Liquid Chromatography Coupled With Mass Spectrometry Research Articles

Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

Purification, Fractionation and Characterization of Anthocyanin from in vitro Culture of Bridelia retusa (L.) Spreng.

Bridelia retusa is traditionally used as an astringent and for treating rheumatic pains. Present study revealed that 2,4-dichlorophenoxyacetic acid alone or in combination with kinetin-fortified Murashige and Skoog medium showed good response in terms of callus from leaf explants of B. retusa. Growth hormones, pH, light, and carbon source influenced secondary metabolic pathways. The highest callus induction was 98.9 % with 2.5 mg/l N6-benzyladenine+2 mg/l 2,4-dichlorophenoxyacetic acid. The optimal fresh and dry weights of the calli were 1.9±0.04 and 0.45±0.03 g, respectively. The calli incubated in light on Murashige and Skoog medium with 4 % glucose containing 2.5 mg/l benzyladenine and 2 mg/l 2,4-dichlorophenoxyacetic acid at pH 3.5 yielded 2.8 mg/g of anthocyanins. Murashige and Skoog medium with glucose was optimal compared to sucrose for anthocyanin synthesis. Addition of kinetin inhibited anthocyanin accumulation. Pigmented calli transferred to half strength Murashige and Skoog medium with NH+/NO3 (1:4 ratio), 70 g/l sucrose and supplementation with benzyladenine+2,4-dichlorophenoxyacetic acid produced remarkable biomass production with anthocyanin synthesis when compared with the initial culture conditions. Suspension cultures of Murashige and Skoog medium containing 2.5 mg/l 2,4-dichlorophenoxyacetic acid and 2 mg/l benzyladenine at pH 5.0 induced anthocyanin synthesis into the medium with pH 4.4–4.6. HCl-water and HCl-ethanol extractions for 90 min was attempted to obtain the maximum amount of anthocyanin content. Temperature factors and pH against anthocyanin extraction and stability was analyzed. Enhancement in the degradation rate constant with a corresponding decline in the t1/2 values was seen with the increasing temperature at pH 1 and 4. Fractionation of anthocyanin was carried by high performance liquid chromatography coupled mass spectrometry revealed 09 fractions comprising acylated cyanidins and two peonidins. The major compounds were cyanidin 3-p-coumaroyl and feruloyl diglucoside-5-glucosides. Thus, the calli from leaf explants could be a good source for anthocyanin synthesis.

Metabolomics of Vibrio cholerae.

Metabolomics is an "omics" approach to quantitatively measure a large set of metabolites. In this chapter, we describe an example method for performing liquid chromatography coupled to mass spectrometry (LC-MS)-based untargeted metabolomics on a cell extract from Vibrio cholerae.

High-throughput system metabolomics method reveals new mechanistic insights of chlorogenic acid by using liquid chromatography coupled to high resolution mass spectrometry.

It has increasingly been recognized that metabolism is highly interconnected with disease, and system metabolomics studies have aimed to discover metabolic biomarkers and analyze the pathways of metabolome phenotypes. To better understand the metabolic alteration related with disease, a urine metabolic profile using a high-throughput system metabolomics technology approach was applied to probe the underlying molecular mechanisms of alcohol-induced liver injury and the therapeutic effects of chlorogenic acid (CA). In this study, endogenous low-molecular-weight metabolites were characterized using liquid chromatography coupled with mass spectrometry (LC-MS). The acquired data was parsed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential biomarkers. A total of 19 biomarkers were identified in a model of alcohol-induced liver injury rats, and it was found that chlorogenic acid had a regulatory effect on 14 of them, associated with multiple metabolic pathways. Comprehensive pathway analysis suggests that CA has the ability to regulate abnormal metabolic states. In addition, accessory examinations such as biochemical analysis and histopathological observations were also performed that showed similar results. As a natural product agent against ethanol-induced liver injury, CA was validated in the rebalancing of a wide range of metabolic disorders. High-throughput system metabolomics represents a powerful approach for revealing new mechanistic insights of natural products.

