LC-MS Assay Research Articles

2D-LC-MS/MS to measure cleaved high-molecular-weight kininogen in human plasma as a biomarker for C1-INH-HAE.

C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. A novel ion-pair 2D LC-MS/MS assay was developed to measure the 46kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction. The LLOQ was 200ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%. A reagent-free LC-MS assay has been developed for the quantitation of 46kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.

A validated high-resolution accurate mass LC-MS assay for quantitative determination of metoprolol and α-hydroxymetoprolol in human serum for application in pharmacokinetics

To determine metoprolol and its metabolite α-hydroxymetoprolol in human serum we validated a method on an LC system with an Exactive® Orbitrap mass spectrometer (Thermo Scientific) as detector and isotope-labelled metoprolol-d7 as internal standard. A simple sample preparation was used with water-acetonitrile (15:85, v/v) as precipitation reagent. This method has a chromatographic run time of 15 min and linear calibration curves in the range of 5.0-250 µg/L for both metoprolol and α-hydroxymetoprolol. Validation showed the method to be accurate, with a good precision, selective and with a lower limit of quantitation of 2.0 µg/L for metoprolol and 1.0 µg/L for α-hydroxymetoprolol, respectively. This validated LC-Orbitrap MS analysis for metoprolol and α-hydroxymetoprolol can be used for application in human pharmacokinetics.

An LC-MS Assay with Isocratic Separation and On-line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma.

SummaryBackgroundBusulfan (Bu) requires therapeutic drug monitoring (TDM) in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT). To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE) of samples.MethodsA protein precipitation (PP) step was performed before the on-line SPE of Bu from 200 µL of plasma spiked with octa-deuterated Bu (D8-Bu) as the internal standard. Bias was assessed with respect to our routine LC-MS/MS Bu assay with off-line extraction using the Passing-Bablok robust regression. Root cause of bias for individual samples was assessed by analyzing the regression residuals.ResultsThe method was linear in the range 37.75–2,416 ng/mL (r2>0.999), with 19.74 ng/mL LLOQ and 10.5% CV at 20 ng/mL. Precision and accuracy were both within ±5%, and neither appreciable matrix nor carryover effects were observed. The Passing-Bablok regression analysis returned a 0.99 slope (95% CI: 0.97 to 1.01) and –6.82 intercept (95% CI: –15.23 to 3.53). Residuals analysis against the 2.5th–97.5th percentiles range showed four samples with significant bias individually.ConclusionsThe method presented can be successfully employed for the routine analysis of Bu in plasmatic samples, and can replace the LC-MS/MS method with off-line extraction without any statistically significant overall bias. In this regard, samples with individual significant bias were reasonably produced by preanalytical issues which had no relation with the conversion to the on-line SPE extraction.

3-(4-Hydroxyphenyl)propionic acid: the forgotten detection substrate for ligand-binding assay-based bioanalysis.

Ligand-binding assays are ideal for routine bioanalysis, but we reason that the straightforward replacement of the conventional chromogenic horseradish peroxidase substrate, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, of a routinely used preclinical immunoassay to detect hIgG, with the fluorogenic 3-(4-hydroxyphenyl)propionic acid would broaden the narrow dynamic range. The replacement leads to a sensitivity of 0.47 (minimum required dilution [MRD] 10) and 1.02 (MRD 50) ng/ml, and dynamic ranges of 3.3 (MRD 10) and 3.6 (MRD 50) orders of magnitude, and thereby had improved sensitivity and dynamic range compared with other conventional colorimetric ELISAs, other ligand-binding assay technologies or LC-MS assays. Improvements in sensitivity and dynamic range were achieved for the sera of horse, mice and monkeys without assay optimization.

In Vivo Dearomatization of the Potent Antituberculosis Agent BTZ043 via Meisenheimer Complex Formation.

Nitrobenzothiazinones are among the most potent antituberculosis agents. Herein, we disclose an unprecedented in vivo reduction process that affords Meisenheimer complexes of the clinical candidates BTZ043 and PBTZ169. The reduction is reversible, occurs in all mammalian species investigated, has a profound influence on the in vivo ADME characteristics, and has considerable implications for the design and implementation of clinical studies. The reduction was confirmed by chemical studies that enabled the complete characterization of the Meisenheimer complex and its subsequent chemistry. Combination of the in vivo and chemical studies with LC-MS characterization and assay development also provides a basis for rational lead optimization of this very promising class of antituberculosis agents.

