Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Zhou, Ping; Altman, Eleonora; Perry, Malcolm B.; Li, Jianjun

doi:10.1128/aem.03082-09

Cited by 40 publications

(43 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lipid A samples were mixed with an equal volume of 5-chloro-2-mercaptobenzothiazole [20 mg ml 21 in chloroform/methanol/water (4 : 4 : 1, by vol.)] supplemented with 20 mM EDTA/ ammonium hydroxide (Zhou et al, 2010) and 0.5 ml aliquots were spotted onto the target plate. Spectra were acquired in the reflectron mode and analysed using flexAnalysis (Bruker Daltonics).…”

mentioning

confidence: 99%

LPS modification promotes maintenance of Yersinia pestis in fleas

et al. 2015

View full text Add to dashboard Cite

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-Larabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

show abstract

mentioning

confidence: 99%

LPS modification promotes maintenance of Yersinia pestis in fleas

et al. 2015

View full text Add to dashboard Cite

show abstract

“…We evaluated lipid A by MALDI-MS in an amount that was 50-to 100-fold less concentrated than that reported previously (5). We observed strong positive-ion production, but only very limited negative-ion production at this con- centration of lipid A, suggesting that phosphorylated lipid A species do not ionize as readily using this matrix and MALDI-MS. We did not attempt to maximize negative-ion recoveries either by modifying the matrix/lipid A ratio or by supplementing the matrix with other ion-enhancing agents, as reported by others (19,45,48). These issues will be addressed in future work.…”

Section: Resultsmentioning

confidence: 99%

Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

et al. 2012

View full text Add to dashboard Cite

Recent reports indicate that Porphyromonas gingivalis mediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides of P. gingivalis have been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity of P. gingivalis LPS or lipid A to engage TLR2 versus TLR4. In the present study, we first prepared P. gingivalis LPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived from P. gingivalis are contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents of P. gingivalis but contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A of P. gingivalis is not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A of P. gingivalis could actually be mediated by phosphorylated dihydroceramides. Porphyromonas gingivalis, along with many other potential periodontal pathogens, produce virulence factors capable of promoting tissue inflammation, loss of connective tissue attachment, and bone loss. Lipopolysaccharide (LPS) and lipid A are extensively studied microbial virulence factors from the standpoint of periodontal disease pathogenesis (1, 4, 9, 10, 20, 23, 24, 34-36, 38, 40), and both can promote inflammatory reactions and bone loss characteristic of tissue changes observed in chronic periodontitis. However, LPS or lipid A of P. gingivalis has not been demonstrated in diseased periodontal tissues. Instead, we reported that a unique fatty acid constituent of both lipid A and LPS of P. gingivalis, called 3-hydroxy isobranched C 17:0 (3-OH iso C 17: 0), exists in lipid products recovered predominantly from organic solvent extracts of subgingival plaque, periodontally diseased tissues, and diseased teeth, with little recovered in aqueous extracts of these samples (26, 29). Since LPS partitions essentially only into the aqueous phase with this extraction technique (39), we concluded that LPS of P. gingivalis and other related Bacteroidetes bacteria (33), including Prevotella intermedia and Tannerella forsythia, is not present to a signif...

show abstract

“…In contrast, the analysis of lipid A that was prepared using a commercially available LPS extraction kit-based procedure with DHB was preferable to detect aminoarabinose-modified lipid A species (Kawasaki, 2009). Furthermore, it was reported that matrix additives consisting of CMBT matrix and EDTA ammonium salt comatrix produce a highly sensitive MALDI-TOF MS analysis of lipid A, (Zhou, Altman, Perry, & Li, 2010). This matrix system enhances the sensitivity of analyzing diphosphorylated lipid A species by more than 100-fold and also provides tolerance to sodium dodecyl sulfate, sodium chloride, and calcium chloride (Zhou et al, 2010).…”

Section: Recent Improvements In Lipid a Analysis By Matrix-assisted Lmentioning

confidence: 99%

Complexity of lipopolysaccharide modifications in Salmonella enterica: Its effects on endotoxin activity, membrane permeability, and resistance to antimicrobial peptides

Kawasaki

2012

Food Research International

View full text Add to dashboard Cite

Study of Matrix Additives for Sensitive Analysis of Lipid A by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Cited by 40 publications

References 37 publications

LPS modification promotes maintenance of Yersinia pestis in fleas

LPS modification promotes maintenance of Yersinia pestis in fleas

Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

Complexity of lipopolysaccharide modifications in Salmonella enterica: Its effects on endotoxin activity, membrane permeability, and resistance to antimicrobial peptides

Contact Info

Product

Resources

About