Reflectron MALDI TOF and MALDI TOF/TOF mass spectrometry reveal novel structural details of native lipooligosaccharides

Abstract

Lipooligosaccharides (LOS) are powerful Gram-negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LOS is pivotal to understanding the mechanisms of infection at the molecular level. The amphiphilic nature of LOS has been the main obstacle for structural analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Our approach has resolved this important issue and has permitted us to obtain reflectron MALDI mass spectra of LOS to reveal the fine chemical structure with minimal structural variations. The high-quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in-source decay yields B-type ions, which correspond to core oligosaccharide(s), and Y-type ions, which are related to lipid A unit(s). MALDI tandem time-of-flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS.

IntracellularShigellaremodels its LPS to dampen the innate immune recognition and evade inflammasome activation

“…1B). It is worth noting that this information was achieved directly on the intact LPS molecule; in fact, the lipid A MS spectrum was directly obtained on the intact LPS upon in source fragmentation obtained in our optimized conditions (33); in this way, we can exclude any chemical change in lipid A as a result of chemical treatment. In aLPS, the canonical hexaacylated lipid A species was highly prevalent (Fig.…”

“…D'Hauteville et al (28) reported that WT S. flexneri serotype 5 (M90T, the same strain as in the present study) possessed a predominantly hexaacylated lipid A (93%). MALDI TOF/TOF MS performed on LPS of a S. flexneri 5 M90T revealed the presence of lipid A isoforms with the major component being the hexaacylated lipid A, with minor peaks being attributed to pentaacylated and tetraacylated species (33).…”

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

“…Using a thin-layer matrix composed of THAP and nitrocellulose [31], intact LOS can be analyzed by MALDI MS, generally in the negative-ion mode [9,10,12,13,[30][31][32]35]. This method produces an envelope of (M-H) -ions, as well as informative prompt fragments arising from the lipid A and oligosaccharide domains of the molecule.…”

“…Prompt fragments representing both domains of the molecule corroborate proposed compositions of the LOS molecular ions detected. Although previously only conducted in linear mode, analysis of intact LOS by high-resolution, reflectron mode MALDI-TOF MS has now been achieved [12,13,32,35]. The ability to measure monoisotopic molecular ions with good mass accuracy (<20 ppm) has dramatically improved the quality of the information that can be obtained from MALDI-TOF MS analysis of intact LOS.…”

Determination of Lipid A Profile of Gram-Negative Bacteria from Arctic Soils Using Mass Spectrometric Approaches

“…특히 지질 A의 구조에 있어서 아실 사슬 (acyl chain)의 위치와 개수 및 인산기의 존재 유무가 내독소 활성에 큰 영향을 끼친다 [12]. 지질 A 구조 분석을 위한 선행 연구들에 있어서는, 핵자기공명 분광기 (nuclear magnetic resonance (NMR) spectroscopy) [13], 전기분무 질량분석기 (electrospray ionization coupled tandem mass spectrometry (ESI-MS/MS)) [14], 모세관 전기영동 질량분석기 (capillary electrophoresis-mass spectrometry (CE-MS)) [15], 및 매트릭스 보조 이온화/탈착 질량분석기 (matrix-assisted laser ionization/desorption time-of-flight mass spectrometry (MALDI-TOF MS)) [16] Fig. 1(a)) [20][21][22][23].…”