Carbohydrate structure characterization by tandem ion mobility mass spectrometry (IMMS)2

Abstract

A high resolution ion mobility spectrometer was interfaced to a Synapt G2 high definition mass spectrometer (HDMS) to produce IMMS-IMMS analysis. The hybrid instrument contained an electrospray ionization source, two ion gates, an ambient pressure linear ion mobility drift tube, a quadrupole mass filter, a traveling wave ion mobility spectrometer (TWIMS), and a time-of-flight mass spectrometer. The dual gate drift tube ion mobility spectrometer (DTIMS) could be used to acquire traditional IMS spectra but also could selectively transfer specific mobility selected precursor ions to the Synapt G2 HDMS for mass filtration (quadrupole). The mobility and mass selected ions could then be introduced into a collision cell for fragmentation followed by mobility separation of the fragment ions with the traveling wave ion mobility spectrometer. These mobility separated fragment ions are finally mass analyzed using a time-of-flight mass spectrometer. This results in an IMMS-IMMS analysis and provides a method to evaluate the isomeric heterogeneity of precursor ions by both DTIMS and TWIMS to acquire a mobility-selected and mass-filtered fragmentation pattern and to additionally obtain traveling wave ion mobility spectra of the corresponding product ions. This new IMMS(2) instrument enables the structural diversity of carbohydrates to be studied in greater detail. The physical separation of isomeric oligosaccharide mixtures was achieved by both DTIMS and TWIMS, with DTIMS demonstrating higher resolving power (70-80) than TWIMS (30-40). Mobility selected MS/MS spectra were obtained, and TWIMS evaluation of product ions showed that isomeric forms of fragment ions existed for identical m/z values.

Figure 1

Schematics of the instrument, showing the electrospray ionization (ESI) source, the ambient pressure, dual gate ion mobility drift tube, and the Synapt G2 high definition mass spectrometry unit that includes a quadrupole, a traveling wave ion mobility region and a dual stage reflectron time-of-flight mass spectrometer. The inserted window shows the detailed interface connection.

Figure 2

(a) TWIMS separation of a disaccharide-alditol mixture (precursor m/z [M + Na⁺] = 367. (b) DTIMS separation of the same disaccharide-alditol mixture. (c) Overlaid individual spectra of d-Gal-α-1-4-d-Gal-ol (blue) and d-Glc-β-1-6-d-Glc-ol (red) obtained on TWIMS. (d) Overlaid individual spectra of d-Gal-α-1-4-d-Gal-ol (blue) and d-Glc-β-1-6-d-Glc-ol (red) obtained on DTIMS. (e) Mobility selected MS/MS spectrum of mobility peak 1 (from panel b) using a 24.5–25.2 ms window on the dual gate DTIMS. The structure and fragmentation pathway for d-Gal-α-1-4-d-Gal-ol are shown on the right. (f) Mobility selected MS/MS spectrum of mobility peak 2 using a 25.5–26.5 ms window on the dual gate DTIMS. The structure and fragmentation pathway for d-Glc-β-1-6-d-Glc-ol are shown on the right. Note: (a) and (c) were obtained with the system operating in mode 1; (b) and (d) were collected using operation mode 2; (e) and (f) were acquired with mode 3 as described in the experimental section.

Figure 3

(a) TWIMS separation of a trisaccharide mixture (precursor m/z [M + Na⁺] = 527. (b) DTIMS separation of the same mixture. (c) Overlaid individual spectra of raffinose (blue) and maltotriose (red) obtained on TWIMS. (d) Overlaid individual spectra of raffinose (blue) and maltotriose (red) obtained on DTIMS. (e) Mobility selected MS/MS spectrum of mobility peak 1 using the drift time window of 31–31.5 ms on DTIMS (panel b). The structure and fragmentation pathway for raffinose are shown on the right. (f) Mobility selected MS/MS spectrum of mobility peak 2 using the drift time window of 32–32.5 ms on DTIMS (panel b). The structure and fragmentation pathway for maltotriose are shown on the right. Note: (a) and (c) were obtained with the system operating in mode 1; (b) and (d) were collected using mode 2; (e) and (f) were acquired with operation mode 3.

Figure 4

(a) TWIMS separation of product ions derived from mobility selected peak 1 (raffinose) shown in Figure 3, panel b, fragmentation in panel e. (b) TWIMS separation of product ions for mobility selected peak 2 (maltotriose) shown in Figure 3, panel b, fragmentation in panel f. (c) TWIMS separation of raffinose and its product ions acquired from the standard run individually where the system was operated in mode 1. (d) TWIMS separation of maltotriose and its product ions acquired from the standard run individually (mode 1). The corresponding m/z values for the major resolved product ion mobility peaks were labeled in Figures 4a and 4b.

Figure 5

(a) TWIMS separation of a pentasaccharide mixture (precursor m/z [M + Na⁺] = 851). (b) DTIMS separation of the same pentasaccharide mixture. (c) Overlaid individual spectra of cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green) obtained on the TWIMS. (d) Overlaid individual spectra of cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green) obtained on the DTIMS. Note: (a) and (c) were collected with the system operating in mode 1 and (b) and (d) were obtained using operation mode 2 as explained in the experimental section.

Figure 6

Mobility-selected MS/MS spectra of pentasaccharides separated on the drift tube ion mobility region of the instrument. (a) Mobility-selected MS/MS spectrum of peak 1 (Figure 5b) using the drift time window of 38–38.7 ms on DTIMS. The structure and proposed fragmentation pathway of cellopentaose are shown on the right. (b) Mobility-selected MS/MS spectrum of peak 2 (Figure 5b) using the drift time window of 39.5–40.3 ms on DTIMS. The structure and fragmentation pathway of maltopentaose are shown on the right. and is not shown here. (c) Mobility-selected MS/MS spectrum for peak 3 (Figure 5b) using the drift time window of 40.6–41.5 ms on DTIMS. The structure and proposed fragmentation pathway of branched (Man)₅ are displayed on the right.

Figure 7

Overlaid TWIMS separation of product ions derived from isomeric pentasaccharide precursor ions at m/z 851. (a) Overlaid TWIMS separation of product ion m/z 509 for cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green). (b) Overlaid TWIMS separation of product ion m/z 527 for cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green). (c) Overlaid TWIMS separation of product ion m/z 671 for cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green). (d) Overlaid TWIMS separation of product ion m/z 689 for cellopentaose (blue), maltopentaose (red) and branched (Man)₅ (green).