Analytical and Bioanalytical Chemistry

, Volume 406, Issue 30, pp 7925–7935 | Cite as

Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies

  • Sarah M. Totten
  • Lauren D. Wu
  • Evan A. Parker
  • Jasmine C. C. Davis
  • Serenus Hua
  • Carol Stroble
  • L. Renee Ruhaak
  • Jennifer T. Smilowitz
  • J. Bruce German
  • Carlito B. LebrillaEmail author
Research Paper


Glycomic analysis is the comprehensive determination of glycan (oligosaccharide) structures with quantitative information in a biological sample. Rapid-throughput glycomics is complicated due to the lack of a template, which has greatly facilitated analysis in the field of proteomics. Furthermore, the large similarities in structures make fragmentation spectra (as obtained in electron impact ionization and tandem mass spectrometry) less definitive for identification as it has been in metabolomics. In this study, we develop a concept of rapid-throughput glycomics on human milk oligosaccharides, which have proven to be an important bioactive component of breast milk, providing the infant with protection against pathogenic infection and supporting the establishment of a healthy microbiota. To better understand the relationship between diverse oligosaccharides structures and their biological function as anti-pathogenic and prebiotic compounds, large human studies are needed, which necessitate rapid- to high-throughput analytical platforms. Herein, a complete glycomics methodology is presented, evaluating the most effective human milk oligosaccharide (HMO) extraction protocols, the linearity and reproducibility of the nano-liquid chromatography chip time-of-flight mass spectrometry (nano-LC chip-TOF MS) method, and the efficacy of newly developed, in-house software for chromatographic peak alignment that allows for rapid data analysis. High instrument stability and retention time reproducibility, together with the successful automated alignment of hundreds of features in hundreds of milk samples, allow for the use of an HMO library for rapid assignment of fully annotated structures.

Graphical Abstract


Human milk oligosaccharides Rapid-throughput glycomics Nano-flow liquid chromatography-mass spectrometry 



The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Research reported in this publication was supported by the National Institute of Child Health and Human Development, National Institute of General Medicine, and National Center of Complementary and Alternative Medicine of the National Institutes of Health under award numbers R01HD061923, R01GM049077, R01 AT007079, and 1U24DK097154.

Supplementary material

216_2014_8261_MOESM1_ESM.pdf (138 kb)
ESM 1 (PDF 138 kb)


