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. 2012 May 15;424(2):87-96.
doi: 10.1016/j.ab.2012.02.012. Epub 2012 Feb 25.

Coupling Flash Liquid Chromatography With Mass Spectrometry for Enrichment and Isolation of Milk Oligosaccharides for Functional Studies

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Coupling Flash Liquid Chromatography With Mass Spectrometry for Enrichment and Isolation of Milk Oligosaccharides for Functional Studies

John S Strum et al. Anal Biochem. .
Free PMC article

Abstract

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.

Figures

Figure 1
Figure 1
Diagram showing plumbing connections between the flash system and the mass spectrometer. The eluate is split twice to reduce the flow rate. Additional organic solvent and acid are added through a mixing-t just before the eluate reaches the mass spectrometer.
Figure 2
Figure 2
Comparison of electrospray ionization of malto-oligosaccharides with degree of polymerization (DP) 4 through 7 in the presence of acid (A) versus sodium acetate (B). The sodiated oligosaccharides do not fragment appreciably in the source due to a higher energy activation barrier. The signals for DP8 and higher in A are a result of in-source rearrangements that are common for protonated oligosaccharides analyzed by ESI-MS.
Figure 3
Figure 3
Figure 3A. Pooled BMO signal is suppressed by GOS. 3B. Representative neutral BMO fraction that enriches for two BMOs. 3C. Representative acidic BMO fraction that is highly enriched for 2Hex 1NeuAc.
Figure 4
Figure 4
Separation of a 12 mg mixture of HMOs and lactose. Some of the base peaks eluted with greater than 90% purity. The EIC’s shown here are tabulated in Table 1. The first compound in Table 1 (3Hex 1GlcNAc 2dHex) is the purple line. The red line is 2Hex 2dHex. The blue line is 3Hex 1GlcNAc 2dHex. The green line is 3Hex 1GlcNAc 1dHex. The light orange line is 3Hex 1GlcNAc. The dark orange line is 4 Hex 2GlcNAc 1dHex. The off-green line is 4Hex 2GlcNAc.
Figure 5
Figure 5
Representative mass spectrum and tandem mass spectrum (MS2) of an HMO separated from a pool of Human milk oligosaccharides. The tandem data was vital for recognizing sugars due to the low mass accuracy of the instrument. All mass-to-charge ratios are singly protonated species.
Figure 6
Figure 6
Abundant HMOs less than 800 Da were significantly separated from HMOs greater than 1200 Da with size-exclusion chromatography. A. Extracted ion chromatograms for five HMOs obtained with a quadrupole ion trap on-line with flash LC. Extracted ion chromatograms for four HMOs larger than 1200 Da are shown as black lines: (in descending order from the most abundant chromatographic peak) m/z 1241.4 (4Hex 2GlcNAc 1dHex), 1387.5 (4Hex 2GlcNAc 2dHex), 1095.4 (4Hex 2GlcNAc) and 1971.5 (6Hex 4GlcNAc 1dHex). The red extracted ion chromatogram is m/z 708.0 (3Hex 1GlcNAc, predominantly the protonated form of LNT, typically the most abundant HMO). B. and C. Representative mass spectra of high and low mass fractions collected on-line with flash LC.
Figure 7
Figure 7
Extracted ion chromatogram for HMO isomers with m/z 1022.7 and a composition of 3Hex 1GlcNAc2dHex. Isomer separation was achieved on a 12 mg mixture of HMOs and lactose mixture with a 4 g self-packed graphitized carbon column. The smaller peaks eluting next to each major peak are probably anomers, a common observation with the high performance porous-graphitized carbon columns used in analytical scale separations.

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