Characterization of free oligosaccharides from garden cress seed aqueous exudate using PGC LC-MS/MS and NMR spectroscopy.

Garden cress seeds produces mucilage that has found various food applications, however, there is little information on the free oligosaccharides (FOS) contents in these seeds. Herein, we explored the presence of FOS in cress seed aqueous exudate. PGC-LC MS/MS analysis indicated the presence of mainly hexose containing oligosaccharides such as raffinose, stachyose and verbascose belonging to raffinose family of oligosaccharides (RFOs). In addition, minor fraction of planteose, isomeric tri- and tetrasaccharides were also observed. Further, the structural confirmation of the abundant tri- and tetrasaccharide were obtained through 1D and 2D NMR analysis. Thus, the RFOs presence in cress seeds would enhance its bio-functionalities.

Discrimination of raffinose and planteose based on porous graphitic carbon chromatography in combination with mass spectrometry.

Raffinose and planteose are non-reducing, isomeric trisaccharides present in many higher plants. Structurally, they differ in the linkage of α-D-galactopyranosyl to either glucose C(6) or to C (6') of fructose, respectively and thus differentiating each other is very challenging. The negative ion mode mass spectrometric analysis is shown to distinguish planteose and raffinose. However, to facilitate the robust identification of planteose in complex mixtures, herein, we have demonstrated the use of porous graphitic carbon (PGC) chromatography combined with QTOF-MS2 analysis. The separation of planteose and raffinose was achieved on PGC, wherein both have recorded different retention time. Detection through MS2 analysis revealed the specific fragmentation patterns for planteose and raffinose that are distinctive to each other. The applicability of this method on oligosaccharides pool extracted from different seeds showed clear separation of planteose that allowed unambiguous identification from complex mixtures. Therefore, we propose PGC-LC-MS/MS can be employed for sensitive, throughput screening of planteose from wider plant sources.

N-Glycoprofiling of immunoglobulin G and lactoferrin with site-specificity from goat milk using RP-UHPLC MS/MS.

Glycans present in glycoproteins are structurally diverse and contribute to the carbohydrate pool of the milk. Goat milk is a leading non-bovine milk source, wherein glycan diversity of several glycoproteins remains unexplored. Herein, site-specific N-glycoprofiling of two major glycoproteins - immunoglobulin G (IgG) and lactoferrin (Lf) from goat milk was performed through RP-UHPLC Q-Tof MS/MS approach. IgG revealed diverse complex glycans that were predominantly biantennary type with differential core fucosylation, bisecting GlcNAc, and mono/di- sialylation (NeuAc/NeuGc). The N-glycan repertoire of Lf at four sites indicated the range of high mannose, complex and hybrid types with varying abundances. High mannose glycans were specifically observed at N252NT and N564DT sites. Majorly complex glycans with fully sialylated were found at N387VT site. While N495QT site revealed complex and hybrid types with differential core fucosylation and sialylation. The glycan features observed in these glycoproteins would pave way for effective utilization as bioactive ingredients.

Sequence and N-glycan diversity analysis of immunoglobulin G from buffalo milk using RP-UHPLC MS/MS.

Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid sequence and glycan diversity using reverse phase liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. Amino acid sequence analysis of heavy chain constant region revealed the presence of two IgG subtypes namely IgG1 and IgG3, with IgG1 being the abundant. The complete light chain constant region sequence was also determined. N-glycan sequence analysis at a highly conserved site Asn-Ser-Thr revealed the presence of mainly biantennary complex type with core fucosylation (34%), bisecting GlcNAc (19%) and sialylation with both Neu5Ac and Neu5Gc (14%). The observed glycan diversity in buffalo milk IgG is in part comparable with bovine colostrum as well as human, bovine, goat serum counterparts.