Identification of N‐linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry

Breastfeeding is now generally recognized as a critical factor in protecting newborns against infections. An important mechanism responsible for the antibacterial and antiviral effects of breast milk is the prevention of pathogen adhesion to host cell membranes mediated by a number of glycoconjugates, also including glycoproteins. A number of approaches to describe the complexity of human milk proteome have provided only a partial characterization of restricted classes of N‐linked glycoproteins. To achieve this objective, profiling N‐linked glycoproteins of human milk was performed by Hydrophilic Interaction LC (HILIC) and MS analysis. Glycopeptides were selectively enriched from the protein tryptic digest of human milk samples. Oligosaccharide‐free peptides obtained by peptide N‐glycosidase F (PNGase F) treatment were characterized by a shotgun MS‐based approach, allowing the identification of N‐glycosylated sites localized on proteins. Using this strategy, 32 different glycoproteins were identified and 63 N‐glycosylated sites encrypted in them were located. The glycoproteins include immunocompetent factors, membrane fat globule‐associated proteins, enzymes involved in lipid degradation and cell differentiation, specific receptors, and other gene products with still unknown functions.

[1]  L. Wells,et al.  A potential pitfall in 18O-based N-linked glycosylation site mapping. , 2007, Rapid communications in mass spectrometry : RCM.

[2]  S. Davis,et al.  Characterisation of host defence proteins in milk using a proteomic approach. , 2007, Journal of proteome research.

[3]  D. Newburg,et al.  Protection of the Neonate by the Innate Immune System of Developing Gut and of Human Milk , 2007, Pediatric Research.

[4]  S. Nishimura,et al.  Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography. , 2006, Journal of separation science.

[5]  W. Ens,et al.  Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis. , 2006, Analytical chemistry.

[6]  K. Yamauchi,et al.  Bovine lactoferrin: benefits and mechanism of action against infections. , 2006, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[7]  J. Brand,et al.  Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column , 2006, Proteomics.

[8]  W. Hancock,et al.  Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry. , 2006, Glycobiology.

[9]  Martin R Larsen,et al.  Analysis of posttranslational modifications of proteins by tandem mass spectrometry. , 2006, BioTechniques.

[10]  Joseph A Loo,et al.  Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry. , 2006, Journal of proteome research.

[11]  G. Cooper,et al.  Human colostrum: Identification of minor proteins in the aqueous phase by proteomics , 2006, Proteomics.

[12]  Ronald J Moore,et al.  Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment. , 2006, Journal of proteome research.

[13]  R. Aebersold,et al.  Isolation of glycoproteins and identification of their N-linked glycosylation sites. , 2006, Methods in molecular biology.

[14]  M. Betenbaugh,et al.  Controlling N-linked glycan site occupancy. , 2005, Biochimica et biophysica acta.

[15]  Ronald J Moore,et al.  Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. , 2005, Journal of proteome research.

[16]  J. Gebler,et al.  A rapid sample preparation method for mass spectrometric characterization of N-linked glycans. , 2005, Rapid communications in mass spectrometry : RCM.

[17]  L. Hammarström,et al.  Carbohydrate phenotyping of human and animal milk glycoproteins , 2005, Glycoconjugate Journal.

[18]  Ruedi Aebersold,et al.  High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry *S , 2005, Molecular & Cellular Proteomics.

[19]  David J Harvey,et al.  Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry , 2005, Expert review of proteomics.

[20]  Prof. P. Ehrlich.  Ueber Immunität durch Vererbung und Säugung , 1892, Zeitschrift für Hygiene und Infektionskrankheiten.

[21]  M. Giuffrida,et al.  The proteomic approach to analysis of human milk fat globule membrane. , 2004, Clinica chimica acta; international journal of clinical chemistry.

[22]  Jakob Bunkenborg,et al.  A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. , 2004, Journal of proteome research.

[23]  A. Nasi,et al.  Casein proteolysis in human milk: tracing the pattern of casein breakdown and the formation of potential bioactive peptides. , 2004, The Journal of dairy research.

[24]  A. Podtelejnikov,et al.  Screening for N‐glycosylated proteins by liquid chromatography mass spectrometry , 2004, Proteomics.

[25]  B. Lönnerdal.  Human Milk Proteins , 2004 .

[26]  T. Cleary.  Human milk protective mechanisms. , 2004, Advances in experimental medicine and biology.

[27]  B. Lönnerdal.  Human milk proteins: key components for the biological activity of human milk. , 2004, Advances in experimental medicine and biology.

[28]  B. Lönnerdal.  Nutritional and physiologic significance of human milk proteins. , 2003, The American journal of clinical nutrition.