Low levels of serum serotonin and amino acids identified in migraine patients

Migraine is a highly disabling primary headache associated with a high socioeconomic burden and a generally high prevalence. The clinical management of migraine remains a challenge. This study was undertaken to identify potential serum biomarkers of migraine. Using Liquid Chromatography coupled to Mass Spectrometry (LC-MS), the metabolomic profile of migraine was compared with healthy individuals. Principal component analysis (PCA) and Orthogonal partial least squares-discriminant analysis (orthoPLS-DA) showed the metabolomic profile of migraine is distinguishable from controls. Volcano plot analysis identified 10 serum metabolites significantly decreased during migraine. One of these was serotonin, and the other 9 were amino acids. Pathway analysis and enrichment analysis showed tryptophan metabolism (serotonin metabolism), arginine and proline metabolism, and aminoacyl-tRNA biosynthesis are the three most prominently altered pathways in migraine. ROC curve analysis indicated Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine are potential sensitive and specific biomarkers for migraine. Our results show Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine may be as specific or more specific for migraine than serotonin which is the traditional biomarker of migraine. We propose that therapeutic manipulation of these metabolites or metabolic pathways may be helpful in the prevention and treatment of migraine.

Design and validation of an automated solid phase extraction liquid chromatography coupled mass spectrometry method for the quantification of propofol in plasma

Propofol concentration in human plasma can be quantified by liquid chromatography coupled mass spectrometry. Sample preparation usually requires solid phase extraction to remove matrix components and enrich the analyte. To facilitate user-independent measurements and speed extraction, we developed and validated a fully automated high throughput in-line sample preparation system with direct injection into liquid chromatography coupled mass spectrometry. We assessed linearity of each method over the clinically relevant concentration range from 0.5μg/mL to 8μg/mL plasma concentration. R2 values were 0.99 for the automated process and 0.98 for manual sample preparation. The limit of detection was 6ng/mL and the lower limit of quantification was 18ng/mL for the automated method; for the manual process, the limit of detection was 1.58ng/mL and the lower limit of quantification was 4.79ng/mL. Intra-day precision for low, medium and high concentration range of the automated method was validated 4.14%, 9.68% and 3.04% relative standard deviation and 0.29%, 0.12% and 0.52% for the manual process. Carry over was 0.4% with the automated method, whereas there was no carry over with the manual method. Stability of plasma samples was tested with the manual method at concentrations of 1, 4, and 6μg/mL propofol and found to be stable over 150days at −20°C. The manual sample preparation method has successfully been transferred to a fully automated process with appropriate sensitivity and precision but the automatization failed with regard to trueness and working time due to lengthy sample preparation runtime. Therefore it is not suitable for daily use in a hospital laboratory e.g. for brain death diagnosis in the intensive care unit.

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

Mass spectrometry in untargeted liquid chromatography/mass spectrometry metabolomics: Electrospray ionisation parameters and global coverage of the metabolome.

Liquid chromatography coupled to mass spectrometry (LC/MS) is a dominant analytical platform in metabolomics, because of the high sensitivity and resolution, thus enabling large-scale coverage of metabolomes. Correspondingly, electrospray ionisation (ESI) is the favoured ionisation method in untargeted LC/MS metabolomics given the ability to produce large numbers of ions. In the workflow of LC/ESI-MS metabolomics, maximising the ionisation efficiency over a wide mass range is inevitably an essential and determining step, subsequently defining the extent of coverage of the metabolome under investigation. Thus in this study, electronic factors related to the functioning of the ESI source, namely the capillary and sample cone voltages, were explored to investigate the influence on the data acquired in metabolomic investigations. Hydromethanolic samples from an untargeted study (sorghum plants responding dynamically to fungal infection) were analysed on a high-resolution/definition LC/ESI-MS system. Here the capillary and sample cone voltages of the ZSpray™ ESI source were varied between 1.5-3.0kV and 10.0-40.0V, respectively. The acquired data were processed with MarkerLynx™ software and analysed using central composite design response surface methodology and chemometric approaches (principal component analysis and orthogonal projection latent structures-discriminant analysis). The results evidently demonstrate that both capillary and sampling cone voltages not only significantly influence the recorded MS signals with regard to the number and abundance of features, but also the overall structure of the collected data. This consequently impacts on the information extracted from the data and thus affects coverage of the metabolome. The observations postulate in that, untargeted LC/MS metabolomics, 'what you see is what you ionise'. Although there is convergence of collected data under different ESI conditions, the nuances observed indicate that the exploration of different ion source settings could be the best trade-off in expanding and maximising the metabolome coverage in untargeted metabolomic experiments.

