Skip to main content
Log in

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 μL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex2Fuc2 and Hex3HexNAc1Fuc2, as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a − b+), Le(a + b−), and Le(a − b−), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.

MALDI-TOF-MS HMO profiles of Le(a − b+) colostrum samples obtained in the positive-ion mode

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

ACN:

Acetonitrile

ATT:

6-Aza-2-thiothymine

DMB:

2-Diamino-4,5-methylenedioxybenzene

Fuc:

Fucose

FucT:

Fucosyltransferase

Hex:

Hexose

HexNAc:

N-acetylhexosamine

HMO:

Human milk oligosaccharide

LID:

Laser-induced dissociation

LNFP I:

Lacto-N-fucopentaose I (Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc)

LNFP II:

Lacto-N-fucopentaose II (Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc)

LNnT:

Lacto-N-neotetraose

LNT:

Lacto-N-tetraose

MALDI-TOF-MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MS/MS:

Tandem mass spectrometry

PGC:

Porous graphitic carbon

Sia:

Sialic acid

SPE:

Solid-phase extraction

TFA:

Trifluoroacetic acid

References

  1. Schack-Nielsen L, Michaelsen KF (2007) Advances in our understanding of the biology of human milk and its effects on the offspring. J Nutr 137:503S–510S

    CAS  Google Scholar 

  2. Kunz C, Rudloff S, Baier W, Klein N, Strobel S (2000) Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annu Rev Nutr 20:699–722

    Article  CAS  Google Scholar 

  3. Montreuil J, Mullet S (1960) Etude des variations des constituents glucidiques du lait de femme au cours de la lactation. Bull Soc Chim Biol 42:365–377

    CAS  Google Scholar 

  4. Newburg DS, Ruiz-Palacios GM, Altaye M, Chaturvedi P, Meinzen-Derr J, Guerrero Mde L, Morrow AL (2004) Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants. Glycobiology 14:253–263

    Article  CAS  Google Scholar 

  5. Sharon N, Ofek I (2000) Safe as mother's milk: carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconj J 17:659–664

    Article  CAS  Google Scholar 

  6. Angeloni S, Ridet JL, Kusy N, Gao H, Crevoisier F, Guinchard S, Kochhar S, Sigrist H, Sprenger N (2005) Glycoprofiling with micro-arrays of glycoconjugates and lectins. Glycobiology 15:31–41

    Article  CAS  Google Scholar 

  7. Bode L (2009) Human milk oligosaccharides: prebiotics and beyond. Nutr Rev 67:S183–S191

    Article  Google Scholar 

  8. Donovan SM (2009) Human milk oligosaccharides—the plot thickens. Br J Nutr 101:1267–1269

    Article  CAS  Google Scholar 

  9. Kuntz S, Kunz C, Rudloff S (2009) Oligosaccharides from human milk induce growth arrest via G2/M by influencing growth-related cell cycle genes in intestinal epithelial cells. Br J Nutr 101:1306–1315

    Article  CAS  Google Scholar 

  10. Fuhrer A, Sprenger N, Kurakevich E, Borsig L, Chassard C, Hennet T (2010) Milk sialyllactose influences colitis in mice through selective intestinal bacterial colonization. J Exp Med 207:2843–2854

    Article  CAS  Google Scholar 

  11. Gyorgy P, Norris RF, Rose CS (1954) Bifidus factor. I. A variant of Lactobacillus bifidus requiring a special growth factor. Arch Biochem Biophys 48:193–201

    Article  CAS  Google Scholar 

  12. Sela DA, Mills DA (2010) Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides. Trends Microbiol 18:298–307

    Article  CAS  Google Scholar 

  13. Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez-Munguia B, Newburg DS (2003) Campylobacter jejuni binds intestinal H(O) antigen (Fuc α1, 2Gal β1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem 278:14112–14120

