Matrix-assisted Laser Ionization Research Articles

Miniaturization of Instrumental Planar Chromatography with Focus on Mass Spectrometry

The universal trends to miniaturization and to automation of technologies affect the further development of analytical instrumentation and methodologies. In planar chromatography, these trends are characterized by decreasing dimensions and thickness of the stationary phase and the development of respectively tailored equipment. The miniaturization of instrumental planar chromatography is reflected in the interdisciplinary office chromatography concept, in which achievements in office technologies (print and media technologies) and miniaturized layer developments are integrated. The office chromatography concept stimulates research towards miniaturization and will at last implement a fully online miniaturized system. The use of print techniques is still at its infancy in separation science, whereas printing of materials already became an accepted tool in the life sciences. Commercially, available printers were used for sample application on miniaturized plates and were stepwise modified for a precise and quantitative performance. Further miniaturization attempts led to miniaturized layers with different physical and chromatographic properties. The method transfer to such ultrathin-layer chromatography (UTLC) plates demonstrated the synergetic potential of this instrumental concept. Detection and evaluation tools have to be adjusted and integrated, too. Since the beginning of this millennium, several ionization techniques have been introduced or improved, and paved the way for mass spectrometry (MS) directly from miniaturized surfaces. UTLC was already coupled to MS via several ion source interfaces, such as secondary ionization, matrix-assisted laser desorption and ionization, electrospray ionization, desorption electrospray ionization and direct analysis in real time. However, the hyphenation to targeted, scanning or imaging MS still requires adaptations and methodic efforts to utilize UTLC layers.

Inflammation-associated changes in lipid composition and the organization of the erythrocyte membrane

BackgroundReduced erythrocyte survival and deformability may contribute to the so-called anemia of inflammation observed in septic patients. Erythrocyte structure and function are affected by both the membrane lipid composition and the organization. We therefore aimed to determine whether these parameters are affected during systemic inflammation. MethodsA sensitive matrix-assisted laser desorption and ionization time-of-flight mass spectrometric method was used to investigate the effect of plasma components of 10 patients with septic shock and of 10 healthy volunteers subjected to experimental endotoxemia on erythrocyte membrane lipid composition. ResultsIncubation of erythrocytes from healthy control donors with plasma from patients with septic shock resulted in membrane phosphatidylcholine hydrolysis into lysophosphatidylcholine (LPC). Plasma from volunteers undergoing experimental human endotoxemia did not induce LPC formation. The secretory phospholipase A2 IIA concentration was enhanced up to 200-fold in plasma of septic patients and plasma from endotoxin-treated subjects, but did not correlate with the ability of these plasmas to generate LPC. Erythrocyte phosphatidylserine exposure increased up to two-fold during experimental endotoxemia. ConclusionsErythrocyte membrane lipid remodeling as reflected by LPC formation and/or PS exposure occurs during systemic inflammation in a secretory phospholipase A2 IIA-independent manner. General significanceSepsis-associated inflammation induces a lipid remodeling of the erythrocyte membrane that is likely to affect erythrocyte function and survival, and that is not fully mimicked by experimental endotoxemia.

Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.

The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A.solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0-10.0 and a temperature range of 45-50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na(+), K(+)) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A.solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-β-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His(57), His(130), His(169), and Cys(123)). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing.

Determination of phospholipids in milk using a new phosphodiester stationary phase by liquid chromatography-matrix assisted desorption ionization mass spectrometry

A methodology employing high performance liquid chromatography coupled with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry has been utilized to determine the quality of phospholipid classes. Home-made phosphoester chemically bonded stationary phase containing diol, phosphate and octadecyl groups (Diol-P-C18) has been employed in the separation of polar lipids from milk. Each phospholipid fraction was collected manually and identified by MALDI-TOF MS.