A new LC-MS assay for determination of triterpene glycosides in Cimicifuga racemosa avoiding overestimation problems during stability testing

A new LC-MS assay for determination of triterpene glycosides in Cimicifuga racemosa avoiding overestimation problems during stability testing

In Vivo Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1 Analyzed by Ligand-Binding Mass Spectrometry Combined with Quantitation by ELISA.

The in vivo biotransformation of a novel fusion protein tetranectin/apolipoprotein A1 (TN-ApoA1) was investigated by ligand-binding mass spectrometry (LB-MS) in support of enzyme-linked immunosorbent assays (ELISA). The main focus was on catabolites formed by proteolysis of the fusion protein in rabbit following intravenous administration of lipidated TN-ApoA1. The drug and its catabolites were isolated from rabbit plasma by immunocapture with a monoclonal antibody (mAb) binding to the fusion region of TN-ApoA1. The captured drug and catabolites were released from the streptavidin-coated magnetic beads, separated by monolithic RP capillary HPLC, and online detected by high-resolution mass spectrometry. In addition, the same extract was digested with LysN to confirm or further narrow down the structure of the found catabolites. Two pharmacologically active catabolites were identified with conserved fusion region. The major catabolite [3-285] was formed by truncation of AP at the N-terminus and the minor catabolite [29-270] by truncations of either side of the TN-ApoA1 sequence. Since the ELISA determined the sum of TN-ApoA1, along with its two main catabolites, the individual PK profiles of all three components could be derived by applying their mass peak composition for each sampling point. Parent drug accounted for 25% of drug-related material, whereas that of the catabolites [3-285] and [29-270] accounted for 66% and 9%, respectively. This result could be obtained without catabolite specific ELISAs or quantitative LC-MS assays. It was also confirmed that all relevant functional molecules of TN-ApoA1 in the plasma samples were quantified by the ELISA, which provided a good relationship for pharmacokinetic/pharmacodynamic evaluations.

Multi-platform metabolomics assays for human lung lavage fluids in an air pollution exposure study.

Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry.

Generalized multiple internal standard method for quantitative liquid chromatography mass spectrometry

In this contribution, a multiplicative effects model for generalized multiple-internal-standard method (MEMGMIS) was proposed to solve the signal instability problem of LC–MS over time. MEMGMIS model seamlessly integrates the multiple-internal-standard strategy with multivariate calibration method, and takes full use of all the information carried by multiple internal standards during the quantification of target analytes. Unlike the existing methods based on multiple internal standards, MEMGMIS does not require selecting an optimal internal standard for the quantification of a specific analyte from multiple internal standards used. MEMGMIS was applied to a proof-of-concept model system: the simultaneous quantitative analysis of five edible artificial colorants in two kinds of cocktail drinks. Experimental results demonstrated that MEMGMIS models established on LC–MS data of calibration samples prepared with ultrapure water could provide quite satisfactory concentration predictions for colorants in cocktail samples from their LC–MS data measured 10days after the LC–MS analysis of the calibration samples. The average relative prediction errors of MEMGMIS models did not exceed 6.0%, considerably better than the corresponding values of commonly used univariate calibration models combined with multiple internal standards. The advantages of good performance and simple implementation render MEMGMIS model a promising alternative tool in quantitative LC–MS assays.

The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC–MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC–MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.

Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE).