Differential lipidome remodeling during postharvest of peach varieties with different susceptibility to chilling injury.

Peaches ripen and deteriorate rapidly at room temperature. Therefore, refrigeration is used to slow these processes and to extend fruit market life; however, many fruits develop chilling injury (CI) during storage at low temperature. Given that cell membranes are likely sites of the primary effects of chilling, the lipidome of six peach varieties with different susceptibility to CI was analyzed under different postharvest conditions. By using liquid chromatography coupled to mass spectrometry (LC-MS), 59 lipid species were detected, including diacyl- and triacylglycerides. The decreases in fruit firmness during postharvest ripening were accompanied by changes in the relative amount of several plastidic glycerolipid and triacylglyceride species, which may indicate their use as fuels prior to fruit senescence. In addition, levels of galactolipids were also modified in fruits stored at 0°C for short and long periods, reflecting the stabilization of plastidic membranes at low temperature. When comparing susceptible and resistant varieties, the relative abundance of certain species of the lipid classes phosphatidylethanolamine, phosphatidylcholine and digalactosyldiacylglycerol correlated with the tolerance to CI, reflecting the importance of the plasma membrane in the development of CI symptoms and allowing the identification of possible lipid markers for chilling resistance. Finally, transcriptional analysis of genes involved in galactolipid metabolism revealed candidate genes responsible for the observed changes after cold exposure. When taken together, our results highlight the importance of plastids in the postharvest physiology of fruits and provide evidence that lipid composition and metabolism have a profound influence on the cold response.

Mass Spectrometric Approaches for the Analysis of Phytosterols in Biological Samples.

Plant sterols (phytosterols) are important structural components of plant cellular membranes, and they play a major role during development and metabolism. They have health-associated benefits, especially in lowering blood cholesterol levels. Because of their many health claims, there is a growing interest in their analysis. Although various analytical strategies have been employed in analyzing phytosterols, chromatography linked to mass spectrometry (MS) is superior due to its sensitivity. Furthermore, specificity and selectivity are enhanced by utilizing tandem mass spectrometry (MS/MS). This article reviews the various mass spectrometric strategies used for the analysis of phytosterols. It highlights the applications and limitations associated with each MS strategy in various sample matrixes such as plant, human, animal, food, and dietary supplements. GC-MS was historically the method of choice for analysis; however, the derivatization step rendered it tedious and time-consuming. On the other hand, liquid chromatography coupled to MS (LC-MS) simplifies the analysis. Many ionization techniques have been used, namely, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). APCI showed superiority in terms of ion intensity and consistency in ion formation, primarily forming [M + H - H2O]+ ions rather than [M + H]+. In addition, matrix assisted laser desorption ionization (MALDI) as well as ambient mass spectrometry such as direct analysis in real time (DART) have also been evaluated.