Tobacco Smoke-Induced Alterations in Hepatic Lipid Profiles Demonstrated by Imaging Mass Spectrometry

To account for differences in susceptibility to alcoholic liver disease (ALD), tobacco smoking should be evaluated as a potential cofactor given the very high percentage of heavy drinkers also smoke, and the NNK tobacco-specific nitrosamine was shown to cause steatohepatitis and exacerbate molecular and biochemical effects of alcohol on the liver. Since one of the key factors linked to ALD progression is dysregulated lipid metabolism, we examined effects of cigarette smoke (CS) exposures on hepatic lipid profiles using matrix-assisted laser desorption and ionization imaging mass spectrometry (MALDI-IMS). Adult male A/J mice were exposed to air (8 weeks; A8), CS for 4 (CS4) or 8 (CS8) weeks; or CS8 with 2 weeks recovery (CS8+R). MALDI-IMS demonstrated broad CS-associated reductions in hepatic phospholipids that were partly ameliorated by short-term recovery. Principal component analysis revealed CSassociated shifts in phospholipid profiles that also partly normalized with recovery. Heatmaps demonstrated striking effects of CS with graded responses to exposure duration and recovery. Importantly, several of the CS-induced lipid profile alterations persisted after air recovery, suggesting that the responses had become permanent, whereas others worsened with CS exposure duration and were either sustained or revered with recovery

Analysis of Glycosaminoglycan Oligosaccharides by Combined HPTLC/MALDI-TOF MS: Reduced Silica Gel Thickness Leads to Improved Spectral Qualities and Reduced Side Reactions

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method which can be applied to virtually all natural products including oligosaccharides. Unfortunately, however, the unequivocal identification of a TLC spot is normally difficult. Fortunately, this problem can be minimized when mass spectrometry (MS) such as matrix-assisted laser desorption and ionization time-of-flight is used to identify the TLC spots. This work is dedicated to the TLC/MS analysis of oligosaccharides derived from native chondroitin sulfate and hyaluronan. We will show that the thickness of the silica gel layer (200 versus 100 µm) has a tremendous influence on the quality of the mass spectra: a reduced silica gel thickness enhances the spectral quality and, in particular, improves the achievable signal-to-noise ratio. Additionally, unwanted formylation of the GAG oligosaccharides (which occurs due to the high moiety of formic acid in the mobile phase) can also be minimized if MS-grade HPTLC plates are used.

Conlinin in flaxseed (Linum usitatissimum L.) gum and its contribution to emulsification properties

Flaxseed gum (FG) is a mixture of natural polysaccharide and protein derived from flaxseed (Linum usitatissimum L.) that has potential for thickening foods, stabilizing emulsions, and gelling solutions. The composition and identities of proteins in FG have never been reported. In this study, gum prepared from whole flaxseed was deglycosylated by treating it with trifluoromethanesulfonic acid (TFMS). The resultant proteins were separated by 2D-gel electrophoresis. The major protein spots with estimated molecular weights (MW) of 10–11 kDa and 11–12 kDa were excised, digested with trypsin, and analyzed using matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Peptide MS of tryptic digestion fragments was compared to MS of peptides from gene models available through National Center for Biotechnology Information (NCBI) database. Fragments consistent with the seed storage protein conlinin, the low-molecular-mass 2S storage flaxseed protein, were identified as the major spot constituents. Emulsification properties of FG were determined before and after protease hydrolysis with emulsion activity index (EAI) and emulsion stability (ES) as indicators. Both EAI and ES decreased from 98.7 ± 5.4 to 59.9 ± 3.2 m2/g and from 66.4 ± 1.1 to 42.1 ± 2.0%, respectively, after protease treatment. Conlinin is the major protein associated with FG and it plays a fundamental role in determining FG emulsification properties.

Effects of a Terrified-Sound Stress on Serum Proteomic Profiling in Mice.

The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P < 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.

MALDI-mass spectrometry imaging identifies vitronectin as a common constituent of amyloid deposits.

Amyloids are pathological intra- and extracellular fibrillar aggregates of polypeptides with a cross-β-sheet structure and characteristic tinctorial properties. The amyloid deposits commonly enclose several non-fibrillar components of the extracellular matrix. Their potential to regulate the formation and aggregation process of amyloid fibrils is still poorly understood. For a better understanding of the role of the extracellular matrix in amyloidosis, it is essential to gain deeper insights into the composition of amyloid deposits. Here, we utilized matrix-assisted laser desorption and ionization mass spectrometry imaging to identify extracellular matrix compounds in amyloid deposits. Using this technique, we identified and determined the spatial distribution of vitronectin within AApoAI-, ALλ-, ATTR- and AIns amyloid deposits and, using immunohistochemistry, validated the spatial overlap of vitronectin with amyloids in 175 cases with diverse types of amyloid in several different tissues.