The analysis of oxidative stress-induced post-translational modifications remains challenging due to the chemical diversity of these modifications, the possibility of the presence of positional isomers and the low stoichiometry of the modified proteins present in a cell or tissue proteome. Alcoholic liver disease (ALD) is a multifactorial disease in which mitochondrial dysfunction and oxidative stress have been identified as being critically involved in the progression of the disease from steatosis to cirrhosis. Ethanol metabolism leads to increased levels of reactive oxygen species (ROS), glutathione depletion and lipid peroxidation. Posttranslational modification of proteins by electrophilic products of lipid peroxidation has been associated with governing redox-associated signaling mechanisms, but also as contributing to protein dysfunction leading to organelle and liver injury. In particular the prototypical α,β-unsaturated aldehyde, 4-hydroxy-2-nonenal (HNE), has been extensively studied as marker of increased oxidative stress in hepatocytes. In this study, we combined a LC-MS label-free quantification method and affinity enrichment to assess the dose-dependent insult by HNE on the proteome of rat liver mitochondria. We used a carbonyl-selective probe, the ARP probe, to label HNE-protein adducts and to perform affinity capture at the protein level. Using LC-MS to obtain protein abundance estimates, a list of protein targets was obtained with increasing concentration of HNE used in the exposure studies. In parallel, we performed affinity capture at the peptide level to acquire site-specific information. Examining the concentration-dependence of the protein modifications, we observed distinct reactivity profiles for HNE-protein adduction. Pathway analysis indicated that proteins associated with metabolic processes, including amino acid, fatty acid, and glyoxylate and dicarboxylate metabolism, bile acid synthesis and TCA cycle, showed enhanced reactivity to HNE adduction. Whereas, proteins associated with oxidative phosphorylation displayed retardation toward HNE adduction. We provide a list of 31 protein targets with a total of 61 modification sites that may guide future targeted LC-MS assays to monitor disease progression and/or intervention in preclinical models of ALD and possibly other liver diseases with an oxidative stress component.

CLSI C62-A: A New Standard for Clinical Mass Spectrometry.

Until recently, there existed minimal guidance on the use of LC-MS for clinical diagnostics. The Clinical and Laboratory Standards Institute (CLSI)2 has now presented a standardized approach for LC-MS assay development and verification in its new guidance document, CLSI C62-A. This document should aid in the harmonization of LC-MS methods and thus have significant ramifications for the evolution of this technology in the clinical laboratory. The ability to accurately identify and quantify a measurand with high sensitivity and specificity by use of selective reaction monitoring has been the driving force for the adoption of LC-MS in the clinical laboratory. LC-MS offers analytical specificity superior to that of widespread diagnostic methodologies such as immunoassays and enzymatic assays. Whereas immunoassays are commonplace in the clinical laboratory, they are not available for all analytes of interest, are plagued by specificity issues, and require the time-consuming production and evaluation of antibodies during development. Just as immunoassays have evolved since their development >50 years ago and have found their place as the leading automated technological approach to diagnostic testing, mass spectrometry (MS) will continue to progress and influence the clinical laboratory landscape. Therefore, standardization of the use of LC-MS is essential. Mass spectrometers were initially used in clinical laboratories only for specialized testing by highly trained and experienced operators. Today they are essential for the diagnosis of metabolic disorders, screening of diseases, quantification of hormone concentrations, monitoring of drug therapies, identification of microbial organisms, and recognition of drug toxicity and poisonings. Without the current clinical quantitative LC-MS methods, metabolic disorders would go undetected at birth, the accuracy of testosterone measurements for women and children would be equivalent to an educated guess, and monitoring of a variety of chemotherapeutics and immunosuppressants would be limited. Despite its widespread use, there is only 1 quantitative LC-MS assay …

Metabolism and pharmacokinetics of major polyphenol components in rat plasma after oral administration of total flavonoid tablet from Anemarrhenae Rhizoma.

Total flavonoid tablet from Anemarrhenae Rhizoma (Zhimu tablet), which was made of total polyphenol components extracted from the dried rhizome of Anemarrhena asphodeloides Bge. (Zhimu in Chinese), is a novel traditional Chinese medicine prescribed for the treatment of diabetes. Mangiferin (MF) and neomangiferin (NMF) are the two main components detected and determined in Zhimu tablet, accounting for 8.9% of the total weight of each tablet. In the present study, high performance liquid chromatography (HPLC) coupled with time-of-flight (TOF) tandem mass spectrometry (MS) was applied to characterize the metabolites of MF and NMF in rat plasmas collected at different time points after oral administration of Zhimu tablet at a dose of 3.63g/kg (corresponding to 270mg/kg MF). Accurate mass measurement was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of identified metabolites. Time points of appearance of some metabolites, such as isomers, were also taken into account during the structure confirmation. A total of 21 potential metabolites were found in rat plasma at different time points, and the metabolic pathways in vivo were involved in hydrolysis, methylation, glucuronide conjugation, glycoside conjugation, sulphation, dehydration and isomerisation. Furthermore, a selective and accurate LC-MS assay method was developed and validated for the quantification of MF in plasma. Semi-quantification of main conjugated metabolites was also performed in order to describe the dynamic metabolism profiles of polyphenol components in Zhimu tablet. MF concentration in plasma reached 1.36±0.47μgmL(-1) about 5.0h after oral administration of Zhimu tablet, which showed a 3.24- and 4.91-fold increase in plasma maximum concentration and area under the concentration-time curve (AUC) from 0 to 24h of MF compared with those for rats administered with free MF, respectively. The results indicated that the pharmacokinetic processes and bioavailability of MF in rats would be affected by other components in Zhimu tablet.