    Article  CAS  Google Scholar 

  14. Falk PG, Bry L, Holgersson J, Gordon JI (1995) Expression of a human alpha-1,3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of Leb-containing glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection. Proc Natl Acad Sci U S A 92:1515–1519

    Article  CAS  Google Scholar 

  15. Rudloff S, Pohlentz G, Diekmann L, Egge H, Kunz C (1996) Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula. Acta Paediatr 85:598–603

    Article  CAS  Google Scholar 

  16. Bode L, Kunz C, Muhly-Reinholz M, Mayer K, Seeger W, Rudloff S (2004) Inhibition of monocyte, lymphocyte, and neutrophil adhesion to endothelial cells by human milk oligosaccharides. Thromb Haemost 92:1402–1410

    CAS  Google Scholar 

  17. Kobata A (2010) Structures and application of oligosaccharides in human milk. Proc Jpn Acad Ser B Phys Biol Sci 86:731–747

    Article  CAS  Google Scholar 

  18. Urashima T, Fukuda K, Kitaoka M, Ohnighi M, Terabayashi T, Kobata A (2011) Milk Oligosacharides. Noca Science Publishers, Inc., New York

    Google Scholar 

  19. Stahl B, Thurl S, Zeng J, Karas M, Hillenkamp F, Steup M, Sawatzki G (1994) Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem 223:218–226

    Article  CAS  Google Scholar 

  20. Wu S, Grimm R, German JB, Lebrilla CB (2010) Annotation and Structural Analysis of Sialylated Human Milk Oligosaccharides. J Proteome Res 10:856–868

    Article  CAS  Google Scholar 

  21. Wu S, Tao N, German JB, Grimm R, Lebrilla CB (2010) The development of an annotated library of neutral human milk oligosaccharides. J Proteome Res 8:4138–4151

    Article  Google Scholar 

  22. Grollman EF, Ginsburg V (1967) Correlation between secretor status and the occurrence of 2'-fucosyllactose in human milk. Biochem Biophys Res Commun 28:50–53

    Article  CAS  Google Scholar 

  23. Grollman EF, Kobata A, Ginsburg V (1969) An enzymatic basis for Lewis blood types in man. J Clin Invest 48:1489–1494

    Article  CAS  Google Scholar 

  24. Grollman EF, Kobata A, Ginsburg V (1970) Enzymatic basis of blood types in man. Ann N Y Acad Sci 169:153–160

    Article  CAS  Google Scholar 

  25. Kobata A, Ginsburg V (1969) Oligosaccharides of human milk. II. Isolation and characterization of a new pentasaccharide, lacto-N-fucopentaose 3. J Biol Chem 244:5496–5502

    CAS  Google Scholar 

  26. Kobata A, Ginsburg V, Tsuda M (1969) Oligosaccharides of human milk. I. Isolation and characterization. Arch Biochem Biophys 130:509–513

    Article  CAS  Google Scholar 

  27. Egge H (1993) The diversity of oligosaccharides in human milk. In: Renner B, Sawatzki G (eds) New perspectives in infant nutrition. Thieme, New York, pp 12–26

    Google Scholar 

  28. Kobata A (1977) Milk glycoproteins and oligosaccharides. In: Horowitz M, Pigman W (eds) The glycoconjugates. Academic, New York, pp 423–440

    Chapter  Google Scholar 

  29. Le Pendu J (2004) Histo-blood group antigen and human milk oligosaccharides: genetic polymorphism and risk of infectious diseases. Adv Exp Med Biol 554:135–143

    Article  Google Scholar 

  30. Almeida R, Amado M, David L, Levery SB, Holmes EH, Merkx G, van Kessel AG, Rygaard E, Hassan H, Bennett E, Clausen H (1997) A family of human β4-galactosyltransferases. Cloning and expression of two novel UDP-galactose: β-N-acetylglucosamine β1,4-galactosyltransferases, β4Gal-T2 and β4Gal-T3. J Biol Chem 272:31979–31991

