Trihydroxyacetophenone Research Articles

Identification of 2',4',6'-trihydroxyacetophenone as promising cysteine conjugate beta-lyase inhibitor for preventing cisplatin-induced nephrotoxicity.

Cisplatin is a chemotherapeutic agent used to treat many types of malignant tumors. However, irrespective of its potent anticancer properties and efficacy, nephrotoxicity is the dose-limiting factor of cisplatin treatment. Cisplatin infiltrates renal tubular cells in the kidneys and is metabolized by cysteine conjugate-beta lyase 1 (CCBL1) to form highly reactive thiol-cisplatin; this may mediate cisplatin's nephrotoxicity. Therefore, CCBL1 inhibition may prevent cisplatin-induced nephrotoxicity. Using a high-throughput screening assay, we identified 2',4',6'-trihydroxyacetophenone (THA) as an inhibitor of CCBL1. THA inhibited human CCBL1 beta-elimination activity in a concentration-dependent manner. We further investigated the preventive effect of THA on cisplatin-induced nephrotoxicity. THA attenuated the effect of cisplatin on the viability of confluent renal tubular cells (LLC-PK1 cells) but had no effect on cisplatin-induced reduction of proliferation in the tumor cell lines (LLC and MDA-MB-231). THA pre-treatment significantly attenuated cisplatin-induced increases in blood urea nitrogen, creatinine, cell damage score, and apoptosis of renal tubular cells in mice in a dose-dependent manner. Furthermore, THA pre-treatment attenuated cisplatin-induced nephrotoxicity without compromising its anti-tumor activities in mice bearing subcutaneous syngeneic LLC tumors. THA could help prevent cisplatin-induced nephrotoxicity and may provide a new strategy for cisplatin-inclusive cancer treatments.

AKTIVITAS ANTIKANKER SENYAWA ASILFLOROGLUSINOL DARI BIJI Melicope incana TERHADAP SEL KANKER PAYUDARA MCF-7

Melicope incana is one of the plant species that belongs to family Rutaceae. Melicope produces phenolic compounds, including alkaloids, acylphloroglucinol, flavonoids, and coumarins. This study aims to isolate the acyl phloroglucinol derivatives found in M. incana seeds. Extraction of acylphloroglucinol derivatives with methanol using the maceration method at room temperature. Separation and purification using a combination of gravity column chromatography, and radial chromatography. Two acylphloroglucinol compounds have been isolated, namely 3'-geranyl-2',4',6'-trihydroxy acetophenone ( 1 ) and 3'-isoprenyl-2',4',6'-trihydroxy acetophenone ( 2 ). The structure of the two compounds was determined based on UV, IR, 1D, and 2D NMR spectroscopic analysis. Anticancer activity of compounds 1-2 against breast cancer cells MCF-7 showed IC 50 values of 48.98 and 4.30 µg /mL, respectively.

Lithium Hydroxide Hydrolysis Combined with MALDI TOF Mass Spectrometry for Rapid Sphingolipid Detection.

Sphingolipids have diverse structural and bioactive functions that play important roles in many key biological processes. Factors such as low relative abundance, varied structures, and a dynamic concentration range provide a difficult analytical challenge for sphingolipid detection. To further improve mass-spectrometry-based sphingolipid analysis, lithium adduct consolidation was implemented to decrease spectral complexity and combine signal intensities, leading to increased specificity and sensitivity. We report the use of lithium hydroxide as a base in a routine hydrolysis procedure in order to effectively remove common ionization suppressants (such as glycolipids and glycerophospholipids) and introduce a source of lithium into the sample. In conjunction, an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone (THAP) is used to facilitate lithium adduct consolidation during the MALDI process. The result is a robust and high-throughput sphingolipid detection scheme, particularly of low-abundance ceramides. Application of our developed workflow includes the detection of differentially expressed liver sphingolipid profiles from a high-fat-induced obesity mouse model. We also demonstrate the method's effectiveness in detecting various sphingolipids in brain and plasma matrices. These results were corroborated with data from UHPLC HR MS/MS and MALDI FT-ICR, verifying the efficacy of the method application. Overall, we demonstrate a high-throughput workflow for sphingolipid analysis in various biological matrices by the use of MALDI TOF and lithium adduct consolidation.

Development and Validation of an LC-MS/MS Assay to Quantitate 2',4',6'-Trihydroxyacetophenone in Rat and Dog Plasma and its Application to a Pharmacokinetic Study.

