Nano-LC Chip–QTOF-MS/MS analysis was performed with an Agilent 6520 accurate-mass Quadrupole-Time-of-Flight (Q-TOF) LC/MS with a microfluidic nano-electrospray chip containing an enrichment and an analytical column packed with porous graphitized carbon (Agilent Technologies, Santa Clara, CA, USA). Nano-LC QTOF MS/MS parameters were as described previously (28 (link)) and a targeted porcine milk OS library was built using the OS identified by MS/MS; each sample was analyzed in triplicate.
Composition of eluted OS is listed as a set of the five individual monomers composing the OS using the following identification nomenclature: Hex_HexNAc_Fuc_Neu5Ac_Neu5Gc. Abbreviations for the components are as follows: Hex, hexose (glucose or galactose); HexNAc, N-acetylhexososamine; Fuc, fucose; Neu5Ac, N-acetylneuramic acid; Neu5Gc, N-glycolylneuramic acid. The number of each individual monomer present within an identified or quantified OS is represented using the aforementioned nomenclature. It should be noted that the analysis by Nano-LC Chip–QTOF MS separated multiple isomers for each OS; however, here we indicate the relative percentage of composition 3_1_0_0_0 (3 Hex, 1 HexNAc) as the sum of LNT and LNnT. Similarly, the relative percentage of composition 4_2_0_0_0 (4 Hex, 2 HexNAc) was the sum of LNH and LNnH due to close eluting times of the isomer pairs (i.e., co-elution).
Composition of eluted OS is listed as a set of the five individual monomers composing the OS using the following identification nomenclature: Hex_HexNAc_Fuc_Neu5Ac_Neu5Gc. Abbreviations for the components are as follows: Hex, hexose (glucose or galactose); HexNAc, N-acetylhexososamine; Fuc, fucose; Neu5Ac, N-acetylneuramic acid; Neu5Gc, N-glycolylneuramic acid. The number of each individual monomer present within an identified or quantified OS is represented using the aforementioned nomenclature. It should be noted that the analysis by Nano-LC Chip–QTOF MS separated multiple isomers for each OS; however, here we indicate the relative percentage of composition 3_1_0_0_0 (3 Hex, 1 HexNAc) as the sum of LNT and LNnT. Similarly, the relative percentage of composition 4_2_0_0_0 (4 Hex, 2 HexNAc) was the sum of LNH and LNnH due to close eluting times of the isomer pairs (i.e., co-elution).