Comprehensive Lipidome-Wide Profiling Reveals Dynamic Changes of Tea Lipids during Manufacturing Process of Black Tea

As important biomolecules in Camellia sinensis L., lipids undergo substantial changes during black tea manufacture, which is considered to contribute to tea sensory quality. However, limited by analytical capacity, detailed lipid composition and its dynamic changes during black tea manufacture remain unclear. Herein, we performed tea lipidome profiling using high resolution liquid chromatography coupled to mass spectrometry (LC-MS), which allows simultaneous and robust analysis of 192 individual lipid species in black tea, covering 17 (sub)classes. Furthermore, dynamic changes of tea lipids during black tea manufacture were investigated. Significant alterations of lipid pattern were revealed, involved with chlorophyll degradation, metabolic pathways of glycoglycerolipids, and other extraplastidial membrane lipids. To our knowledge, this report presented most comprehensive coverage of lipid species in black tea. This study provides a global and in-depth metabolic map of tea lipidome during black tea manufacture.

Improved liquid chromatographic method for quality control of spiramycin using superficially porous particles

This article describes the development and validation of a liquid chromatographic method for spiramycin using a column with superficially porous particles. Gradient elution was applied and the mobile phase consisted of phosphate buffer (0.2M; pH 8.3) – H2O – acetonitrile in a ratio 10:60:30 (v/v/v) for mobile phase A and 10:30:60 (v/v/v) for mobile phase B. UV detection was performed at 232nm. Compared to previous methods, the analysis time was about two times faster and impurities were better separated. Furthermore, impurities which were present above 0.25% were characterized using liquid chromatography coupled with mass spectrometry (LC/MS).

Isolation of berberine tannate using a chromatography activity-guided fractionation from root bark of Berberis hispanica Boiss. & Reut.

ABSTRACTRoot bark of Berberis hispanica Boiss. & Reut. constitutes an important source of alkaloids and is traditionally used in Algeria and Morocco in many herbal formulations particularly for the treatment of stomach infections and colon cancer. Activity-guided fractionation of Berberis extract is developed by step-gradient elution on column chromatography followed by preparative HPLC to isolate the most antistaphylococcal compound. Solvent extraction-activity screening indicates that ethanol should be used as the extracting solvent as it has exhibited the highest activity against Staphylococcus aureus(Rosenbach ATCC6538) followed by water extract, whereas no activity is recorded for acetone and hexane extracts. Screening of collected fractions from chromatographic separations indicates that the fraction presenting the highest antistaphylococcal activity is identified as berberine tannate by UV spectrum, melting point measurement and confirmed by liquid chromatography coupled to mass spectrometry (LC/MS). The minimal inhibition concentration value of berberine tannate against S. aureus is evaluated at 5 µg mL−1.

Fast pure ion chromatograms extraction method for LC-MS

Liquid chromatography coupled with mass spectrometry (LC-MS) has shown great potential in analysis complex samples. However, informative feature extraction is still challenge since the electrospray ionization in LC-MS tends to produce ninety percent or more ions not originated from compounds of interest. The concept of pure ion chromatogram (PIC) is effective to extract informative ions, but tradition PIC methods are time-consuming because of their theories and programming languages. In this study, we present a novel method, called Fast Pure Ions Chromatograms (FPIC), for extracting PICs from raw LC-MS dataset effectively and quickly. This method can search ion of PIC from its maximum bi-directionally and adaptively, which can improve the stability and reduce the computation time drastically. A further speedup has been achieved by exploiting modern software engineering techniques. FPIC was validated by analyzing four LC-MS datasets: MM14 and MM48, simulated MM48 and quantification (MTBLS234) datasets. Results show that FPIC outperformed traditional methods in the recall, precision and F-score, and it has good reliability of quantification. Furthermore, the method is very fast with few adjustable parameters, which leads to an approximately 125-fold speedup over PITracer and 18-fold speedup over XCMS. An open source implementation of the FPIC method is available at https://github.com/zmzhang/pymass.

Enhancing the Mass Spectrometry Sensitivity for Oligonucleotide Detection by Organic Vapor Assisted Electrospray.

