharides^21–24. However, conventional mass spectrometry methods only determine part of the structures of oligosaccharides, i.e., compositions and linkage positions; they do not provide information on anomericity (differentiation of the α and β anomeric configurations of glycosidic bonds) and monosaccharide stereoisomers (e.g., differentiation of glucose, galactose, and mannose, or N-acetylglucosamine and N-acetylgalactosamine). Consequently, the milk oligosaccharides whose structures have been completely determined are mainly high in abundance.

A new mass spectrometry technique, logically derived sequence tandem mass spectrometry (LODES/MSⁿ), for oligosaccharide structural determination, was recently developed^50–55. In this method, oligosaccharides are dissociated into desired monosaccharide and disaccharide fragments through sequential collision-induced dissociation (CID) in mass spectrometer, and the structures of these monosaccharides and disaccharides are determined by subsequent CID in the same mass spectrometer. In each step of CID, a fragment based on dissociation mechanism is chosen as the precursor ion for the next step of CID^56–60. The structures of oligosaccharides are then reconstructed by combining the structures of various monosaccharides and disaccharides. Details of the method have been reported^50–56. This method can determine the linkage positions, identify the α and β anomeric configurations, and differentiate stereoisomers in oligosaccharides. LODES/MSⁿ has been applied to determine the structures of polysaccharide⁶¹, oligosaccharides in glycosphingolipids⁶², N-glycans released from proteins^{63, 64}, and the free oligosaccharides in milk⁶⁵. Because of the high sensitivity of mass spectrometry, LODES/MSⁿ can be used to determine the oligosaccharides in trace amount which cannot be determined using conventional enzyme digestion and nuclear magnetic resonance spectroscopy. In this study, we applied this method to determine the neutral trisaccharides in caprine colostrum and mature milk, and many new oligosaccharides were found. Particularly, many of them do not have the conventional structures of milk oligosaccharides in which the reducing end is lactose (Galβ-(1–4)-Glc). The results suggest the existence of undiscovered biosynthetic pathways.

(A) Isomer purification and structural determination

The free oligosaccharides extracted from caprine colostrum were separated into different sizes using size exclusion chromatography. The eluents of trisaccharides from size exclusion chromatography were then sent into a HPLC with an amide-80 column for the first dimension separation. The chromatograms of the trisaccharides, consisting of two hexoses and one fucose (Hex)₂Fuc, three hexoses (Hex)₃, and two hexoses and one N-acetylhexosamine (Hex)₂HexNAc separated by the amide-80 column and detected by mass spectrometry of selected ions m/z 511, 527, and 568 (sodium ion adducts of (Hex)₂Fuc, (Hex)₃, and (Hex)₂HexNAc, respectively) are shown in Fig. 1. Separation and structural determination of the isomers of (Hex)₂Fuc, (Hex)₃, and (Hex)₂HexNAc are described separately in the following three sections.

(Hex)₂HexNAc

There are many isomers of (Hex)₂HexNAc covered by the curves of ions m/z 568 in Fig. 1(b), but these isomers were not separated well from each other. The eluents from the amide-80 column were collected every 30 seconds. The collected fractions were concentrated and individually injected into an HPLC with a porous graphitic carbon (PGC) column for further separation. The chromatograms of (Hex)₂HexNAc using a PGC column are illustrated in Fig. 2(a)–(h). Because isomers were partially separated by the amide-80 column, the isomer distribution in the front part of the curve of (Hex)₂HexNAc in Fig. 1(b) is different from the isomer distribution in the rear part of the same curve, as illustrated by the changes in the relative abundances of these isomers in the chromatograms of Fig. 2(a)–(h). Notably, we used intact oligosaccharides (i.e., no reduction at the reducing end), therefore one isomer may appear as two peaks in a chromatogram if the α and β anomeric configurations of the sugar at the reducing end of a given isomer were separated by the PGC column. Although the relative abundance of a given isomer changed in these chromatograms, the relative intensities of the α and β anomers of a given isomer remain the same because the α and β anomers of the same isomer reached equilibrium in solution through mutarotation. Consequently, a comparison of the relative intensities in these chromatograms provided a crude idea which two peaks in chromatogram belong to the same isomer. For example, the peaks at retention time t = 39.1 and 47.6 min in Fig. 2(a) and 2(b) may belong to one isomer, and peaks at retention time t = 27.0 and 32.6 min in Fig. 2(b)–(d) may belong to another isomer.

