ELISA reagent coverage evaluation by affinity purification tandem mass spectrometry

Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been used to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput. There are, however, inherent difficulties reconciling the protein-centric results of MS characterization with ELISA, or providing assurance that ELISA has acceptable coverage against all process-specific HCP impurities that could pose safety or efficacy risks. Here, we describe efficient determination of ELISA reagent coverage by proteomic analysis following affinity purification with a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications can be compared with the actual downstream process impurities for a given process to enable a highly focused assessment of ELISA reagent suitability. We illustrate the utility of this approach by performing coverage evaluation of an anti-HCP polyclonal against both an HCP immunogen and the downstream HCP impurities identified in a therapeutic monoclonal antibody after Protein A purification. The overall goal is to strategically implement affinity-based mass spectrometry as part of a holistic framework for evaluating HCP process clearance, ELISA reagent coverage, and process clearance risks. We envision coverage analysis by AP-MS will further enable a framework for HCP impurity analysis driven by characterization of actual product-specific process impurities, complimenting analytical methods centered on consideration of the total host cell proteome.     

Profiling Eph receptor expression in cells and tissues: A targeted mass spectrometry approach

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

Morpheus Spectral Counter: A computational tool for label‐free quantitative mass spectrometry using the Morpheus search engine

Label‐free quantitative MS based on the Normalized Spectral Abundance Factor (NSAF) has emerged as a straightforward and robust method to determine the relative abundance of individual proteins within complex mixtures. Here, we present Morpheus Spectral Counter (MSpC) as the first computational tool that directly calculates NSAF values from output obtained from Morpheus, a fast, open‐source, peptide‐MS/MS matching engine compatible with high‐resolution accurate‐mass instruments. NSAF has distinct advantages over other MS‐based quantification methods, including a greater dynamic range as compared to isobaric tags, no requirement to align and re‐extract MS1 peaks, and increased speed. MSpC features an easy‐to‐use graphic user interface that additionally calculates both distributed and unique NSAF values to permit analyses of both protein families and isoforms/proteoforms. MSpC determinations of protein concentration were linear over several orders of magnitude based on the analysis of several high‐mass accuracy datasets either obtained from PRIDE or generated with total cell extracts spiked with purified Arabidopsis 20S proteasomes. The MSpC software was developed in C# and is open sourced under a permissive license with the code made available at http://dcgemperline.github.io/Morpheus_SpC/ .     

A new derivative for oxosteroid analysis by mass spectrometry

Here we report a new method for oxosteroid identification utilizing “tandem mass tag hydrazine” (TMTH) carbonyl-reactive derivatisation reagent. TMTH is a reagent with a chargeable tertiary amino group attached through a linker to a carbonyl-reactive hydrazine group. Thirty oxosteroids were analysed after derivatisation with TMTH by electrospray ionization mass spectrometry (ESI-MS) and were found to give high ion-currents compared to underivatised molecules. ESI-tandem mass spectrometry (MS/MS) analysis of the derivatives yielded characteristic fragmentation patterns with specific mass reporter ions derived from the TMT group. A shotgun ESI-MS method incorporating TMTH derivatisation was applied to a urine sample.     

Quantitative analysis of biomarkers,drugs and toxins in biological samples by immunoaffinity chromatography coupled to mass spectrometry or tandem mass spectrometry: A focused review of recent applications

Immunoaffinity chromatography (IAC), mass spectrometry and especially tandem mass spectrometry (MS/MS) represent the most efficient and reliable analytical techniques for specific isolation, unequivocal identification and accurate quantification of numerous natural and synthetic substances in biological samples. This review article focuses on the combined use of these outstanding methodologies in basic and clinical research and in life sciences for the quantitative analysis of low- and high-molecular mass biomarkers, drugs and toxins in urine, plasma or serum samples, in tissue and other biologicals systems published in the last decade. The analytes discussed in some detail include the biomarkers of oxidative stress 15(S)-8-iso-prostaglandin F_2α {15(S)-8-iso-PGF_2α} and 3-nitrotyrosine, the major urinary metabolite of the lipid mediators cysteinyl leukotrienes, i.e., the leukotriene E₄ (LTE₄), melatonin, and the major collagen type II neoepitope peptide in human urine.     

Simultaneous analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol enantiomers in rat plasma by high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)‐trans‐T and (−)‐trans‐T. The main metabolic pathways are O‐demethylation and N‐demethylation, producing trans‐O‐desmethyltramadol ( M1 ) and trans‐N‐desmethyltramadol ( M2 ) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)‐trans‐T and (+)‐ M1 and to the monoaminergic action of (+/−)‐trans‐T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans‐T, M1 , and M2 enantiomers. The analytes were resolved on a Chiralpak® AD column using hexane:ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans‐T and M1 and 0.1 ng/ml for M2 . The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans‐T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans‐T and M2 was enantioselective (AUC_(+)/(−) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans‐T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans‐T pharmacokinetics. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

A new rapid and comprehensive peptidome analysis by one-step direct transfer technology for 1-D electrophoresis/MALDI mass spectrometry

We have developed a new target plate for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This target plate enables direct electric transfer of analytes from the 1-dimensional gel electrophoresis (1-DE) gel to the target plate in one step. Incorporated with a one-step direct transfer technique, this novel 1-DE/MALDI-MS (1-DE/MS) system eliminates staining, extracting, loading, and many other time-consuming intermediate processes, thereby greatly reducing analysis time while providing high throughput proteome analysis. Furthermore, in peptidome analysis, during the 1-DE step this system separates or removes the high molecular weight plasma proteins in blood and the various low molecular weight substances in tissue extracts, which interfere with mass spectrometry. This system can therefore be used for peptide profiling of any biological sample without special pretreatment. In view of these advantages, the 1-DE/MS system will greatly improve the usefulness of current peptidomic modalities in the discovery and validation of biomarker molecules in various body fluids and tissue extracts, permitting early detection, diagnosis, and treatment of diseases.     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.     

Liquid chromatographic and tandem mass spectrometric assay for evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors: application of biomarkers in drug discovery

A simple and selective assay for the evaluation of in vivo inhibition of rat brain monoamine oxidases (MAO) A and B following a single dose of MAO inhibitors was developed through the simultaneous determination of endogenous 5-hydroxy tryptamine, 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophane, and 2-phenethylamine (PEA) in rat brain using liquid chromatography-tandem mass spectrometry (LC/MS/MS). These analytes were separated on a Zorbax SB-C18 column using a gradient elution with acetonitrile and 0.2% formic acid and detected on an electrospray ionization mass spectrometer in positive-ion multiple-reaction-monitoring mode. The susceptibility and variability of these analytes as potential biomarkers in response to MAO inhibition in vivo were evaluated after application to three MAO inhibitors, tranylcypromine, clorgyline, and pargyline. A dramatic increase (about 40-fold) in PEA brain level and a decrease in 5-HIAA by more than 90% were observed after administration of 15 mg/kg of the nonselective MAO inhibitor tranylcypromine. As expected, the brain level of PEA escalated to about 6-fold, while the 5-HIAA level remained unchanged following a dose of the MAO B inhibitor pargyline at 2mg/kg. In contrast, the brain level of 5-HIAA reduced by approximately 53%, but the PEA level was unaffected following the same dose of the MAO A inhibitor clorgyline. The results indicated that 5-HIAA and PEA were susceptible and effective biomarkers in the rat brain in response to MAO A and B inhibition, respectively. The LC/MS/MS method is useful not only for the determination of inhibitory potency but also for the differentiation of the selectivity of a MAO inhibitor against rat brain MAO A and B in vivo.     

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.