Using Vitek MALDI-TOF mass spectrometry to identify species belonging to the Acinetobacter calcoaceticus–Acinetobacter baumannii complex: a relevant alternative to molecular biology?

Acinetobacter baumannii belongs to the Acinetobacter calcoaceticus–baumannii complex (Acb) containing 2 other pathogenic species: Acinetobacter pittii and Acinetobacter nosocomialis. Identification of these bacteria remains problematic despite the use of matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here, we enriched the SARAMIS™ database of the Vitek MS® plus mass spectrometer to improve the identification of species of the Acb complex. For each species, we incremented reference spectra. Then, a SuperSpectrum was created based on the selection of 40 specific masses. In a second step, we validated reference spectra and SuperSpectra with 100 isolates identified by rpoB gene sequencing. All the isolates were correctly identified by MALDI-TOF MS with the database we created as compared to the identifications obtained by rpoB sequencing. Our database enabled rapid and reliable identification of the pathogen species belonging to the Acb complex. Identification by MALDI-TOF MS with our database is a good alternative to molecular biology.

Proteomic analysis of vestibular schwannoma: conflicting role of apoptosis on the pathophysiology of sporadic vestibular schwannoma.

In this study, we investigated the pathophysiology and mechanism underlying sporadic forms of vestibular schwannoma (VS) by comparing VS tissue with normal nerve tissue using proteomics. Proteomic analysis by two-dimensional electrophoresis and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry facilitates identification and characterization of specific proteins related to the pathogenesis of various diseases. Proteins were extracted from two vestibular nerve specimens and two VS specimens and analyzed in parallel using two-dimensional electrophoresis. We then analyzed 29 spots that were differentially expressed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Upregulated proteins associated with apoptosis were confirmed by Western blot analysis and immunohistochemistry. Twenty-nine proteins showing significant changes in the expression level between VS tissue and normal nerve tissue were identified. Of these, seven proteins were related to apoptosis. Our findings indicate that apoptosis is associated in a complex manner with the pathophysiology of VS. The suppression of apoptosis is presumably involved in tumor occurrence and, conversely, increased apoptotic expression may contribute to the slow tumor growth rate and may be correlated with the Antoni type B area.

Application of matrix-assisted laser desorption and ionization time of flight mass spectrometry to the study of the proteinaceous binders in paint: blue paint composition in the series "The Life of Virgin" by Alonso Cano (17th century) as a case study.

The identification of proteinaceous materials in paint constituents provides very valuable information regarding the techniques used by the painter and the most suitable procedures for conserving and restoring their works. Although the analysis of proteinaceous materials is nowadays a common task when dealing with works of art, the reliable detection and identification of protein traces is still complicated, particularly when very small samples can be taken that may contain a mixture of different organic materials (oils, waxes, resins, gums etc.). We therefore proposed a proteomic approach to investigate protein materials in paintings at trace levels in order to obtain a better understanding of the painter's technique. After trypsin digestion of the paint samples, mass spectra were obtained by matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF-MS) and they were compared with the Mascot database and with theoretical digested proteins. This study contributes to the knowledge about the technique used by Alonso Cano (Granada, Spain, 1601-1667), one of the most original and brilliant artists from the Spanish Golden Age (17th century), in the series called the Life of the Virgin (six paintings), part of the iconographic program about the life of the Virgin Mary, nowadays seen in the main chapel of Granada Cathedral. The objective of the present study was to test the use of proteinaceous material, mainly egg yolk, in the paint used by Cano, as suggested in previous research, although this would have been unusual at that time when most artists used oil paints. Based on the results of the analysis here presented, the use of protein in the binding media can most likely be excluded.