To confirm which two peaks belong to one isomer, eluents from the PGC column were collected in fractions every 30 seconds, ensuring that different peaks in Fig. 2(a)-(h) were collected into separate fractions. These eluents were stored at room temperature for several hours before being reinjected separately into the same PGC column. If two peaks in Fig. 2(a)-(h) are from the same isomer, reinjecting the eluents into the same PGC column would show two peaks again in the chromatogram, even though each fraction initially contained only one anomer. This is because the α and β anomers of the same isomer change to each other and reach equilibrium through mutarotation during the storage time.

Eluents from amide-80 column in tubes 12–15 [Figure 2(a)–(d)] had similar isomer distributions, they were combined and sent into HPLC with a PGC column for further isomer separation. The chromatogram is illustrated in Fig. 2(i). Then, the eluents collected from the PGC column at different retention time in Fig. 2(i) were reinjected into the same PGC column again. Parts of the chromatograms are illustrated in Fig. 2 (j)–(n). Each chromatogram in Fig. 2 (j)–(n) represents a chromatogram of a pure isomer collected from PGC column at different retention time. For example, isomer shown in Fig. 2(j) was collected at the retention time 27.0–27.5 min in Fig. 2(i). Although only the peak at t = 27.3 min was collected initially, the chromatogram in Fig. 2(j) shows two peaks, t = 27.3 and 32.9 min, indicating these two peaks belong to one isomer.

For the isomer shown in Fig. 2(n) which was collected at the retention time t = 40.5–41.0 min, it was contaminated by the isomer in Fig. 2(m) because one peak of the isomer shown in Fig. 2(m) had a retention time similar to that of isomer shown in Fig. 2(n). To purify the isomer shown in Fig. 2(n), repeated injections of the eluent collected at t = 40.5–41.0 min into PGC column followed by the collection at t = 40.5–41.0 min were performed until no contamination was found. These isomers were then individually transported into a linear ion trap mass spectrometer for structural determination.

The structures of oligosaccharides were determined using two methods. The first method involved comparing them to the oligosaccharides extracted from human, bovine, and caprine milk in our previously studied⁶⁵. In this method, the structures of oligosaccharides extracted from caprine colostrum were determined using the following three criteria. (1) Comparison of the chromatogram retention times of the selected m/z value to those of the oligosaccharides extracted from human, bovine, and caprine milk. Both the retention times of α and β-anomers of a given isomer must match those of the oligosaccharides extracted from human, bovine, and caprine milk. (2) Comparison of one MS² and two MS³ mass spectra at the corresponding chromatogram retention times to those of the oligosaccharides extracted from human, bovine, and caprine milk. (3) Comparison of the relative intensities of α and β-anomers of a given isomer to those of the oligosaccharides extracted from human, bovine, and caprine milk. Structures were assigned only if the aforementioned three criteria were satisfied simultaneously.

The second method for structural determination is LODES/MSⁿ. If the aforementioned three criteria were not satisfied, the peaks in the chromatogram represent oligosaccharides not reported in our previous work. The structures of these oligosaccharides were determined using LODES/MSⁿ. LODES/MSⁿ involves sequential collision induced dissociation (CID) of oligosaccharide sodium (or lithium) adducts in an ion trap mass spectrometer^50–55. The selected fragments for the next stage CID in the sequences of MSⁿ are derived from carbohydrate dissociation mechanisms^56–60. The mechanisms of oligosaccharide sodium ion adducts used in this study are summarized as the following three propensity rules.

(1) Dehydration mainly takes place at the reducing end of oligosaccharides.

(2) Cross-ring dissociation mainly takes place at the reducing end of oligosaccharides and follows the rules of retro-aldol reaction. Fragmentation patterns of retro-aldol reaction are used to determine the linkage position of the sugar at the reducing end. Details of fragmentation patterns are illustrated in Figure S1 and S2 of the Supplementary Information.

(3) Cleavages of the glycosidic bond can occur at any glycosidic bond (i.e., not limited to the reducing end).

