Human milk is regarded as the gold standard for infant feeding. INTRODUCTION EXPERIMENTAL CONCLUSIONS [1] Why Human Milk Oligosaccharides Are the Next Big Breakthrough? www.nutrition news.abbott Connie A. Remoroza 1 , Yuxue Liang 1 , Tytus D. Mak 1 , Sergey L. Sheetlin 1 , Yuri A. Mirokhin 1 , Joice San Andres 2 , Michael L. Power 3 , Stephen E. Stein 1 1 Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg MD 20899 2 Central Luzon State University, Maharlika Highway Nueva Ecija 3120, Philippines 3 Nutrition Laboratory, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, D.C., USA TANDEM MASS SPECTRAL ANALYSIS OF FREE OLIGOSACCHARIDES IN MILK OF VARIOUS MAMMALS Human milk oligosaccharides feed good bacteria in the gut where 70% of the immune system exists [1]. This study focuses on the identification and molecular fingerprinting of oligosaccharides derived from the milk of mammals using the Hydrophilic Interaction Liquid Chromatography and Mass Spectrometry for building NIST prebiotics/glycans MS library. Human milk SRM 1953 ➢Removal of lipids & proteins Sample reduction & clean up ➢SPE removes salts. 20% and 40% acetonitrile separates neutral and acidic oligosaccharides. Analysis of HMOs & BMOs by HILIC- ESI-MS ➢Identification of neutral and acidic oligosaccharides. Molecular structure analysis using spectral library and SIM Glycan Building of NIST oligos MS Library NIST SRMs Nomenclature of oligosaccharide RESULTS I. Separation based on size, net charge & type of oligosaccharides II. Data analysis using different analytical platforms NIST Tandem MS Library Glucose Galactose GlcNAc Fucose NeuAc ➢High resolution MS 2 values ➢negative and positive mode ➢Different collision energies ➢Different adducts ➢Score or match factor (MF) provides the degree of structure similarity. ➢Error tolerance of below 10 ppm. ➢ In summary, 140 of human milk oligosaccharides and over 70 non human oligosaccharides have been identified in milk of various mammals. ➢ The HILIC-ESI-MS method in combination with NIST MS search engine can now be used to accurately determine various fucosylated and sialylated oligosaccharides present in infant formula and other domestic animal milk. Email address: [email protected] III. Hybrid MS Library Search facilitates the identification of unknown oligosaccharides HILIC MS profile of oligosaccharides present in African lion milk. IV. Quantitative analysis of oligosaccharides in milk of various mammals 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Time (min) Isoglobotriose LNnH N3020 LNnT LDFT N4010 LNFP III N6020 N5020 N5120a N5120b N4130 N5020 511.35 0.00 0.00 0.00 N1110 N4000b N4000a N4000 c LNFP III Relative Abundance 3’-SL 3’-GL 6’-GSL 3’-GDL 3’-DSL DP 3 DP 4 DP 5 DP 6 DP 7 DP 8 N4120 672.51 0.00 0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6’-SL 3’-GSL N2010 6’-DSL 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 N3120 N4220 N4020 3’- GL African Lion milk Goat milk Buffalo milk Bovine milk A B D Isotopic masses Monoisotopic mass 0 20 40 60 80 100 3'-SL 6'-SL 3'-GL 6'-GL 3'-SLN A3001 A4001 a A4001 b A3011 a A3011 b A3002 S-LNnH Relative abundance Lion Goat Buffalo SRM1549a α2-3 β1-4 α2-6 β1-4 α2-6 β1-4 β1-4 α2-3 β1-4 α2-3 β1-4 β1-4 α2-3 β1-3 α1-4 α2- β1-3 α1-4 β1-4 β1-3 β1-4 β1-6 β1-4 α2-6 β1-4 B β1-4 0 20 40 60 80 100 Relative abundance Lion Goat Bufallo SRM1549a A β1-4 α1-2 β1-4 α1-3 β1-4 α1-3 β1-4 β1-3 β1-4 β1-6 β1-4 b1-4 a1-3 β1-3 b1-4 β1-4 β1-4 β1-6 Smithsonian National Zoo Milk Repository DeltaMass= 41.027 sMF score = 50 hMF score = 922 m/z 730.2378 SRM 1953 Glycan 3,4 ,3-Galactotetraose Head to Tail MF=922 RMF=931 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 0 50 100 50 100 255.2315 275.0736 305.0840 347.0947 365.1055 365.1049 388.1214 406.1317 406.1316 429.3186 437.1251 500.2826 527.1575 550.1741 568.1842 599.1783 610.1959 640.2050 670.2163 671.1996 712.2268 712.2263 509.1470 0,2 A 4 Y 2 B 3 Y 3 2,4 A 4 2,4 A 2 C 2 B 2 Z 2 B 1 2,4 A 4 B 2 Y 2 B 3 Z 2 2,4 A 2 0,4 A 4 Y 3 Z 2 Y 2 B 3 Y 3 B 3 Y 2 B 2 C D m/z= 878.3116 SRM 1953 Glycan LNFP III Head to Tail MF=826 RMF=855 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750 780 810 840 870 0 50 100 50 100 289.0898 367.1214 388.1204 450.1585 534.1795 550.1729 638.2261 670.2146 696.2323 696.2303 732.2533 756.2506 816.2723 818.2886 858.2824 860.2980 DeltaMass= 2.016 sMF score = 309 hMF score = 826 Y 2 Y 3a B 3 A B Y 3 Y 2 B 2 /Y 3 730.2357 Y 2 Y 3a B 3 B 2 B 2 Y 3 B 3 0,4 X 0 2,4 A 4 B 3 /Y 3 552.1901 - 18 Da - 18 Da Reduced B 2 Human milk reference material ➢Ion plot of identified compounds. This demonstrates the robust identification of unknown compounds using tandem mass spectral libraries. ➢Comparison of spectra based on MS 2 similarity between oligosaccharides by IsoPique.