Investigating Interactions between the Lectin-Like Domain of Thrombomodulin and Complement Component 3

Protein-protein interactions are vital to the proper functioning of numerous biological systems. Thrombomodulin (TM) is a protein that is involved in the down-regulation of coagulation induced by the clotting protein thrombin. Complement component 3 (C3) is a vital component of the complement system, which is involved in innate immunity against bacteria and viruses. However, dysregulation of C3 can lead to the degradation of host cells. Evidence suggests that the lectin-like domain of TM (TMD1) may interact with active C3 (C3b) to inactivate it, thus preventing host cell degradation. The research conducted herein required the expression and purification of TMD1 in yeast cells and the isolation and purification of C3 from bovine blood plasma. A protein pull-down assay was used to verify that the two proteins interact. To aid in specific immobilization of the TMD1, two lysine residues were converted to methionine by site-directed mutagenesis via polymerase chain reaction leaving only one free amine available for future reactions. The wild type and mutant TMD1 proteins were characterized by mass spectrometry and urea-induced unfolding. The proteins were also both used in pull-down assays with C3. Once interactions are confirmed, hydrogen/deuterium exchange followed by matrix assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) will be performed to determine which regions of each protein are involved in binding. Additionally, interactions between the two proteins will be characterized using fluorescence resonance energy transfer (FRET) experiments.

Rapid recovery of cyanobacterial pigments in desiccated biological soil crusts following addition of water.

We examined soil surface colour change to green and hydrotaxis following addition of water to biological soil crusts using pigment extraction, hyperspectral imaging, microsensors and 13C labeling experiments coupled to matrix-assisted laser desorption and ionization time of flight-mass spectrometry (MALD-TOF MS). The topsoil colour turned green in less than 5 minutes following water addition. The concentrations of chlorophyll a (Chl a), scytonemin and echinenon rapidly increased in the top <1 mm layer while in the deeper layer, their concentrations remained low. Hyperspectral imaging showed that, in both wet and dehydrated crusts, cyanobacteria formed a layer at a depth of 0.2–0.4 mm and this layer did not move upward after wetting. 13C labeling experiments and MALDI TOF analysis showed that Chl a was already present in the desiccated crusts and de novo synthesis of this molecule started only after 2 days of wetting due to growth of cyanobacteria. Microsensor measurements showed that photosynthetic activity increased concomitantly with the increase of Chl a, and reached a maximum net rate of 92 µmol m−2 h−1 approximately 2 hours after wetting. We conclude that the colour change of soil crusts to green upon water addition was not due to hydrotaxis but rather to the quick recovery and reassembly of pigments. Cyanobacteria in crusts can maintain their photosynthetic apparatus intact even under prolonged periods of desiccation with the ability to resume their photosynthetic activities within minutes after wetting.

The functional proteomics analysis of VEGF-treated human epithelial ovarian cancer cells.

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factor, can impact the tumor cell proliferation and invasion, but the mechanism remains unclear. This study is to investigate the key proteins which may play an important role in the VEGF-induced progress of ovarian cancer cells. The total protein from HO-8910 cells was separated by two-dimensional electrophoresis (2-DE), and differentially expressed proteins were identified by matrix-assisted laser desorption and ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS) and PDQuest image analysis software. Furthermore, real-time PCR, Western blot, and immunocytochemistry were also used to confirm different expression levels of differential proteins. Morphological changes and invasion capability were evaluated by electron microscope and Matrigel invasion assay, respectively. The highly reproducible and well-resolved 2-DE patterns of both HO-8910/VEGF and HO-8910 cells were acquired. A total of 17 expressed differential proteins were identified, 8 proteins were upregulated (ACTB, TIM, PDIA3, PDIA1, DCTN2, KIC17, SIAS, and KIC10) and 9 downregulated (KIC18, GRP78, CAPG, PPIA, ROA2, LMNA, EZRI, ADRM1, and ENOA). Ultrastructure of VEGF-treated group showed more malignant characteristic compared with control group, an obvious increase in the number of cells penetrating the Matrigel membrane in VEGF-treated group (P < 0.05). These results suggested that VEGF could impact ovarian cancer's malignant progression by regulating expression of associated proteins.

Proteomic Analysis of Fruit Bending in Cucumber (Cucumis sativus L.)