The dissociation mechanisms of lithium ion adducts are similar to those of sodium ion adducts. However, cross-ring dissociation and dehydration occurring at the nonreducing end cannot be neglected. Therefore, in some cases, the O1 atom of the monosaccharide at the reducing end had to be labeled with ¹⁸O when lithium ion adducts were used for structural determination.

Here, we used the trisaccharide Galα-(1–3)-Galβ-(1–4)-GlcNAc [Figure 2(k)] as an example to illustrate how the structure was determined using LODES/MSⁿ. The mass spectrum, presented on the left side of Fig. 3(a), shows the fragments produced from CID of the precursor ion m/z 568 [sodium adduct of (Hex)₂HexNAc]. The loss of neutral m = 101 from the precursor ion, resulting in the fragment ion m/z 467, represents the cross-ring dissociation of a HexNAc at the reducing end (rule 2). The glycosidic bond linkage(s) between Hex and HexNAc can be 1–4 or 1–6 for linear trisaccharides, or (1–4, 1–6) for branched trisaccharides, according to the retro-aldol reaction (refer to Figure S1-S3 of Supplementary Information). The fragment ions m/z 365 (sodium ion adduct of (Hex)₂) and m/z 347 (sodium ion adduct of (Hex)₂-H₂O) found in the same CID spectrum suggested two hexoses were connected together. Therefore, the trisaccharide must be linear, i.e., Hex-Hex-HexNAc, and HexNAc is located at the reducing end. The corresponding CID sequence and the structures of fragments are illustrated in the middle of Fig. 3(a), with the possible precursor structures derived from these fragments are illustrated on the right side of Fig. 3(a).

In the next step, the large intensity of the fragment ion m/z 275 (compared to ions m/z 305 and 245) found in the CID sequence 568 (sodium ion adduct of precursor)→365(sodium ion adduct of (Hex)₂)→fragments [left side of Fig. 3(b)] indicated that the linkage between two hexoses is 1→3, according to the retro-aldol reaction (refer to Figure S1 and S2 of Supplementary Information). In the third step [Figure 3(c)], comparison of the CID spectrum 568→244 (sodium ion adduct of HexNAc)→fragments [Figure 3(c)] to the HexNAc monosaccharide database (refer to Figure S4 of Supplementary Information) suggested that the HexNAc is GlcNAc.

To determine the stereoisomer of each hexose, the hexose lithium ion adducts, ions m/z 187 produced from the following two CID sequences, 552 (lithium ion adduct of precursor)→331 (lithium ion adduct of (Hex)₂-H₂O)→187, and 552 (lithium ion adduct of precursor)→390 (lithium ion adduct of Hex-HexNAc)→187, were used in the subsequent CID spectrum measurement. The ion m/z 187 produced from these two sequences represented the hexose at the terminal nonreducing end and center, respectively. The structures of these two hexoses are determined by comparing CID spectra of these two hexose monosaccharides [left side of Fig. 3(d) and 3(e)] to the monosaccharide database in Figure S5 of the Supplementary Information. Spectrum similarities, as shown on right side of Fig. 3(d) and (e), suggest they are Galα and Galβ, respectively because they have the highest similarity score for the hexose at the terminal nonreducing end and center, respectively. In the final step, the CID sequence 568 (sodium ion adduct of precursor)→406 (sodium ion adduct of Hex-HexNAc)→fragments was studied. The intensity of fragment ion m/z 328 (loss of m = 78) was much larger than that of fragment ion m/z 329 (loss of m = 77) in the CID spectrum [left side of Fig. 3(f)]. Comparison to the CID spectra of disaccharides Gal-β-(1–4)-GlcNAc and Gal-β-(1–6)-GlcNAc [Figure S6 of Supplementary Information] suggests the linkage between Gal and GlcNAc was 1–4. Consequently, the entire trisaccharide was determined to be Galα-(1–3)-Galβ-(1–4)-GlcNAc.

The structures of the other purified isomers, illustrated in Fig. 2(j)-(n), (p) were determined using the LODES/MSⁿ analogous to the aforementioned method. The CID spectra used for the structural determination are presented in Figure S11-S15 of Supplementary Information. The other isomers, for which the retention time, MS² and MS³ spectra and relative intensities of anomers were found to be identical to the oligosaccharides found in bovine milk and human milk in our previous study, had their structural determination made by comparison to the oligosaccharides assigned previously in bovine milk and human milk.