  1. 1.
    Bode L (2012) Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 22(9):1147–1162. doi: 10.1093/glycob/cws074 CrossRefGoogle Scholar
  2. 2.
    Bode L, Kuhn L, Kim H-Y, Hsiao L, Nissan C, Sinkala M, Kankasa C, Mwiya M, Thea DM, Aldrovandi GM (2012) Human milk oligosaccharide concentration and risk of postnatal transmission of HIV through breastfeeding. Am J Clin Nutr 96(4):831–839. doi: 10.3945/ajcn.112.039503 CrossRefGoogle Scholar
  3. 3.
    Kunz C, Rudloff S, Baier W, Klein N, Strobel S (2000) Oligosaccharides in human milk: structural, functional, and metabolic aspects. Annu Rev Nutr 20(1):699–722. doi: 10.1146/annurev.nutr.20.1.699 CrossRefGoogle Scholar
  4. 4.
    Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS (2005) Human-milk glycans that inhibit pathogen binding protect breast-feeding infants against infectious diarrhea. J Nutr 135(5):1304–1307Google Scholar
  5. 5.
    Newburg DS, Ruiz-Palacios GM, Morrow AL (2005) Human milk glycans protect infants against enteric pathogens. Annu Rev Nutr 25(1):37–58. doi: 10.1146/annurev.nutr.25.050304.092553 CrossRefGoogle Scholar
  6. 6.
    Hua S, Lebrilla C, An HJ (2011) Application of nano-LC-based glycomics towards biomarker discovery. Bioanalysis 3(22):2573–2585. doi: 10.4155/bio.11.263 CrossRefGoogle Scholar
  7. 7.
    Costello CE, Contado-Miller JM, Cipollo JF (2007) A glycomics platform for the analysis of permethylated oligosaccharide alditols. J Am Soc Mass Spectrom 18(10):1799–1812. doi: 10.1016/j.jasms.2007.07.016 CrossRefGoogle Scholar
  8. 8.
    Erney RM, Malone WT, Skelding MB, Marcon AA, Kleman-Leyer KM, O’Ryan ML, Ruiz-Palacios G, Hilty MD, Pickering LK, Prieto PA (2000) Variability of human milk neutral oligosaccharides in a diverse population. J Pediatr Gastroenterol Nutr 30(2):181–192CrossRefGoogle Scholar
  9. 9.
    Leo F, Asakuma S, Nakamura T, Fukuda K, Senda A, Urashima T (2009) Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography. J Chromatogr A 1216(9):1520–1523. doi: 10.1016/j.chroma.2009.01.015 CrossRefGoogle Scholar
  10. 10.
    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(7):795–799. doi: 10.1023/A:1018529703106 CrossRefGoogle Scholar
  11. 11.
    De Leoz M, Wu S, Strum J, Niñonuevo M, Gaerlan S, Mirmiran M, German JB, Mills D, Lebrilla C, Underwood M (2013) A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants. Anal Bioanal Chem 405(12):4089–4105. doi: 10.1007/s00216-013-6817-1 CrossRefGoogle Scholar
  12. 12.
    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(20):7471–7480. doi: 10.1021/jf0615810 CrossRefGoogle Scholar
  13. 13.
    Strum JS, Kim J, Wu S, De Leoz MLA, Peacock K, Grimm R, German JB, Mills DA, Lebrilla CB (2012) Identification and accurate quantitation of biological oligosaccharide mixtures. Anal Chem 84(18):7793–7801. doi: 10.1021/ac301128s CrossRefGoogle Scholar
  14. 14.
    Wu S, Grimm R, German JB, Lebrilla CB (2010) Annotation and structural analysis of sialylated human milk oligosaccharides. J Proteome Res 10(2):856–868. doi: 10.1021/pr101006u CrossRefGoogle Scholar
  15. 15.
    Bao Y, Chen C, Newburg DS (2013) Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry. Anal Biochem 433(1):28–35. doi: 10.1016/j.ab.2012.10.003 CrossRefGoogle Scholar
  16. 16.
    Kottler R, Mank M, Hennig R, Müller-Werner B, Stahl B, Reichl U, Rapp E (2013) Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection. Electrophoresis 34(16):2323–2336. doi: 10.1002/elps.201300016 CrossRefGoogle Scholar
  17. 17.
    Blank D, Gebhardt S, Maass K, Lochnit G, Dotz V, Blank J, Geyer R, Kunz C (2011) High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides. Anal Bioanal Chem 401(8):2495–2510. doi: 10.1007/s00216-011-5349-9 CrossRefGoogle Scholar
  18. 18.
    Wu S, Tao N, German JB, Grimm R, Lebrilla CB (2010) Development of an annotated library of neutral human milk oligosaccharides. J Proteome Res 9(8):4138–4151. doi: 10.1021/pr100362f CrossRefGoogle Scholar
  19. 19.
    Aldredge D, An HJ, Tang N, Waddell K, Lebrilla CB (2012) Annotation of a serum N-glycan library for rapid identification of structures. J Proteome Res 11(3):1958–1968. doi: 10.1021/pr2011439 CrossRefGoogle Scholar
  20. 20.
    Hua S, An HJ, Ozcan S, Ro GS, Soares S, DeVere-White R, Lebrilla CB (2011) Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers. Analyst 136(18):3663–3671. doi: 10.1039/C1AN15093F CrossRefGoogle Scholar
  21. 21.
    Jones E, Oliphant T, Peterson P (2001) Scipy: open source scientific tools for Python. Accessed 07 Jan 2014
  22. 22.
    Hunter JD (2007) Matplotlib: a 2D graphics environment. Comput Sci Eng 9(3):90–95CrossRefGoogle Scholar
  23. 23.
    Smith CA, Want EJ, O’Maille G, Abagyan R, Siuzdak G (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78(3):779–787. doi: 10.1021/ac051437y CrossRefGoogle Scholar
  24. 24.
    Ruhaak LR, Taylor S, Miyamoto S, Kelly K, Leiserowitz G, Gandara D, Lebrilla C, Kim K (2013) Chip-based nLC-TOF-MS is a highly stable technology for large-scale high-throughput analyses. Anal Bioanal Chem 405(14):4953–4958. doi: 10.1007/s00216-013-6908-z CrossRefGoogle Scholar
  25. 25.
    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(4):1548–1557. doi: 10.1021/pr1009367 CrossRefGoogle Scholar
  26. 26.
    Totten SM, Zivkovic AM, Wu S, Ngyuen U, Freeman SL, Ruhaak LR, Darboe MK, German JB, Prentice AM, Lebrilla CB (2012) Comprehensive profiles of human milk oligosaccharides yield highly sensitive and specific markers for determining secretor status in lactating mothers. J Proteome Res 11(12):6124–6133. doi: 10.1021/pr300769g Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Sarah M. Totten
    • 1
  • Lauren D. Wu
    • 1
  • Evan A. Parker
    • 1
  • Jasmine C. C. Davis
    • 1
  • Serenus Hua
    • 1
    • 2
  • Carol Stroble
    • 1
  • L. Renee Ruhaak
    • 1
  • Jennifer T. Smilowitz
    • 3
    • 4
  • J. Bruce German
    • 3
    • 4
  • Carlito B. Lebrilla
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of CaliforniaDavisUSA
  2. 2.Asia Glycomics Reference SiteChungnam National UniversityDaejeonSouth Korea
  3. 3.Foods for Health InstituteUniversity of CaliforniaDavisUSA
  4. 4.Department of Food Science & TechnologyUniversity of CaliforniaDavisUSA

Personalised recommendations