In cucumber, fruit shape is an important quality criterion, and fruit bending is known to limit growth, yield, and taste. To investigate the post-transcriptional changes that regulate fruit bending and to better understand the underlying molecular mechanisms, we generated a proteomic profile of the abdomen and back of cucumber bending fruit. Two-dimensional gel electrophoresis (2-DE) allowed the detection of approximately 900 distinct protein spots in each gel, 32 of which were differentially expressed in the abdomen and back of bending cucumber fruit. Ten of the differentially expressed proteins were analyzed using matrix-assisted laser ionization time of flight mass spectrometry (MALDI-TOF/MS). A search of primary databases showed that the identified proteins are involved in various metabolic processes and cellular responses, including photosynthesis metabolism, energy metabolism, defense and stress response, and regulation. The identified proteins included large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase, which are involved in photosynthesis and photorespiratory metabolism, and isocitrate dehydrogenase, which is involved in the tricarboxylic acid cycle. It is possible that imbalances in catabolic and anabolic processes directly affect the bending of cucumber fruit. The predicted function of the cobalamin-independent methionine synthase isozyme is closely related to ethylene biosynthesis; fruit bending may be regulated by ethylene, or by ethylene signaling crosstalk during fruit development. The 14-3-3 protein is usually considered to be a regulation-related protein, which plays a role in regulating cell hyperplasia, cell differentiation during growth, and apoptosis during senescence. Involvement of guanosine triphosphate (GTP)-binding proteins in signal transmission is known to regulate the development of cells in cucumber fruits and to play a role in fruit shape variation. Patterns of protein expression showed high repeatability. We hypothesize that these proteins may play an important role in growth and bending of cucumber fruits. The results of our study provide insight into the genetic mechanism underlying fruit bending in cucumber, and may help to promote cultivation of new varieties with superior fruits.

Serum Proteomic Profiling of Lung Adenocarcinoma with Brain Metastasis Based on Matrix-Assisted Laser Ionization Time of Flight Mass Spectrometry Analysis

Background: Once patients presented with brain metastasis, the prognosis is poor with shortened survival of up to 6 months, and therefore early recognition of brain metastasis may be beneficial to outcomes of patients. The present work focused on serum proteomic biomarkers that represent the status of lung adenocarcinoma especially for patients with brain metastasis. Methods: 100 serum samples including 25

Utilization of matrix‐assisted laser desorption and ionization time‐of‐flight mass spectrometry for identification of infantile seborrheic dermatitis‐causing Malassezia and incidence of culture‐based cutaneous Malassezia microbiota of 1‐month‐old infants

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI-TOF-MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1-month-old infants using this technique. A Malassezia species-specific MALDI-TOF-MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1-month-old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI-TOF-MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI-TOF-MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia.

Identification of lactic acid bacteria in suancai, a traditional Northeastern Chinese fermented food, and salt response of Lactobacillus paracasei LN-1

Suancai is a traditional fermented food that is still popular in northeastern China. Twenty-four bacterial isolates, obtained from 10 samples of naturally fermented suancai broth via screened cultivation, were found to be lactic acid bacteria by physiological and biochemical testing and 16S rDNA-sequence analysis. Among the isolates, 21 rod-shaped strains were classified as Lactobacillus plantarum (eight strains), Lactobacillus sakei (six strains), Lactobacillus curvatus (five strains), or Lactobacillus paracasei (two strains), while three cocci-shaped isolates were identified as Leuconostoc mesenteroides. These lactic acid bacteria are subjected to salt stress during the fermentation of suancai. In the present study, we examined Lactobacillus paracasei LN-1’s display of salt tolerance. To understand the mechanism involved, a proteomics-based, two-dimensional electrophoresis analysis was undertaken to reveal the response of LN-1 during growth in medium with or without NaCl. Out of 23 protein spots that showed differential changes in expression, seven were identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Further analysis showed that chaperone proteins (Hsp 60 and Hsp 70) and a fatty acid biosynthesis enzyme (Fab G) possibly play important roles in the ability of Lactobacillus paracasei LN-1 to resist salt stress.

Influence of Lipid A Acylation Pattern on Membrane Permeability and Innate Immune Stimulation

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.