Oligosaccharides in caprine colostrum and mature milk have been studied extensively in previous research.^{40, 41, 45, 47, 48, 65–77}. Among the isomers of (Hex)₂HexNAc, two isomers, GalNAcα-(1–3)-Galβ-(1–4)-Glc and GlcNAcβ-(1–3)-Galβ-(1–4)-Glc have been reported before. Three isomers, GalNAcα-(1–3)-Galβ-(1–4)-Glc, GalNAcβ-(1–3)-Galβ-(1–4)-Glc, and Galβ-(1–4)-[GalNAcβ-(1–2)]-Glc were reported in our previous study, and they were also found in this study. Additionally, six isomers were newly discovered in this study: Manα-(1–6)-Manβ-(1–4)-GlcNAc, Galα-(1–3)-Galβ-(1–4)-GlcNAc, GalNAcα-(1–4)-Glcβ-(1–4)-Glc, GalNAcβ-(1–3)-Glcβ-(1–4)-Glc, Galβ-(1–3)-Glcβ-(1–4)-GlcNAc, and Galβ-(1–4)-Glcβ-(1–4)-GlcNAc. The isomer Manα-(1–6)-Manβ-(1–4)-GlcNAc may be produced from the degradation of N-glycans, while the other isomers cannot be explained by the biosynthesis from lactose or degradation from large oligosaccharides.

In Fig. 2(e)-(g), there are some peaks at retention time 36.8, 37.2, and 40.6 min. However, the intensities of these peaks are very small, and/or the separation from other isomers is difficult. The structures of these peaks were not determined. Therefore, the total number of (Hex)₂HexNAc isomers are greater than what we have identified.

The comparison of (Hex)₂HexNAc isomers in colostrum and mature milk is illustrated in Fig. 4. In general, the structural diversity of oligosaccharides in colostrum is greater than that in mature milk. Many oligosaccharides, which do not have lactose at the reducing end, are more abundant in colostrum than in mature milk.

(Hex)₃

The purification and structural determination of (Hex)₃ isomers are similar to those of (Hex)₂HexNAc. Figure 5 (a)-(h) show the chromatograms of isomers collected from the eluents of the amide-80 column at different retention time, separated by the PGC column. Most of the isomers can be identified by comparing their retention time, MS² and MS³ spectra, and relative intensities of anomers to the oligosaccharides identified in bovine and human milk in our previous study. For the isomers that cannot be identified by comparing to the oligosaccharides found in bovine and human milk, they were purified by separating from the other isomers. The pure isomers collected from the PGC column at different retention time were reinjected into the PGC column separately to check their purity. The chromatograms of these isomers are illustrated in Fig. 5(i)-(m). The structures of these isomers are then identified using LODES/MSⁿ.

Here we used the isomer shown in Fig. 5(l) as an example to demonstrate the structural determination. The mass spectrum, presented on the left side of Fig. 6(a), shows the fragments produced from CID of the precursor ion m/z 527 [sodium adduct of (Hex)₃]. The loss of neutral m = 60 from the precursor ion, resulting in the fragment ion m/z 467, represents the cross-ring dissociation of a Hex at the reducing end (rule 2), and the glycosidic bond linkage(s) between the Hex at reducing end and the other Hex can be 1–4 (for linear trisaccharide), or (1–4, 1–6) for branched trisaccharide, according to the retro-aldol reaction (Figure S1 and S2 of Supplementary Information). The CID sequence and structures of fragments are illustrated in the middle of Fig. 6(a), while the possible precursor structures derived from these fragments are illustrated on the right side of Fig. 6a. The fragment ion m/z 347 (sodium ion adduct of (Hex)₂-H₂O) in the CID spectrum of 527(sodium ion adduct of precursor)→467(cross-ring dissociation from the Hex at the reducing end)→fragments [left side of Fig. 6(b)] suggest that the two hexoses not at the reducing end are connected together. Therefore, the trisaccharide must be linear with a 1–4 linkage at the reducing end, i.e., Hex-Hex-(1–4)-Hex. The ion m/z 275 found in the CID sequence 527 (sodium ion adduct of precursor) →467(cross-ring dissociation from the Hex at the reducing end)→365 (sodium ion adduct of (Hex)₂ at nonreducing end)→fragments [left side of Fig. 6(c)] indicated that the linkage between two hexoses at nonreducing end is 1–3, according to the retro-aldol reaction. Therefore, the linear trisaccharide is Hex-(1–3)-Hex-(1–4)Hex. To determine the stereoisomer of each hexose, lithium ion adduct of ¹⁸O labelled trisaccharide was used in CID. The hexose lithium ion adducts, ion m/z 187 or 189, produced through the CID sequences 513→451→331→187, 513→351→187, and 513→351→189 represented the hexose at the terminal nonreducing end, center, and reducing end, respectively. The CID spectra of these hexoses [Figure 6(d), 6(e), 6(f)] were compared to the CID spectra of the monosaccharide database provided in Figure S5 of Supplementary Information to determine the stereoisomers of these hexoses. Spectrum similarities, as shown in Fig. 6, show that Galβ and Glcβ, and Glc have the highest similarity scores for the hexose at the terminal nonreducing end, center, and reducing end, respectively. Consequently, the entire trisaccharide was determined to be Galβ-(1–3)-Glcβ-(1–4)-Glc.