In the present study, a simple, rapid, and reliable bioanalytical method was developed using liquid chromatography with tandem-mass spectrometry (LC-MS/MS) to quantify 2′,4′,6′-trihydroxyacetophenone (THAP) in rat and dog plasma with 2′,4′,6′-trihydroxybenzaldehyde as an internal standard (IS). The LC-MS/MS instrument was operated in the multiple reaction monitoring (MRM) mode to detect THAP at m/z transition 166.89 > 82.8 and IS at 152.89 > 82.8, respectively. A simple, one-step protein precipitation (PP) method was employed with acetonitrile for sample preparation. Utilizing a Gemini C18 column, THAP and IS were separated with an isocratic mobile phase consisting of 10 mM ammonium acetate and methanol (10:90, v/v) at a flow rate of 0.2 mL/min. Total chromatographic run time was 2.5 min per sample injection. The standard calibration curve for THAP was linear (r2 ≥ 0.9987) over the concentration range of 0.1 to 100 µg/mL with the lower limit of quantitation (LLOQ) of 0.1 µg/mL (S/N ratio > 10). According to the regulatory guidelines from the U.S. Food and Drug Administration (FDA) and the Korea Ministry of Food and Drug Safety (MFDS), our newly developed biomedical analytical method was fully and adequately validated in terms of selectivity, sensitivity, linearity, intra- and inter-day precision and accuracy, recovery, matrix effect, stability, and dilution integrity. Our validated assay was successfully utilized in a nonclinical pharmacokinetic study of THAP in rats and dogs.

Phloretin cytoprotection and toxicity

Phloretin (Phl) is a dihydrochalcone flavonoid with significant cytoprotective properties; e.g., free radical trapping, electrophile scavenging. Based on this, it has been suggested that Phl might be useful in the treatment of pathogenic processes and prevention of drug toxicities. Therefore, we determined the ability of Phl to provide route- and dose-dependent hepatoprotection in a mouse model of acetaminophen (APAP) overdose. Intraperitoneal (i.p.) administration of Phl produced a bimodal effect; i.e., the highest dose (2.40 mmol/kg) did not prevent APAP-induced lethality, whereas lower doses (0.2–0.4 mmol/kg) afforded modest hepatoprotection. When given alone, the highest i.p. Phl dose was lethal within 24 h, whereas the lower doses were not toxic. Oral Phl (0.40–2.40 mmol/kg) did not prevent APAP-induced hepatotoxicity. The highest oral dose given alone (2.4 mmol/kg) produced 64% lethality, whereas lower doses were not lethal. This toxicity profile was reflected in a study using APAP-exposed isolated mouse hepatocytes, which showed that the Phl pharmacophores, 1,3,5-trihydroxyacetophenone (PG) and 2′,4′,6′-trihydroxyacetophenone (THA) where protective. Corroborative cell free studies showed that polyphenol protectants prevented glutathione loss mediated by the APAP metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Thus, in spite of possessing cytoprotective attributes, Phl was generally toxic in our APAP models. These and earlier findings suggest that Phl is not a candidate for drug design. In contrast, we have found that the enol-forming pharmacophores, THA and PG, are potential platforms for pharmacotherapeutic development.

Structure analysis of geopolymers synthesized from clay originated from Serbia

The paper presents chemical and structural analysis of geopolymer materials which are obtained by alkali-activated calcined clay (metakaolin) originated from Serbia under strictly defined conditions. Characterization of the metakaolin and geopolymers molecular structure has been done using X-ray diffraction, scanning electron microscope, Fourier transform infrared spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The paper presents the possibility of obtaining geopolymer structure and differences in chemical and structural characterization of these materials taking into account the concentration of NaOH as a variable parameter. The results of MALDI analysis of metakaolin and synthesized geopolymer structures using various matrix system: 2,4,6 trihydroxyacetophenone (THAP), α-cyano-4-hydroxycinnamic, 2,6 dihydroxyacetophenone and laser desorption/ionization, have shown that THAP matrix is the most appropriate for analysing these aluminosilicate materials.

Enolate-Forming Phloretin Pharmacophores: Hepatoprotection in an Experimental Model of Drug-Induced Toxicity.