[29]  Enrico Bertino,et al.  Structural proteome of human colostral fat globule membrane proteins , 2003, Proteomics.

[30]  Ruedi Aebersold,et al.  Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry , 2003, Nature Biotechnology.

[31]  Y. Yuki,et al.  Identification of low‐abundance proteins of bovine colostral and mature milk using two‐dimensional electrophoresis followed by microsequencing and mass spectrometry , 2002, Electrophoresis.

[32]  P. Ferranti,et al.  Interallelic recombination is probably responsible for the occurrence of a new alpha(s1)-casein variant found in the goat species. , 2002, European journal of biochemistry.

[33]  M. Dwek,et al.  Use of proteomic methodology for the characterization of human milk fat globular membrane proteins. , 2002, Analytical biochemistry.

[34]  R. Ceriani,et al.  Structural and functional aspects of three major glycoproteins of the human milk fat globule membrane. , 2001, Advances in experimental medicine and biology.

[35]  S. Patton.  MUC1 and MUC-X, epithelial mucins of breast and milk. , 2001, Advances in experimental medicine and biology.

[36]  J. Lord,et al.  Glycoprotein degradation: Do sugars hold the key? , 2000, Current Biology.

[37]  R. Kiwan,et al.  Protective function of human milk: the milk fat globule. , 1999, Seminars in perinatology.

[38]  M. Mann,et al.  18O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching. , 1999, Analytical chemistry.

[39]  E. Nordhoff,et al.  Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. , 1999, Journal of mass spectrometry : JMS.

[40]  D. Newburg.  Human milk glycoconjugates that inhibit pathogens. , 1999, Current medicinal chemistry.

[41]  Y. Yuki,et al.  Identification of minor proteins of human colostrum and mature milk by two‐dimensional electrophoresis , 1998, Electrophoresis.

[42]  M. Hamosh.  Protective Function of Proteins and Lipids in Human Milk , 1998, Neonatology.

[43]  M. Hamosh,et al.  Glycoproteins of the Human Milk Fat Globule in the Protection of the Breast-Fed Infant against Infections , 1998, Neonatology.

[44]  Michael R. Taylor,et al.  Role of human-milk lactadherin in protectoin against symptomatic rotavirus infection , 1998, The Lancet.

[45]  V. Calderone,et al.  The Unusual Amino Acid Triplet Asn–Ile–Cys Is a Glycosylation Consensus Site in Human α-Lactalbumin , 1997, Journal of protein chemistry.

[46]  Andrew Gooley,et al.  Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1 , 1997, The Journal of Biological Chemistry.

[47]  P. Ferranti,et al.  Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein. , 1997, European journal of biochemistry.

[48]  D. Segal,et al.  Multiple Dimeric Forms of Human CD69 Result from Differential Addition of N-Glycans to Typical (Asn-X-Ser/Thr) and Atypical (Asn-X-Cys) Glycosylation Motifs* , 1997, The Journal of Biological Chemistry.

[49]  E. De Clercq,et al.  Antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro. , 1995, The Journal of infectious diseases.

[50]  S. Meri,et al.  Shedding and enrichment of the glycolipid-anchored complement lysis inhibitor protectin (CD59) into milk fat globules. , 1995, Immunology.

[51]  M. Bissell,et al.  Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments. , 1995, Journal of cell science.

[52]  T. Takao,et al.  A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in 18O- , 1992, Analytical biochemistry.

[53]  Ylva Gavel,et al.  Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering , 1990, Protein engineering.

[54]  N. Sharon,et al.  Adhesins as lectins: specificity and role in infection. , 1990, Current topics in microbiology and immunology.

[55]  Tadao Saito,et al.  Chemical structure of neutral sugar chains isolated from human mature milk κ-casein , 1988 .

[56]  E. Berger,et al.  Structure identification of the complex-type, asparagine-linked sugar chains of beta-D-galactosyl-transferase purified from human milk. , 1986, Carbohydrate research.

[57]  P. Stahl,et al.  Identification of the macrophage mannose receptor as a 175-kDa membrane protein. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[58]  P. Vilja,et al.  Lectin binding affinities of human milk fat globule (HMFG) membrane antigens. , 1986, Molecular immunology.

[59]  S. Ahlstedt,et al.  Protective factors in milk and the development of the immune system. , 1985, Pediatrics.

[60]  A. Antony,et al.  Isolation, characterization, and comparison of the solubilized particulate and soluble folate binding proteins from human milk. , 1982, The Journal of biological chemistry.

[61]  H. Donat.  [Total protein and immunoglobulin content of human milk]. , 1976, Zentralblatt fur Gynakologie.