    Article  CAS  Google Scholar 

  31. Isshiki S, Togayachi A, Kudo T, Nishihara S, Watanabe M, Kubota T, Kitajima M, Shiraishi N, Sasaki K, Andoh T, Narimatsu H (1999) Cloning, expression, and characterization of a novel UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase (β3Gal-T5) responsible for synthesis of type 1 chain in colorectal and pancreatic epithelia and tumor cells derived therefrom. J Biol Chem 274:12499–12507

    Article  CAS  Google Scholar 

  32. Sasaki K, Kurata-Miura K, Ujita M, Angata K, Nakagawa S, Sekine S, Nishi T, Fukuda M (1997) Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis. Proc Natl Acad Sci U S A 94:14294–14299

    Article  CAS  Google Scholar 

  33. Ma B, Simala-Grant JL, Taylor DE (2006) Fucosylation in prokaryotes and eukaryotes. Glycobiology 16:158R–184R

    Article  CAS  Google Scholar 

  34. Erney R, Hilty M, Pickering L, Ruiz-Palacios G, Prieto P (2001) Human milk oligosaccharides. A novel method provides insight into human genetics. Adv Exp Med Biol 501:285–297

    CAS  Google Scholar 

  35. Johnson PH, Watkins WM (1992) Purification of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to type 1 and lactose-based oligosaccharide chains. Glycoconj J 9:241–249

    Article  CAS  Google Scholar 

  36. Kobata A (2000) A journey to the world of glycobiology. Glycoconj J 17:443–464

    Article  CAS  Google Scholar 

  37. Oriol R, Le Pendu J, Mollicone R (1986) Genetics of ABO, H, Lewis, X and related antigens. Vox Sang 51:161–171

    Article  CAS  Google Scholar 

  38. Thurl S, Henker J, Siegel M, Tovar K, Sawatzki G (1997) Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides. Glycoconj J 14:795–799

    Article  CAS  Google Scholar 

  39. Viverge D, Grimmonprez L, Cassanas G, Bardet L, Solere M (1990) Discriminant carbohydrate components of human milk according to donor secretor types. J Pediatr Gastroenterol Nutr 11:365–370

    Article  CAS  Google Scholar 

  40. Kunz C, Bode L, Rudloff S (2003) Genetic variability of human milk oligosaccharides: are there biologic consequences? In: Bachmann C, Koletzko B (eds) Genetic expression and nutrition. Nestle Nutrition Workshop Series, Pediatric Program, Nestec Ltd. Vevey/Lippincott Williams & Wilkins, Philadelphia, pp 137–152

    Google Scholar 

  41. Thurl S, Munzert M, Henker J, Boehm G, Muller-Werner B, Jelinek J, Stahl B (2010) Variation of human milk oligosaccharides in relation to milk groups and lactational periods. Br J Nutr 104:1261–1271

    Article  CAS  Google Scholar 

  42. Morrow AL, Meinzen-Derr J, Huang P, Schibler KR, Cahill T, Keddache M, Kallapur SG, Newburg DS, Tabangin M, Warner BB, Jiang X, (2011) Fucosyltransferase 2 Non-Secretor and Low Secretor Status Predicts Severe Outcomes in Premature Infants. J Pediatr 158:745–751

    Article  CAS  Google Scholar 

  43. Albrecht S, Schols HA, van den Heuvel EG, Voragen AG, Gruppen H (2009) CE-LIF-MSn profiling of oligosaccharides in human milk and feces of breast-fed babies. Electrophoresis 31:1264–1273

    Article  Google Scholar 

  44. Ninonuevo MR, Park Y, Yin H, Zhang J, Ward RE, Clowers BH, German JB, Freeman SL, Killeen K, Grimm R, Lebrilla CB (2006) A strategy for annotating the human milk glycome. J Agric Food Chem 54:7471–7480