Drug-induced toxicity is often mediated by electrophilic metabolites, such as bioactivation of acetaminophen (APAP) to N-acetyl-p-benzoquinone imine (NAPQI). We have shown that APAP hepatotoxicity can be prevented by 2-acetylcyclopentanone (2-ACP). This 1,3-dicarbonyl compound ionizes to form an enolate nucleophile that scavenges NAPQI and other electrophilic intermediates. In this study, we expanded our investigation of enolate-forming compounds to include analyses of the phloretin pharmacophores, 2',4',6'-trihydroxyacetophenone (THA) and phloroglucinol (PG). Studies in a mouse model of APAP overdose showed that THA provided hepatoprotection when given either by intraperitoneal injection or oral administration, whereas PG was hepatoprotective only when given intraperitoneally. Corroborative research characterized the molecular pharmacology (efficacy, potency) of 2-ACP, THA, and PG in APAP-exposed isolated mouse hepatocytes. For comparative purposes, N-acetylcysteine (NAC) cytoprotection was also evaluated. Measurements of multiple cell parameters (e.g., cell viability, mitochondrial membrane depolarization) indicated that THA and, to a lesser extent, PG provided concentration-dependent protection against APAP toxicity, which exceeded that of 2-ACP or NAC. The enolate-forming compounds and NAC truncated ongoing APAP exposure and thereby returned intoxicated hepatocytes toward normal viability. The superior ability of THA to protect is related to multifaceted modes of action that include metal ion chelation, free radical trapping, and scavenging of NAPQI and other soft electrophiles involved in oxidative stress. The rank order of potency for the tested cytoprotectants was consistent with that determined in a parallel mouse model. These data suggest that THA or a derivative might be useful in treating drug-induced toxicities and other conditions that involve electrophile-mediated pathogenesis.

Direct analysis of lipids from blood using matrix-assisted laser desorption ionization (MALDI) mass spectrometry

Introduction: Lipids are essential biological pathways involved in metabolism, signalling and energy storing. However, abnormal lipid levels, such as cholesterol, play a crucial role in heart diseases and type 2 diabetes mellitus. Knowing cholesterol levels is therefore essential in monitoring the metabolic functions of the individual and preventing chronic and lethal diseases. Objectives: To evaluate the potential of matrixassisted laser desorption ionization (MALDI) mass spectrometry as a fast and reliable method for the direct screening of patients. Materials and methods: An aliquot consisting of 15– 20μl of human blood was collected from the finger. Lipids were extracted from blood either by direct extraction or after a preliminary centrifugation and plasma separation. An Axima Performance MALDI-ToF-ToF (Shimadzu, Manchester, UK) equipped with a 337nm nitrogen laser was used in the analysis. Measurements were performed in reflectron positive mode using an acceleration voltage of 20kV. Red phosphorus was used as the calibrant. Results: A method for direct lipid blood analysis was developed and described as follows. A volume of 10μl of blood was mixed 1:1 v/v with a solution of CH3 OH/ CHCl3 (1:1 v/v). The solution was vortexed and left to separate for 10 minutes, resulting in 6μ l of recoverable solution. After we obtained the blood and made the solution, 10μl of blood was centrifuged for 4 minutes (30k relative centrifugal force) and 4μl of plasma recovered. The plasma was then diluted 1:2 v/v with the CH3 OH-CHCl3 solution. Stirring and extraction were carried out as above and 4μl from the organic phase was collected and subjected to MALDI analysis. THAP (2′ ,4′ ,6′ -Trihydroxyacetophenone) (10mg/ml) was dissolved in 7:3 v/v CH3 OH/H2 O (50mMammonium citrate) and used as the matrix. Samples were mixed 1:1 v/v with the matrix solution and 1μl spotted on the MALDI target. Conclusions: By using only 10μ l of blood, it was possible to obtain a fingerprint of plasma lipids for diagnostic assessment.

Enhanced Visualization of Small Peptides Absorbed in Rat Small Intestine by Phytic-Acid-Aided Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry

Enhanced visualization of small peptides absorbed through a rat intestinal membrane was achieved by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-IMS) with the aid of phytic acid as a matrix additive. Penetrants through intestinal peptide transporter 1, i.e., glycyl-sarcosine (Gly-Sar, 147.1 m/z) and antihypertensive dipeptide, Val-Tyr (281.2 m/z), were chosen for MALDI-IMS. The signal-to-noise (S/N) ratios of dipeptides Gly-Sar and Val-Tyr were seen to increase by 2.4- and 8.0-fold, respectively, when using a 2',4',6'-trihydroxyacetophenone (THAP) matrix containing 5.0 mM phytic acid, instead of the THAP matrix alone. Owing to the phytic-acid-aided MALDI-IMS method, Gly-Sar and Val-Tyr absorbed in the rat intestinal membrane were successfully visualized. The proposed imaging method also provided useful information on intestinal peptide absorption; to some extent, Val-Tyr was rapidly hydrolyzed to Tyr by peptidases located at the intestinal microvillus during the absorption process. In conclusion, the strongly acidic additive, phytic acid, is beneficial for enhancing the visualization of small peptides using MALDI-IMS, owing to the suppression of ionization-interfering salts in the tissue.

