Separation and Structural Sequence Analysis of Sialylated HMOs via Tandem Mass Spectrometry†

doi:10.7503/cjcu20170593

Abstract

Abstract:

Sialylated human milk oligosaccharides(SHMOs), important components of human milk oligosaccharides, play a key role in the growth and development of the newborn. Herein, based on the difference of polarity, spatial structure and charge density of sialylated oligosaccharides, a semi-preparative porous graphitized carbon(PGC) chromatography was employed to separate SHMOs in high purity. An off-line electrospray ionization tandem mass spectrometry(ESI-MSⁿ) was performed to identify the sequential structures of eight oligosaccharides including 6'-SL, 3'-SL, LSTb, LSTc, F-LSTc, F-LSTb, DSLNT and FS-LNnH. This study provides a useful reference for the further structure-activity analysis and application development of SHMOs.

Key words: Sialylated human milk oligosaccharides, Porous graphitized carbon separation, Tandem MS, Sequence analysis

CLC Number:

O657
O629.1

TrendMD:

LANG Yinzhi, LIU Shilong, WANG Chen, ZHANG Xiao, LÜ Youjing, CAI Chao, LI Guoyun, YU Guangli. Separation and Structural Sequence Analysis of Sialylated HMOs via Tandem Mass Spectrometry^†[J]. Chem. J. Chinese Universities, 2018, 39(4): 645.

Figures/Tables 8

Fig.1 Determination of sialic acids in NHMOs and SHMOs via an improved RP-HPLC methoda. Standard; b. SHMOs; c. NHMOs.

Fig.2 Separation of eight sialylated human milk oligosaccharides by PGC-HPLC method(A) and their ESI-MS spectra(B)

Table 1 Structural determination of eight sialylated human milk oligosaccharides by ESI-MSn

Fraction	Molecular ion	Theoretical M_w	DP^*	Composition	Glycan name
S1	632.19[M-H]^-	633.21	3	Neu5Ac₁Gal₁Glc₁	6'-SL
S2	632.19[M-H]^-	633.21	3	Neu5Ac₁Gal₁Glc₁	3'-SL
S4	997.31[M-H]^-	998.34	5	Neu5Ac₁Gal₂Glc₂	LSTb
S6	997.31[M-H]^-	998.34	5	Neu5Ac₁Gal₂Glc₂	LSTc
S5	1143.36[M-H]^-	1144.40	6	Fuc₁Neu5Ac₁Gal₂Glc₂	F-LSTc
S7	1143.36[M-H]^-	1144.40	6	Fuc₁Neu5Ac₁Gal₂Glc₂	F-LSTb
S3	643.70[M-2H]^2-	1289.44	6	Neu5Ac₂Gal₂Glc₂	DSLNT
S8	1508.48[M-H]^-; 771.74[M+2H₂O-2H]^2-	1509.53	8	Fuc₁ Neu5Ac₁Gal₃Glc₃	FS-LNnH

Fig.3 ESI-MS/MS spectra of monosialylated lactose species from S1(A) and S2(B) using [M-H]-(m/z 632.19) as the precursor ions

Fig.4 ESI-MS/MS spectra of monosialylated tetrasaccharide species from S4(A) and S6(B) using [M-H]-(m/z 997.31) as the precursorsα: The large branch; β: the small branch.

Fig.5 ESI-MS/MS spectra of monofucosylated and monosialylated tetrasaccharide species from S7(A) and S5(B) using [M-H]-(m/z 1143.36) as the precursor ionsα: The large branch; β: the small branch.

Fig.6 ESI-MS/MS product ion spectra and ESI-MS3 product ion spectrum of disialylated tetrasaccharide from S3 fraction using [M-H]-(m/z 1288.40)(A) and Y1α(m/z 997.31)(B) as the precursor,respectivelyα: The large branch; β: the small branch.

Fig.7 ESI-MS/MS and ESI-MS3 spectrum of monofucosylated and monosialylated hexasaccharide from S8 fraction using[M-H]-(m/z 1508.48) and D2b-4(m/z 673.21) as the precursor, respectivelyα, The large branch. β, the small branch; β', the large branch of β; β'', the small branch of β.

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