Online 2D-LC-MS/MS assay to quantify therapeutic protein in human serum in the presence of pre-existing antidrug antibodies.

The formation of antidrug antibodies (ADA) can interfere with the accurate quantitation of therapeutic proteins, leading to significantly underestimated drug concentrations and confounded pharmacokinetic (PK) data interpretation. Although highly desirable, development of ADA-tolerant bioanalytical methods enabling unbiased measurement of both free and ADA-bound drug presents a considerable challenge. We report herein the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. Unlike ligand-binding assay, our method is not prone to interferences from ADA, allowing accurate and precise measurement of the IgAP in the range of 0.05 to 10 μg/mL in 25 μL of human serum with a wide range of anti-IgAP antibody levels. The intra- and inter-run precision (coefficient of variation (CV%)) was within 11.5% and 10.5%, respectively, and the bias was within ±7.1% for all quality control (QC) concentrations. With little modification, the described method can readily be applicable to the quantitation of other biotherapeutic proteins in the ADA-positive clinical matrices.

O16. Marinobufagenin as a promising preeclampsia risk assessment marker: purification from toad venom and LC–MS identification in human plasma

Introduction Marinobufagenin (MBG) is an endogenous bufadienolide cardiac inotrope which is demonstrating growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE) and end-stage renal disease hypertension. Mammalian MBG is an inhibitor of the α 1 isoform of Na + , K + -ATPase with vasoconstrictive and cardiotonic properties, resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1,2,3]. The rise of endogenous MBG appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential target in the biomarker panel for PE. A sensitive and accurate analytical method is needed to assess MBG in as lower level as possible in plasma. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Objective Our aim is to develop a MBG assay using a more specific and easy to access technique, such as LC–MS/MS. An algorithm dealing with the MBG plasma levels might be established by clinicians in the future, in order to predict, in combination with other clinical and biological markers, the risk for preeclampsia in pregnant women. Methods As the major source for MBG is located in the parotid glands of the Bufo Marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. A pre-extraction procedure was elaborated to concentrate and clean the plasma sample prior to its analysis. A LC–MS based assay designed to determine MBG in human plasma is being optimized, giving us the opportunity to investigate MBG in non-pregnant healthy volunteers plasma as well as in early pregnant women plasma. Results and discussion Pure MBG has been successfully extracted from the B. Marinus toad venom and the identity of the compound has been confirmed. An extraction procedure for MBG from plasma has been set up by use of solid phase extraction cartridges. Preliminary results allowed us to authenticate the presence of MBG by LC–MS/MS in non-pregnant women as well as in early pregnancy. Further optimization and validation of the LC–MS assay are needed to quantify MBG plasma levels. However, these pioneering observations, are giving the clinicians a promising perspective for early preeclampsia risk assessment in pregnant women.

Quantitative mass spectrometric analysis of dipeptides in protein hydrolysate by a TNBS derivatization-aided standard addition method

The aim of this study was to establish, through a standard addition method, a convenient quantification assay for dipeptides (GY, YG, SY, YS, and IY) in soybean hydrolysate using 2,4,6-trinitrobenzene sulfonate (TNBS) derivatization-aided LC–TOF-MS. Soybean hydrolysate samples (25.0mgmL−1) spiked with target standards were subjected to TNBS derivatization. Under the optimal LC–MS conditions, five target dipeptides derivatized with TNBS were successfully detected. Examination of the standard addition curves, with a correlation coefficient of r2>0.979, provided a reliable quantification of the target dipeptides, GY, YG, SY, YS, and IY, in soybean hydrolysate to be 424±20, 184±9, 2188±199, 327±16, and 2211±133μgg−1 of hydrolysate, respectively. The proposed LC–MS assay is a reliable and convenient assay method, with no interference from matrix effects in hydrolysate, and with no requirement for the use of an isotope labeled internal standard.

The alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta.

The present study aimed at investigating the alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta. Animal retroperitoneal inflammatory model in vivo and macrophagocyte release inflammatory factor model in vitro were used to detect the effect of alum processing on raphides and lectin. Scanning electron microscopy was used to observe the change in raphides during processing; HPLC method was used to determine the correlation between the dissolution and corrosion of raphides and ion in the alum solution; (27)Al-NMR technology was used to detect the relationship between aluminum oxalate complex formation and the dissolved and corrosion of raphides. The change in protein peptide sequence of lectin during the processing of alum solution was determined by Shotgun LC-MS assay. Raphides induced severe rabbit conjunctival edema and an intraperitoneal injection of lectin increased PGE2 and protein in mice peritoneal exudate, while decreased after treatment with alum solution processing. During the processing raphides was dissolved and corroded, then its structure was damaged. Raphides was soaked in the alum solution and significantly decreased the oxalate content, and the effect was related with Al(3+) in the alum. Al(3+) in the alum combined with C2O4(2-) of raphides into a stable complex compound promoted the dissolution of calcium oxalate. Raphides soaked in the alum made lectin proteins dissolve, whereas protein peptide sequence of lectin was changed and the protein structure was damaged. Alum solution could decrease the toxicity of P. ternata (Thunb.) Breit. and P. pedatisecta Schott. Since it made a special crystal structure of raphides damage and the protein of lectin dissolve. The structure of toxic substances significantly changed, which decreased the inflammatory effect.

LC–UV/MS methods for the analysis of prochelator—Boronyl salicylaldehyde isonicotinoyl hydrazone (BSIH) and its active chelator salicylaldehyde isonicotinoyl hydrazone (SIH)

Salicylaldehyde isonicotinoyl hydrazone (SIH) is an intracellular iron chelator with well documented potential to protect against oxidative injury both in vitro and in vivo. However, it suffers from short biological half-life caused by fast hydrolysis of the hydrazone bond. Recently, a concept of boronate prochelators has been introduced as a strategy that might overcome these limitations. This study presents two complementary analytical methods for detecting the prochelator-boronyl salicylaldehyde isonicotinoyl hydrazone-BSIH along with its active metal-binding chelator SIH in different solution matrices and concentration ranges. An LC–UV method for determination of BSIH and SIH in buffer and cell culture medium was validated over concentrations of 7–115 and 4–115μM, respectively, and applied to BSIH activation experiments in vitro. An LC–MS assay was validated for quantification of BSIH and SIH in plasma over the concentration range of 0.06–23 and 0.24–23μM, respectively, and applied to stability studies in plasma in vitro as well as analysis of plasma taken after i.v. administration of BSIH to rats. A Zorbax-RP bonus column and mobile phases containing either phosphate buffer with EDTA or ammonium formate and methanol/acetonitrile mixture provided suitable conditions for the LC–UV and LC–MS analysis, respectively. Samples were diluted or precipitated with methanol prior to analysis. These separative analytical techniques establish the first validated protocols to investigate BSIH activation by hydrogen peroxide in multiple matrices, directly compare the stabilities of the prochelator and its chelator in plasma, and provide the first basic pharmacokinetic data of this prochelator. Experiments reveal that BSIH is stable in all media tested and is partially converted to SIH by H2O2. The observed integrity of BSIH in plasma samples from the in vivo study suggests that the concept of prochelation might be a promising strategy for further development of aroylhydrazone cytoprotective agents.

Improvement of the performance of targeted LC-MS assays through enrichment of histidine-containing peptides.