Novel Ionic Matrices for Enhanced Ionization of Oligosaccharides/Glycopeptides during MALDI-MS Analysis

本研究发展了四种基于三羟基苯乙酮（THAP）的新型离子液体基质,即2′,3′,4′-THAP/二甲基苯胺（DMA）、2′,4′,6′-THAP/DMA、2′,3′,4′-THAP/吡啶（Py）及2′,4′,6′-THAP/Py,用于提高寡糖/糖肽在MALDI-TOF MS中的离子化效率.与传统的固体基质2,5-二羟基苯甲酸（DHB）和2′,4′,6′-三羟基苯乙酮（2′,4′,6′-THAP）相比,新型离子液体体系分析不同类型的寡糖链,均可获得更高的检测灵敏度.可使葡聚糖（dextran 1000）和环状寡聚糖β-环糊精在MALDI质谱中的信噪比提高10倍以上,RNase B的复杂寡糖链也实现了高灵敏度的检测.在糖肽分析中,2′,3′,4′-THAP/DMA离子液体高灵敏度地检测到辣根过氧化酶的7条糖肽,而2′,3′,4′-THAP作为基质时却无法检测到任何信号.

Sublimation of New Matrix Candidates for High Spatial Resolution Imaging Mass Spectrometry of Lipids: Enhanced Information in Both Positive and Negative Polarities after 1,5-Diaminonapthalene Deposition

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Rapid screening of anthocyanins in berry samples by surfactant‐mediated matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been used for the identification of flavonoids from three berry extracts: lowbush blueberry (Vaccinium angustifolium), lingonberry (Vaccinium vitis-idaea), and blackberry (Rubus armeniacus). The addition of the surfactant led to suppression of matrix ions from both alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2',4',6'-trihydroxyacetophenone (THAP). This is the first case of this method being successfully employed with a matrix other than CHCA. It was observed that CHCA led to a great deal of fragmentation of the sugar moiety from glycosides, whereas THAP produced more intact glycoside molecules, and thus leads to better characterization of the flavonoids in a berry sample. The flavonoids were characterized and quantified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with UV detection. Although MALDI-TOF-MS did not lead to the identification of as many flavonoids, it did enable us to identify many anthocyanin glycosides. Quantification was achieved and demonstrated that use of the THAP matrix can enable quantification of the intact glycosides with relative standard deviation (RSD) values of less than 10% with surfactant addition. These results are comparable with LC results. MALDI-TOF-MS with THAP matrix thus provided a rapid method for the qualitative screening of these compounds. It took only a few minutes, greatly reducing the analysis time from that in traditional LC/MS methods.

Matrix‐assisted laser desorption/ionization mass spectrometry of polysaccharides with 2′,4′,6′‐trihydroxyacetophenone as matrix

So far, there have been only a few matrices reported for detection of polysaccharides with molecular weight higher than 3000 Daltons by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, we found that 2',4',6'-trihydroxyacetophenone (THAP) is a good matrix for MALDI time-of-flight MS analysis of polysaccharides with broad mass range. Large polysaccharides, dextrans, glycoproteins and polysialic acids have been successfully detected by MALDI-MS with THAP as matrix.

Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.

Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto non-characterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; these were (1) [15.5K-61 K-U4 atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro, and (2) U1 snRNP particles purified from HeLa cells. We show that the use of 2',4',6'-trihydroxyacetophenone (THAP) as MALDI matrix allows analysis of cross-linked peptide-RNA oligonucleotides in the reflectron mode at high resolution, enabling sufficient accuracy to assign unambiguously cross-linked RNA sequences. Most important, post-source decay (PSD) analysis under these conditions was successfully applied to obtain sequence information about the cross-linked peptide and RNA moieties within a single spectrum, including the identification of the actual cross-linking site. Thus, in U4 atac snRNA we identified His 270 in the spliceosomal U4/U6 snRNP-specific protein 61 K (hPrp31p) cross-linked to U 44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U 30 of U1 snRNA. This type of analysis is applicable to any type of RNP complex and may be expected to pave the way for the further analysis of protein-RNA complexes in much lower abundance and/or of cross-links that are obtained in low yield.

Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry.

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.

Characterization of Poly(amidoamine) Dendrimers and Their Complexes with Cu2+ by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers of generation 2−4 (Gn-OH, n = 2, 3, and 4; Mr ∼ 3.7, 6.9, and 14.3 kDa, respectively) and dendrimers encapsulating Cu2+ ions have been investigated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Umbelliferone (7-hydroxycoumarin) and 2‘,4‘,6‘-trihydroxyacetophenone (THAP) were used as the matrices for dendrimers with and without Cu2+ in the interior. The MALDI-TOF results provide accurate molecular weight determinations of all dendrimers and some multiple copper complexes of G2-OH and G3-OH (accuracy <0.01%). More importantly, this approach provides direct characterization of multiple-cation adducts and quantitative evaluation of the complexing capacity of dendrimers hosting transition-metal ions. The results are used to determine dendrimer structure and evaluate polydispersity.

