Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM)

Abstract

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

Keywords: Glycoproteomics; Human milk; MRM; Mass spectrometry; UPLC.

Conflict of interest statement

Conflict of interest The authors declare that there is no conflict of interest.

Figures

Fig. 1

Representative Q-TOF tandem mass spectra of glycopeptides. a MS/MS spectrum of glycopeptide Hex5HexNAc4Fuc1Neu5Ac1-TAGWNIPMGLLF497NQTGSCK from LF. b MS/MS spectrum of Hex5HexNAc4Fuc1Neu5Ac1- TAGWNVPIGTLRPFL156NWTGPPEP-IEAAVAR from LF. Green circles, yellow circles, blue squares, red triangles, and purple diamonds represent mannose, galactose, GlcNAc, fucose, and NeuAc residues, respectively

Fig. 2

Glycan site-heterogeneity of human milk glycoproteins: a LF, b IgG, c IgM, d A1AT, and e sIgA. Green circles, yellow circles, blue squares, red triangles, and purple diamonds represent mannose, galactose, GlcNAc, fucose, and NeuAc residues, respectively

Fig. 3

Total MRM chromatogram for seven standard protein mix using UPLC-C18 chromatography. MRM chromatograms for a peptides and glycopeptides, b peptide with assigned transitions, and c glycopeptides with assigned transitions. The MRM transitions are shown in Table 1. One MRM transition was monitored for each glycopeptide; two MRM transitions were monitored for each peptide

Fig. 4

Peptide calibration curves for protein quantitation. a α-lact, b LF, c IgA1/2, d IgG1234, e IgM, f A1AT, and g LZ. The response can be fitted to a quadratic equation (dashed in blue). The dynamic rang was over 1000. The linear fit (dashed in red) generated an equation with R2 from 0.99 to 0.999. The linear range was more than 100

Fig. 5

Eleven normalized LF glycopeptide abundances monitored from three milk samples (a, b, and c). LF concentration (g/L) of three milk samples on the right of the plot. Normalization was performed with the ratio between glycopeptide signal peak area and the LF peptide peak area. This suggests the dynamic variation on site-specific glycosylation. Error bars are representative of reproducibility from replicates on different days