Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn

Abstract

This report is the first in a series of three focused on establishing congruent strategies for carbohydrate sequencing. The reports are divided into (i) analytical considerations that account for all aspects of small oligomer structure by MSn disassembly, (ii) database support using an ion fragment library and associated tools for high-throughput analysis, and (iii) a concluding algorithm for defining oligosaccharide topology from MSn disassembly pathways. The analytical contribution of this first report explores the limits of structural detail exposed by ion trap mass spectrometry with samples prepared as methyl derivatives and analyzed as metal ion adducts. This data mining effort focuses on correlating the fragments of small oligomers to stereospecific glycan structures, an outcome attributed to a combination of metal ion adduction and analyte conformation. Facile glycosidic cleavage introduces a point of lability (pyranosyl-1-ene) that upon collisional activation initiates subsequent ring fragmentation. Product masses and ion intensities vary with interresidue linkage, branching position, and monomer stereochemistry. Excessive fragmentation is the property of small oligomers where collisional energy within a smaller number of oscillators dissipates through extensive fragmentation. The procedures discussed in this report are unified into a singular strategy using an ion trap mass spectrometer with the sensitivity expected for electron multiplier detection. Although a small set of structures have been discussed, the basic principles considered are fully congruent, with ample opportunities for expansion.

Scheme 1

Example of a retro-Diels—Alder reaction in a 1,4-linked B₂-type Hex-Hex disaccharide, showing formation of an m/z 329 ion from an m/z 445 ion (A). The m/z 139 ion is generally of low abundance or absent, presumably due to the larger fragments stronger affinity for the metal ion charge carrier. (B) shows the generic cross-ring cleavages that may be formed from B-type ions of various linkages. R indicates the location of mono- or oligosaccharide substituents.

Figure 1

ESI-MS² spectra of methylated disaccharide isobars composed of different monosaccharides. (A) Gal-α(1-4) Gal and (B) Glc-α(1-4) Glc. The absence of unsaturation, as in B-ions, results in little fragment specificity.

Figure 2

ESI-MS³ spectral comparison of native (A) and methylated (B) B₂-type fragment of maltotriositol. Comparative results demonstrate importance of methylation and unsaturation (e.g., Figure 1) providing precursor ion favorable to extensive fragmentation.

Figure 3

ESI-MS³ spectral comparison of native (A) and methylated (B) B₂-type fragment of tetrasaccharide milk glycan lacto-N-fucopentaose I. Note the prominent cross-ring cleavage fragment in the permethylated sample.

Figure 4

ESI-MS³ spectral comparison of B₂-type fragments of the tetrasaccharides (A) lacto-_N-tetraose and (B) lacto-_N-neotetraose. Note the different fragments present in each spectra, arising from the different linkages.

Figure 5

ESI-MS³ spectral comparison of B₂-type fragments of reduced, methylated trisaccharides (A) maltotriose and (B) panose. The presence of the cross-ring cleavage fragment at m/z 315 in the Glc-α(1-4)-Glc(1-ene) fragment, along with the m/z 329 fragment, is indicative of a 1-4 linkage, while the presence of only a m/z 329 cross-ring cleavage fragment (and absence of m/z 315) is indicative of a 1-6 linkage in panose.

Figure 6

ESI-MS³ spectral comparison of the effect of monomer identity on B₂-type fragment spectra from reduced, methylated trisaccharides. (A) globotriose vs (B) maltotriose. Note the different pattern of intensities on spectra collected on the same instrument.

Figure 7

ESI-MS³ spectral comparison of effect of monomer identity on B₂-type fragment spectra from reduced, methylated trisaccharides. (A) nigerotriose vs (B) linear B2 trisaccharide. Note the different pattern of intensities seen on spectra obtained with the same instrument.

Figure 8

ESI-MS⁴ spectral comparison of different B₁-type fragment spectra from reduced, methylated trisaccharides. (A) maltotriose (glucose) vs (B) globotriose (galactose). Note the more prominent m/z 109 fragment in the galactose spectra.

Figure 9

ESI-MSⁿ spectral comparison of C₁-type fragments of reduced, methylated maltotriose (A), unreduced, methylated Man-β(1-4)-GlcNAc (B), nigerotriose (C), and mannobiose (D). Note the different pattern of intensities between glucose (A, C) and mannose monomers (B, D) and the similarity of (A) and (C) vs (B) and (D).

Figure 10

ESI-MS³ spectral comparison of anomeric configuration on B₂-type fragment spectra from reduced, methylated trisaccharides. (A) maltotriose vs (B) cellotriose. Note the slightly different pattern of intensities.

Figure 11

ESI-MS³ spectral comparison of anomeric configuration on B₂-type fragment spectra from reduced, permethylated trisaccharides. (A) nigerotriose vs (B) laminaritriose. Note the different pattern of intensities.