MALDI-TOF MS analysis of isoflavones in soy products.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique having a number of advantages for food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify isoflavones in soy samples. 2',4',6'-Trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHB) were both good matrices for isoflavones, but DHB was chosen as the best because it worked well for sample extracts, with good spot-to-spot repeatability. Isoflavones were predominantly ionized in a protonated form with a very small amount of sodium or potassium adduct ions. Fragmentation occurred only through loss of glycosidic residues. Daidzin showed more than twice the response of genistin using MALDI-TOF MS. A simple solid phase extraction of isoflavones from soy samples was developed for MALDI-TOF MS analysis. MALDI-TOF MS can provide an isoflavone profile in 2 min and serves as a powerful tool to identify and study processing changes of isoflavones in soy products.

Analysis of nucleotides directly from TLC plates using MALDI-MS detection

The methodology for the detection of picogram quantities of nucleotides directly from TLC plates without the use of radioactive labeling has been developed. The method couples thin-layer chromatography (TLC) separation with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) detection. The TLC/MALDI coupling protocol was studied and optimized for the separation and detection of deoxyribonucleotides. Several ammonia based solvents were examined as potential extraction solvents for the TLC/MALDI coupling protocol. It was found that in order to obtain maximum MALDI signal intensity and minimal analyte spreading, the extraction solvent should produce Rf-values for the analytes in the range of 0.3–0.4. Rf-values above this range led to extensive analyte spreading and those below this range resulted in poor extraction. Various MALDI matrices and co-matrices were investigated, the best results were obtained using 2′,4′,6′-trihydroxyacetophenone (THA) as a matrix. The extraction solvent chosen was an ammonium hydroxide/methanol (100 mM/30%, Rf = 0.28–0.38) solvent system which was found to provide the best sensitivity, minimal lateral spreading and excellent matrix transfer. Using the optimized coupling protocol, the detection limits for the deoxyribonucleotide monophosphates were established at or better than 10 picograms.

Matrix influence on the formation of positively charged oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry

The ionization efficiency of various ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) matrices was investigated. A site of fixed positive charge was generated on an oligonucleotide by addition of a quaternary ammonium. This quaternary ammonium-tagged oligonucleotide was then used as an internal standard to probe the relative ionization capabilities of 3-hydroxypicolinic acid (3-HPA), 2′,4′,6′-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) in positive-ion mode. MALDI-MS analysis of equimolar mixtures of the quaternary ammonium-tagged oligonucleotide and an unmodified polythymidylic acid, dT 12, found that 3-HPA yielded more abundant protonated dT 12 molecular ions than either THAP or DHBA. These results demonstrate that the low ion yields previously reported for polythymidylic acid are due to the matrix utilized and are not due to the low proton affinity of thymidine. Primary, secondary and tertiary amines were also incorporated into dT 12 to examine the effect of these different amines on the protonation efficiency of the three matrices under investigation. Similar results were obtained, regardless of the amine-tag utilized, with protonation efficiency following the trend 3-HPA > THAP > DHBA. Consideration of the various factors that might influence the overall production of positively charged polythymidylic acid finds that it is the matrix:phosphodiester backbone interaction that might play the important role in determining the optimal MALDI-MS response. These results are a step towards understanding the matrix properties necessary for optimal production of oligonucleotide molecular ions in MALDI-MS.

Effects of sample preparation on matrix-assisted laser desorption/ionization time-of-flight mass spectra for sodium polystyrene sulfonate

Studies have been made on the influence of matrix concentration and the selection of matrix-solvent combinations for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Sodium polystyrene sulfonate (SPS) with five different degrees of polymerization, Mw = 1640, 4950, 7950, 16 600 and 57 500, were studied. The matrix 2′,3′,4′-trihydroxyacetophenone (THAP) was selected and was combined with acetonitrile, methanol, 2-propanol or water as the solvent. The MALDI-TOF experiments in negative ion mode reveal the following observations: (1) The concentration of the matrix must be increased as the degree of the polymerization increases; (2) depending on the degree of polymerization, there are different optimum combinations between the matrix and the polymer samples, and (3) different polarity of the solvents used in the matrix (THAP) preparation result in different MALDI spectra. Copyright © 1999 John Wiley & Sons, Ltd.