UC Davis, Davis, CA
Rapid-throughput method of quantitation of human milk proteins and their glycoforms was developed.
Human milk has long been recognized as not only a source of nutrition but for a number of functional components to the newborn. Milk proteins are highly glycosylated, yet the roles of glycosylation in the function of milk is limited by the lack of tools to quantitate various protein glycoforms. We have developed a new MRM approach to quantitate human milk proteins and their glycosylation, giving the capability to monitor glycosylation at site-specific level. The process involves identifying the best peptides for protein quantitation, mapping the site-specific glycan heterogeneity of the glycoproteins, and characterizing the fragmentation patterns of specific glycopeptides to determine the best transitions for MRM. The method advances our understanding of the role of glycan-conjugates in human health.
Quantitation of human milk proteins and their peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with the state of lactation. To be useful, the method must be rapid throughput. In a 96-well plate, standard protein mixture (200 µg of human lactoferrin, ?-lactalbumin, 100 µg of IgA, and 20 µg of IgG, IgM, antitrysin and lysozyme) and 25 µL of human milk samples were reduced and alkylated prior to trypsin digestion at 37?C for 18 hr. The resulting peptides and glycopeptides were cleaned and enriched via solid phase extraction (SPE) with C-18 cartridges. The mixture was profiled using nano-LC-chip-QTOF mass spectrometer and quantified using UPLC-ESI-QqQ mass spectrometer.
Our strategy allows both the determination of protein concentrations as well as the absolute and relative abundances of the different glycoforms in a given sample. The proteins, human lactoferrin (hLF), ?-lactalbumin, IgA, IgG, IgM, lysozyme and antitrypsin, are the most abundant in milk and represent nearly 80% of the whey protein abundances. For each protein two or three unique peptides were identified as being robust for quantitation. Nano?ow LC?MS analysis with the Q-TOF instrument allowed identi?cation of the glycopeptides (from hLF, IgA, IgM, IgG and antitrypsin) based on the accurate mass and the tandem MS. Glycopeptides were characterized by their fragmentation pattern to obtain the best transitions. For example, hLF has three potential N-glycosylation sites, but only two glycosites were occupied having mainly complex type N-glycans. In total, more than 10 glycoforms were observed at Asn156 and 5 glycoforms were present at Asn497.
UPLC separation allowed rapid-throughput quantitation using a 16-min gradient for monitoring hundreds of peptide and glycopeptide transitions. Over 100 glycopeptides were monitored from the five glycoproteins.
This rapid-throughput platform was tested for its reproducibility and accuracy, and applied to a clinical study with 171 human milk samples from 46 mothers at four time points during lactation. Within a 2-day run, protein concentration and protein glycosylation level were monitored and quantitated. hLF concentration decreased from 2.3 mg/ to 1.5 mg/mL, while ?-lactalbumin remained around 3.8 mg/mL over lactation. Lysozyme was the only protein found increasing concentration from 0.08 mg/mL to 0.16 mg/mL. By normalizing the glycopeptide signals to the protein abundances, hLF glycosylation at both glycosites showed a four-fold decrease during lactation.