The CID spectra used for the structural determination of the purified isomers in Fig. 5(j), (l), (m) are presented in Figure S16-19 of Supplementary Information. For the remaining isomers which the retention time, MS² and MS³ spectra and relative intensities of anomers matched those of oligosaccharides found in bovine milk and human milk in our previous study, the structures were determined based on our earlier assignments.

There are 11 isomers of (Hex)₃ found in caprine colostrum in this work. Among these isomers, Galα-(1–3)-Galβ-(1–4)-Glc, Galβ-(1–3)-Galβ-(1–4)-Glc, and Galβ-(1–6)-Galβ-(1–4)-Glc have been reported before⁴¹, Galβ-(1–4)-Glcβ-(1–4)-Glc, Galβ-(1–4)-[Galβ-(1–2)]-Glc and Galβ-(1–4)-[Glcα-(1–2)]-Glc were reported in our previous study⁶⁵. Three isomers, Galβ-(1–3)-Glcβ-(1–4)-Glc, Galβ-(1–6)-Glcβ-(1–4)-Glc, Galβ-(1–6)-[Hex-(1–4)]-Glc, and a nonreducing trisaccharide Galβ-(1–4)-Glcβ-(1–1)-Gal were newly found in this study. The stereoisomers and anomericities of another isomer, Hex-(1–6)-Hex-(1–4)-Hex, at retention time 25.9 and 27.2 min [Figure 5(a)], were not determined. Peaks labeled by question marks in Fig. 5 represent structures that remain unidentified. The structures of these isomers are difficult to be determined due to low abundance and challenges in separating them from other isomers.

The comparison of (Hex)₃ isomers in colostrum and mature milk is illustrated in Fig. 7. The structural diversity of oligosaccharides in colostrum exceeds that of mature milk, with many oligosaccharides lacking lactose at the reducing end being more abundant in colostrum than in mature milk.

(Hex)₂Fuc

The purification and structural determination of (Hex)₂Fuc are similar to those of (Hex)₂HexNAc and (Hex)₃. Figure 8 (a)-(f) show chromatograms of isomers collected from the eluents of the amide-80 column at different retention time, separated by the PGC column. Tube 15 contains only one isomer [Figure 8(c)]. Isomers at retention time 25.2 and 26.0 min [Figure 8(d)] and the isomer at 13.2 and 14.4 [Figure 8(f)] were separated from other isomers through fraction collection of the PGC column. The pure isomers collected at different retention time were reinjected separately into the PGC column to check the purity. Chromatograms of these isomers are illustrated in Fig. 8(g) and (h). Among the isomers at retention time 29–42 min, there are many isomers [Figure 8(a) and 8(b)]. Figure 8(i)-(o) shows the chromatograms of these isomers separated by PGC column from the fraction collections of Fig. 8(b) for every two minutes. Only one isomer was successfully purified [Figure 8(l)]; the intensities of the other isomers are either too small or they are too difficult to separate from the others. The purified isomer was then identified using LODES/MSⁿ.