Mass spectrometric-based quantification using targeted methods has matured during the past decade and is now commonly used in proteomics. However, the reliability of protein quantification in complex matrixes using selected reaction monitoring is often impaired by interfering signals arising from coelution of nontargeted components. Sample preparation methods resulting in the reduction of the number of peptides present in the mixture minimizes this effect. One solution consists in the selective capture of peptides containing infrequent amino acids. The enrichment of histidine-containing peptides via immobilized metal-ion affinity chromatography loaded with Cu(2+) ions (IMAC-Cu) was applied in a quantitative workflow and found to be a simple and cost effective method for the reduction of sample complexity with high recovery and selectivity. When applied to a series of depleted human plasma digests, the method decreased nonspecific signals, resulting in a more precise and robust protein quantification. The method was also shown to be an alternative to HSA/IgG depletion during plasma protein analysis. This method, used in conjunction with recent improvements in the instrument's peak capacity, addresses a bottleneck generally encountered in quantitative proteomics studies by providing the robustness and throughput required for the analysis of large sample series without compromising the number of proteins monitored.

Abstract 2693: Harnessing MALT1 inhibition for rational combinatorial therapy of ABC-DLBCL

Abstract The MALT1 paracaspase plays a critical role in the proliferation and survival of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL), the most chemo-resistant form of DLBCL. MALT1 mediates activation of the B-cell receptor (BCR) downstream of somatic mutations in signaling components such as: CD79, CARD11, A20 or MYD88, leading to chronically activated NF-κB. MALT1 is the effector enzyme of the CARD11/Bcl10/MALT1 signalosome, a massive, high order structure that functions as an amplifier of BCR signaling to NF-κB. MALT1 is a compelling therapeutic target since: i) it is the only paracaspase in humans, ii) MALT1 knockout mice are viable, and iii) ABC-DLBCLs are biologically dependent on MALT1 activity. MALT1 is only active when forming multimeric complexes. In order to identify potential MALT1 inhibitors we biochemically engineered an obligate dimerized form of MALT1 and an enzymatic assay for high throughput screening. We screened a ∼50,000 compound chemical diversity library and identified and validated 19 distinct chemical scaffolds that inhibited MALT1 with an IC50<20 μM. Three compounds induced selective dose-dependent suppression of MALT1-dependent ABC-DLBCL cells (MI-2, p<0.0001; MI-4, p=0.006; MI-11, p<0.0001). The most potent compound in cell-based assays was MI-2 with a GI25 in the low nanomolar range. MI-2 analogs also displayed nanomolar activity. In depth analysis using LC-MS, NMR and enzymatic kinetic assays revealed that MI-2 binds irreversibly to the active site of MALT1. In DLBCL cells MI-2 inhibited MALT1 cleavage of its targets TNFAIP3, CYLD, BCL10 and RelB, as well as nuclear translocation of c-REL and NF-κB activation. MI-2 inhibited proliferation, induced G1 arrest and ultimately promoted apoptosis in ABC-DLBCLs including those with mutations that bypass BTK inhibitors. MI-2 was non toxic in vivo and potently and specifically inhibited the growth of xenotransplanted ABC-DLBCLs (p=0.014, t-test) but not GCB-DLBCLs. MI-2 selectively killed primary human ABC-DLBCL specimens ex vivo. Given that multiple pathways contribute to ABC-DLBCL pathogenesis we hypothesized that MALT1 inhibitors would be most effective within combinatorial therapy regimens. Along these lines MI-2 strongly enhanced the activity of CHOP chemotherapy drugs against ABC-DLBCL cells. BCR signaling forms a complex network of signaling molecules beyond NF-κB, and accordingly MALT1 targeted therapy was strongly enhanced with small molecules that affect other branches of this pathway, such as PI3K inhibitors (e.g. BKM120). Finally MI-2 synergized with small molecules such as BH3 mimetics (most notably ABT-737) that target fundamental complementary survival pathways to BCR signaling in ABC-DLBCLs. In summary, we identified the first specific MALT1 inhibitor drug and demonstrated a promising role for MALT1 targeted therapy as an anchor of rational combinatorial therapy against ABC-DLBCL. Citation Format: Lorena Fontan, Chenghua Yang, Venkataraman Kabaleeswaran, Laurent Volpon, Michael Osborne, Elena Beltran, Monica Rosen, Rita Shaknovich, Shao N. Yang, Randy D. Gascoyne, Leandro Cerchietti, Jose A. Martinez-Climent, J. Fraser Glickman, Katherine Borden, Hao Wu, Ari Melnick. Harnessing MALT1 inhibition for rational combinatorial therapy of ABC-DLBCL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2693. doi:10.1158/1538-7445.AM2014-2693