Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

Abstract

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. Graphical Abstract ᅟ.

Keywords: Acyl chain; Collisional activation; Lipid A; Tandem mass spectrometry.

Figure 1

(a) Structure of lAA and the corresponding MS/MS spectra of the deprotonated lipid by: (b) CID at NCE 35, (c) HCD at NCE 25, and (d) UVPD at 2.5 mJ for seven pulses. Only the most prominent fragment ions are labeled. ERMS plots are shown in (e) HCD and (f) UVPD. The HCD ERMS plot is shown with error bars in Figure S3 to illustrate the typical standard deviations of these measurements.

Figure 2

Summary of sequential fragmentation of lAA upon increasing HCD energy.

Figure 3

lAC, lAD, and lAE structures with key cleavage sites indicated (a, b, c) and respective HCD ERMS plots (d, e, f) for the deprotonated lipids.

Figure 4

HCD spectra of deprotonated (a) lAD and (b) lAE. MS³ spectra in (c) to (f) reveal the sequential order of 3′α and 2′ε chain cleavage.

Figure 5

Structures of (a) lAF and (b) lAG. The HCD ERMS plots are shown for each doubly deprotonated lipid in (c) and (d), respectively.

Figure 6

lAH, lAI, and lAJ structures with key cleavage sites indicated (a, b, c) and respective HCD ERMS plots (d, e, f) for the deprotonated lipids.