The isomer presented in Fig. 8(g) serves as an example to demonstrate the structural determination process. The mass spectrum, presented on the left side of Fig. 9(a), shows the fragments generated from CID of the precursor ion m/z 511 [sodium adduct of (Hex)₂Fuc] and the CID sequence as well as the structures of fragments are illustrated in the middle. The loss of neutral m = 60 from the precursor ion yields the fragment ion m/z 451, indicating cross-ring dissociation of a Hex or Fuc at the reducing end, according to the retro-aldol reaction. This CID spectrum suggests two possible structures: a linear trisaccharide with a Hex or Fuc at the reducing end and a glycosidic bond linkage between the reducing-end sugar (Hex or Fuc) and the other sugars through 1–4 linkage, or a branched trisaccharide with (1–4, 1–6) linkages with a Hex at the reducing end. Since fragment m/z 365 indicates two connecting hexoses, the possible precursor structures would be those on the right side of Fig. 9(a).

In the CID spectrum of 511 (sodium ion adduct of precursor)→451(cross-ring dissociation from the Hex or Fuc at the reducing end)→fragments [left side of Fig. 9(b)], fragment ions such as m/z 365 (sodium ion adduct of (Hex)₂), m/z 347 (sodium ion adduct of (Hex)₂-H₂O), m/z 349 (sodium ion adduct of HexFuc), and m/z 331 (sodium ion adduct of (HexFuc)-H₂O) were not found, indicating the trisaccharide is branched. Therefore, the trisaccharide must be Hex-(1–4 or 1–6)-[Fuc(1–4 or 1–6)]-Hex. The intensities of fragment ions m/z 289, 259, and 229 in the ratio of 5: 3: 1 found in the CID spectrum through the sequence 511 (sodium ion adduct of precursor)→349 (sodium ion adduct of disaccharide Fuc-Hex)→fragments [left side of Fig. 9(c)] indicated that the linkage between Fuc-Hex is 1–6, according to the retro-aldol reaction. The intensities of fragment ion m/z 305 found in the CID spectrum through the sequence 511 (sodium ion adduct of precursor)→365 (sodium ion adduct of disaccharide Hex-Hex)→fragments [left side of Fig. 9(d)] indicated that the linkage between Hex-Hex is 1–4, according to the retro-aldol reaction. Therefore, the branched trisaccharide is Hex-(1–4)-[Fuc-(1–6)]-Hex. The branched trisaccharide can be cross-checked using ¹⁸O labelled at the reducing end of lithium ion adduct. Fragment ions m/z 351 (lithium ion adduct of ¹⁸O labeled Hex-Hex) and m/z 335 (lithium ion adduct of ¹⁸O labeled Fuc-Hex) found in the CID spectrum 497→fragments in Fig. 9(e) suggest the trisaccharide is branched.

To determine the stereoisomer of each hexose, the lithium ion adduct of ¹⁸O labeled trisaccharide was used in CID. The hexose lithium ion adducts, ion m/z 187 or 189, produced through the CID sequences 497→351→187 and 497→351→189, represent the hexose at nonreducing end and reducing end, respectively. The CID spectra of these hexoses [Figure 9(f) and 9(g)] are compared to the CID spectra of the monosaccharide database provided in Figure S5 of the Supplementary Information to determine the stereoisomers of these hexoses. Spectrum similarities, as shown in Fig. 9(f) and (g), indicate they are Galβ and Glc, respectively. Consequently, the entire trisaccharide was determined to be Galβ-(1–4)-[Fuc(1–6)]-Glc. At this moment, we do not have Fucα-(1–6)-Glc and -Fucβ-(1–6)-Glc disaccharide CID spectra for comparison, and the anomeric configuration of the glycosidic bond between Fuc and Glc is not determined. The CID spectra for structural determination of the other isomers are illustrated in Figure S20-S22 of the Supplementary Information.

Among the isomers of (Hex)₂Fuc, Fucα-(1–2)-Galβ-(1–4)-Glc and Galβ-(1–4)-[Fucα-(1–3)]-Glc in caprine colostrum have been reported before⁴¹, while Fucα-(1–6)-Galβ-(1–4)-Glc and Galβ-(1–4)-[Fuc-(1–6)]-Glc were newly found in this study. At retention time 32–42 min, there are at least four isomers of (Hex)₂Fuc, but only one of them is structurally identified. The abundances of the other isomers are too small or too difficult to separate from other isomers, and the structures remain unidentified.