There are two challenges in oligonucleotide detection by liquid chromatography coupled with mass spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber, and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as an ion-pair reagent. The MS signal intensity was improved 600-fold (for standard oligonucleotide dT15) by the isopropanol vapor assisted electrospray, and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS with few adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced more than 10-fold on average compared with adding acids into the electrospray solution. Finally, oligonucleotides and peptides or histones were simultaneously detected in MS with little interference with each other. Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.

Comparative Electrochemical, Photoelectrochemcal and Photocatalytic Abatement of Emerging Pollutant

Drug residues are unregulated emerging pollutants and are frequently found in the aquatic environment. Due to their extensive use, presence in the aquatic environment and their potential for impacts on wildlife and humans, it is becoming an important environmental issue. The present study evaluates the photo-assisted electrochemical treatment of sulfamethoxazole (SMX), an antibiotic used in the prevention and treatment of human and animal diseases. The experiments were carried out in a photo illuminated flow cell reactor and the effects of current density (10-60 mA cm-1) and concentration of sodium chloride in the supporting electrolyte were determined. The Drug could be removed completely at all current densities while TOC abatement was appreciable during the process. The process followed pseudo-first order kinetics and values of electrical energy-per-order were dependent on current density and sodium chloride concentration. Different reaction intermediates were separated and identified by liquid chromatography coupled with mass spectrometry (LC-MS), thereby providing an insight into the mechanistic details of the degradation pathway. The photo-assisted electrochemical degradation proceeded through competitive reactions such as chlorination, hydroxylation and substitution on the aromatic ring, while further ring opening gave rise to more simple organic compounds and, ultimately, the mineralization of SMX. The possible degradation route were also proposed and discussed. . The efficiency of photoelectrochemical process was compared with photo-catalytic and electrochemical separately, and showed a synergism for TOC removal.

Modulation of Muller cell membrane organization by 24S‐hydroxycholesterol

PurposeMuller glial cells (MGC) are involved in the retinal homeostasis and they are sustaining cholesterol homeostasis. A lack or an excess of cholesterol in neurons can cause neuro‐degeneration. Cholesterol is eliminated from the retina mainly as 24S‐hydroxycholesterol (24S‐OHC). 24S‐OHC is produced by 24S‐hydroxylase (CYP46A1) in retinal ganglion cells (RGC) and is overexpressed during glaucoma. the objective of this study was to determine whether 24S‐OHC triggers MGC membrane dynamics involving lipid rafts and contribute to gliosis at early and late time points.MethodsMGCs were grown in vitro from retinas of young rats. They were treated with 24S‐OHC (10 μM) for 2 min or 6h. Lipid rafts of MGC membranes were obtained after 1% lubrol lysis and 20h‐ultracentrifugation at 180,000 g in a sucrose gradient. The expression of caveolin‐1, Flotillin‐1, GFAP, Vimentin, Connexin‐30, Connexin‐43, phosphorylated and non phosphorylated p38 and p42‐44 MAPK was analyzed by Western‐blotting. High‐performance liquid chromatography coupled with mass spectrometry (LC‐MS) was used to estimate the levels of ganglioside GM3 (monosialodihexosylganglioside), and protein pathways were analyzed by nanoLC‐MS/MS in raft and non raft fractions from MGCs after treatment with 24S‐HOC.ResultsCholesterol, sphingomyelin and saturated fatty acids C15:0, C16:0, C17:0 and C18:0 were enriched in the rafts fractions in MGCs. Structural proteins, Caveolin‐1 and flotillin‐1, and functional proteins, Connexin‐30 and ‐43, were localized in the MGCs rafts. Ganglioside GM3 showed a characteristic enrichment in the raft fractions. Protein implicated in adhesion or oxidative stress pathways in the raft and non‐raft fractions were up and down‐regulated by treatment with 24S‐OHC.ConclusionsOur data showed that 24S‐OHC induced early changes in protein distribution in raft microdomains.

Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis.

This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation.

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC–MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure–property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response.

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p<0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p<0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.