ABSTRACT
Sialylated human milk oligosaccharides (SHMOs) possess unique biological activities. Qualitative and quantitative analyses of SHMOs at different lactation stages are limited by interference from neutral oligosaccharides, glycan structural complexity, and low detection sensitivity. Herein, our previously developed glycoqueuing strategy was improved and applied to enable an isomer-specific quantitative comparison of SHMOs between colostrum milk (CM) and mature milk (MM). A total of 49 putative structures were determined, including 1 α2,6-linked and 13 α2,3-linked isomers separated from seven newly discovered SHMO compositions. The content of most oligosaccharides was more than 50% lower in MM than in CM, and α2,3-sialylation was observed in 43.74% of SHMOs from CM and 22.95% of SHMOs from MM. Finally, the fucosylation level of the SHMOs increased from 16.45 to 22.28% with prolonged lactation. These findings provide the basis for further studies on the structure-activity relationship of SHMOs and a blueprint to improve infant formula.
Subject(s)
Milk, Human , Milk , Infant , Female , Pregnancy , Humans , Animals , Colostrum , Lactation , Infant Formula , Breast Feeding , OligosaccharidesABSTRACT
Sialylated N-glycans are an integral component of whey proteins in human milk and play an irreplaceable role in infant growth and development. Currently, there are few studies on quantitative comparison of sialylated N-glycans in milk obtained at different lactation stages. Here, a preliminary isomer-specific quantification of whey sialylated N-glycans of human colostrum milk (CM) and mature milk (MM) was performed by using our recently developed glycoqueuing strategy. Such a preliminary comparison revealed that the whey sialylated N-glycan content was 86.4% lower in MM than in CM. Twenty-three α2,6-linked sialylated N-glycan isomers were detected with no α2,3-linked isomer observed. For the first time, three mono-sialylated and four bi-sialylated glycan isomers were reported. With the prolongation of lactation, the relative abundance of mono-sialylated glycans increased, whilst the relative abundance of bi-sialylated glycans decreased significantly. These findings contribute to the understanding of the structure-function relationship of sialylated N-glycans in the human whey fraction.
Subject(s)
Colostrum/chemistry , Glycoproteins/chemistry , Milk, Human/chemistry , N-Acetylneuraminic Acid/chemistry , Polysaccharides/chemistry , Sequence Analysis , Whey Proteins/chemistry , Animals , Female , Humans , Isomerism , Lactation , PregnancyABSTRACT
Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (nâ¯=â¯3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16⯵g ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSDâ¯=â¯2.96%, nâ¯=â¯7) and a desirable linear dynamic range from 78â¯nmol/mL to 10⯵mol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M]+ signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na]+ and [M+K]+). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.
Subject(s)
Betaine/analogs & derivatives , Glycomics/methods , Oligosaccharides/chemistry , Polysaccharides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Betaine/chemistry , Blood Proteins/chemistry , Female , Glycoproteins/chemistry , Glycosphingolipids/chemistry , Humans , Milk Proteins/chemistry , Milk, Human/chemistry , Reproducibility of Results , Sialic Acids/chemistryABSTRACT
Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for quantitatively profiling abnormal sialylated O-glycans associated with disease development, but technically hindered by the poor releasing efficiency of O-glycans from glycoprotein or the labile nature of sialic acid residues at glycans. Herein, we developed an isotopic precursor based metabolic amplification and labeling (IPMAL) technique for relative quantitative profiling of the repertoire O-glycans between normal and tumor cells by ESI-MS. Two groups of cells were incubated with peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd0) or a heavy labeled peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd5) precursor respectively to amplify the repertoire of O-glycans as Bnd0/d5-O-glycans which could achieve the quantitative O-glycome analysis by ESI-MS after derivatization. The established method demonstrates desirable feasibility, accuracy (relative error (RE) ≤ 4.20%), reproducibility (coefficient of variation (CV) ≤ 7.61%, n = 3) and good quantitation linearity (R 2 > 0.99, n = 3) for five Bn-O-glycans with 2 orders of magnitude. Finally, the method has been successfully applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell line L02 and human hepatoma cell line SMMC-7721. Moreover, the α-2,3/2,6 sialic acid isomers of Bn-O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid specific derivatization procedure.