    Article  CAS  Google Scholar 

  45. Maass K, Ranzinger R, Geyer H, Von der Lieth CW, Geyer R (2007) "Glyco-Peakfinder"—de novo composition analysis of glycoconjugates. Proteomics 7:4435–4444

    Article  CAS  Google Scholar 

  46. Ceroni A, Maass K, Geyer H, Geyer R, Dell A, Haslam SM (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7:1650–1659

    Article  CAS  Google Scholar 

  47. von der Lieth CW, Freire AA, Blank D, Campbell MP, Ceroni A, Damerell DR, Dell A, Dwek RA, Ernst B, Fogh R, Frank M, Geyer H, Geyer R, Harrison MJ, Henrick K, Herget S, Hull WE, Ionides J, Joshi HJ, Kamerling JP, Leeflang BR, Lutteke T, Lundborg M, Maass K, Merry A, Ranzinger R, Rosen J, Royle L, Rudd PM, Schloissnig S, Stenutz R, Vranken WF, Widmalm G, Haslam SM (2011) EUROCarbDB: an open-access platform for glycoinformatics. Glycobiology 21:493–502

    Article  Google Scholar 

  48. Anumula KR (1994) Quantitative determination of monosaccharides in glycoproteins by high-performance liquid chromatography with highly sensitive fluorescence detection. Anal Biochem 220:275–283

    Article  CAS  Google Scholar 

  49. Wuhrer M, Dennis RD, Doenhoff MJ, Bickle Q, Lochnit G, Geyer R (1999) Immunochemical characterisation of Schistosoma mansoni glycolipid antigens. Mol Biochem Parasitol 103:155–169

    Article  CAS  Google Scholar 

  50. Galuska SP, Geyer H, Bleckmann C, Rohrich RC, Maass K, Bergfeld AK, Mühlenhoff M, Geyer R (2010) Mass spectrometric fragmentation analysis of oligosialic and polysialic acids. Anal Chem 82:2059–2066

    Article  CAS  Google Scholar 

  51. Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y (1987) Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal Biochem 164:138–145

    Article  CAS  Google Scholar 

  52. Tao N, Wu S, Kim J, An HJ, Hinde K, Power ML, Gagneux P, German JB, Lebrilla CB (2011) Evolutionary glycomics: characterization of milk oligosaccharides in primates. J Proteome Res 10:1548–1557

    Article  CAS  Google Scholar 

  53. Kunz C, Rudloff S, Schad W, Braun D (1999) Lactose-derived oligosaccharides in the milk of elephants: comparison with human milk. Br J Nutr 82:391–399

    Article  CAS  Google Scholar 

  54. Donald AS, Feeney J (1988) Separation of human milk oligosaccharides by recycling chromatography. First isolation of lacto-N-neo-difucohexaose II and 3'-galactosyllactose from this source. Carbohydr Res 178:79–91

    Article  CAS  Google Scholar 

  55. Kuhn R, Gauhe A (1958) Über die lacto-difuco-tetraose der Frauenmilch. Justus Liebigs Ann Chem 611:249–253

    Article  CAS  Google Scholar 

  56. Kuhn R, Gauhe A (1960) Über ein kristallisiertes, Lea-aktives Hexasaccharid aus Frauenmilch. Chem Ber 93:647–651

    Article  CAS  Google Scholar 

  57. Domon B, Costello CE (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate J 5:397–409

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Professor G. Bein and Professor H. Jomaa (Institute of Immunology and Transfusion Medicine, University Hospital Giessen-Marburg, Germany) for performing the Lewis blood group determination of blood samples as well as Dr. P. Gilbert (St. Josef’s Hospital Giessen) for the collection of milk and blood samples.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Rudolf Geyer or Clemens Kunz.

Additional information

Dennis Blank, Sabine Gebhardt, and Kai Maass contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blank, D., Gebhardt, S., Maass, K. et al. High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides. Anal Bioanal Chem 401, 2495–2510 (2011). https://doi.org/10.1007/s00216-011-5349-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5349-9

Keywords

Navigation