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Abstract No.: TuP-203
Session: Posttranslational Modifications
Presentation date: Tue, Aug 29, 2006
Presentation time: 09:50 – 11:20

Identification of N-glycoproteins in Human Milk by HILIC Enrichement and Tandem Mass Spectrometry

Gianluca Picariello1, Gianfranco Mamone1, MariaGrazia Calabrese1,2, Simonetta Caira1, Pasquale Ferranti2, Peter Roepstorff3, Francesco Addeo2

1 CNR, Avellino, Italy
2 DSA University Federico II, Portici Napoli, Italy
3 BMB Departement, University of Southern Denmark, , Odense, Denmark

Correspondence address: Gianluca Picariello, CNR, ISA, via Roma 52, Avellino, 83100 Italy.

Keywords: Glycoproteins; MS/MS; Post-translational Modification; Protein Identification.

Novel aspect: Identification of glycosylation sites in complex mixtures of proteins.


Milk contains a range of "protective factors" contributing to protect new born from disease. A growing body of evidence is suggesting that milk oligosaccharides and glycoconjugates, such as glycoproteins, are involved in the protection of infants from pathogenic attack. Here we describe a proteomic approach to profile human milk N-glycoproteins aimed to establish factors involved in the immunity and antimicrobial activity. It consists of four steps: (1) Isolation of milk proteins by precipitation with 12% triclocoacetic acid (TCA), dephosphorylation with alkaline phosphatase and tryptic digestion of proteins; (2) Selective glycopeptide enrichment from a complex peptide mixture via Hydrophilic Interaction Liquid Chromatography (HILIC); (3) Deglycosylation of isolated glycopeptides by Peptide N-glycosidase F (PNGase F); and (4) Sequencing of native and deglycosylated peptides by complementary mass spectrometric techniques (MALDI-TOF/TOF MS/MS, MALDI-Q/TOF MS/MS and micro-LC-ESI-Q/TOF MS/MS) and indentification of parent glycoproteins by correlation of tandem mass spectrometric data with sequence databases. MALDI signals attributed to glycopeptides, on the base of the typical molecular mass differences, not shifting in molecular mass after PNGase F treatment, were assigned to O-glycosylated peptides. By this approach, on the whole 30 sites of N-glycosylation were identified at Asn residues of consensus triplets Asn-X-Ser/Thr (X being any amino acid) belonging to 15 different glycosylated proteins. We identified only one peptide with multiple glycosylation sites. Most of the proteins were of structural or secretory origin such as lactadherin or lactotransferrin, involved in the host defence against infection. Protein glycosylation play also a main role in intermolecular interactions and in protecting the proteins from intra- and extra-cellular degradation. The study revealed the presence in the human milk of N-glycosylated proteins such as immunocompetent complexes, membrane fat globule enzymes, proteins involved in lipid degradation, specific receptors and proteins with a still unknown function. Our results contribute to define on molecular basis the protective role of human milk and correlate specific glycoconjugate components to justify the low incidence of symptomatic gastrointestinal infections observed in breast-fed infants. The described strategy for glycopeptide selective enrichment is suggested for the identification of oligosaccharide moieties of N-glycoproteins from colostrum and mature human milk using specific endo-glycosidases.