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

Abstract

LOS MALDI IMS-MSAbstract. Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source Bprompt^fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis.

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate

“…It is important to emphasize that both the LPS and dLPS samples produced heterogeneous mass spectra, even after crude IMS filtering, presumably due to many different LPS structures in the samples. Other analyses of R-LPS by both ESI-MS and MALDI-MS have demonstrated similar results [34,43]. This means that the activity data shown in the previous section are cumulative and cannot be attributed to a particular R-LPS structure.…”

The Biosynthesis of Lipooligosaccharide from Bacteroides thetaiotaomicron

“…6 ). Although instrument-specific limitations prevented us from obtaining a resolution as high as others have observed, the limited degree of resolution that we achieved was sufficient for confirming the approximate masses of LOS molecules in the sample and comparing them to those of the truncated mutants ( 8 , 48 ).…”

“…We adapted a previously published strategy for analyzing intact LPS/LOS by MALDI-TOF ( 47 , 48 ). LPS/LOS analysis is typically challenging due to difficulties in inducing the glycolipid to ionize because of its size and polarity.…”

“…S3 ). Additionally, every sample has a cluster of peaks around 1,688 m/z representing lipid A, which likely derives from in-source fragmentation ( 48 ).…”

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence

“…MALDI-TOF MS and ion-mobility spectrometry (IMS)-MS analyses were performed on a Synapt G2 high-definition MS system (Waters Corporation, Milford, MA) in sensitivity mode. The instrument is equipped with a T-wave ion-mobility cell (Triwave TM ) (39,40) and was operated in MALDI mode as previously described (41). Spectra were obtained in negative-or positive-ion mode operating a neodymium-doped yttrium aluminum garnet laser at 355 nm and 200 Hz.…”