Figure 10 shows the comparison of oligosaccharides in colostrum and mature milk. Analogous to the (Hex)₂HexNAc and (Hex)₃, the structural diversity of oligosaccharides in colostrum is more than that of mature milk.

In conclusion, we demonstrated that a new mass spectrometry, logically derived sequence tandem mass spectrometry (LODES/MSⁿ), was applied for the structural determination of neutral trisaccharides extracted from caprine colostrum and mature milk. This method does not rely on the oligosaccharide standards, thus it is particularly useful for identifying undiscovered oligosaccharides. New oligosaccharides were found in caprine colostrum, and many of which lack lactose at the reducing end. Instead, they feature Glcβ-(1–4)-Glc or Glcβ-(1–4)-GlcNAc at the reducing end, indicating the existence of undiscovered biosynthetic pathways. These unusual oligosaccharides are more abundant in colostrum than that in mature milk. These newly discovered oligosaccharides indicate undiscovered biosynthetic pathways.

Caprine colostrum and mature milk were purchased from local market. The method used to extract free oligosaccharides from milk and determine their structure was similar to that described in our previous study⁶⁵. In brief, Folch solution and ethanol were used to remove fat and proteins in milk, respectively, followed by solid-phase extractions using two different cartridges. One type of cartridge was C18 cartridges, which were used to remove remaining lipids and proteins, while the other type was PGC cartridges, used to remove monosaccharides, disaccharides, and salt. After solid-phase extraction, oligosaccharides were size-selected through size exclusion chromatography. The fractions collected from the size exclusion were then subjected to two-dimensional high-performance liquid chromatography (HPLC) for isomer separation. Isomers were first separated by an HPLC with a TSKgel amide-80 column (150 mm × 2.0 mm, particle size of 5 µm, Tosoh Bioscience GmbH, Griesheim, Germany), followed by the other HPLC with a PGC Hypercarb column (2.1 mm × 150 mm or 2.1 mm × 100 mm, particle size of 3 µm, Thermo Fisher Scientific, Waltham, MA, USA). The collected isomers were then individually injected into a linear ion trap mass spectrometer (LTQ XL, Thermo Fisher Scientific) for structural determination. The method used to prepare ¹⁸O-labeled oligosaccharide has been described in previous studies⁶⁵. Details of the structural determination using LODES/MSⁿ are presented in the following sections.

Competing Interests

C.K.N.is one of the inventors of a US patent (US 10,796,788 B2); a part of the method described in the patent for determining the carbohydrate structure was used in this work. All other authors declare no competing interests.

Additional Information (including a Competing Interests Statement)

Author Contribution

W.C.W. and H.E.L. extracted and analyzed milk oligosaccharides; C.H.C. and S.C.H. synthesized Hex-HexNAc and HexNAc-Hex disaccharides, K.D., Z.T., and C. H. L. synthesized Fuc-Gal disaccharides; C.K.N designed the experiment, conceived the LODES/MSn method, analyzed data, wrote the manuscript and supplementary information.

Acknowledgements

This work was financially supported by iMATE project (AS-iMATE-111-32), Academia Sinica, Taiwan.

Data Availability

The data supporting the findings of this study are available within the article and its Supplementary Information.

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Competing interest reported. C.K.N.is one of the inventors of a US patent (US 10,796,788 B2); a part of the method described in the patent for determining the carbohydrate structure was used in this work. All other authors declare no competing interests.

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Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

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Introduction

Results and Discussion

(A) Isomer purification and structural determination

(Hex)₂HexNAc

(Hex)₃

(Hex)₂Fuc

Methods

Declarations

Competing Interests

Additional Information (including a Competing Interests Statement)

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Unusual free trisaccharides in caprine colostrum discovered by logically derived sequence tandem mass spectrometry

Status:

Version 1

Abstract

Figures

Introduction

Results and Discussion

(A) Isomer purification and structural determination

(Hex)2HexNAc

(Hex)3

(Hex)2Fuc

Methods

Declarations

Competing Interests

Additional Information (including a Competing Interests Statement)

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

(Hex)₂HexNAc

(Hex)₃

(Hex)₂Fuc