Profiling cholinesterase adduction: a high-throughput prioritization method for organophosphate exposure samples.
AuthorsCarter MD, Crow BS, Pantazides BG, Watson CM, DeCastro BR, Thomas JD, Blake TA, Johnson RC
Journal
PubMed ID23954929
'A high-throughput prioritization method was developed for use with a validated confirmatory method detecting organophosphorus nerve agent exposure by immunomagnetic separation high-performance liquid chromatography tandem mass spectrometry. A ballistic gradient was incorporated into this analytical method to profile unadducted butyrylcholinesterase (BChE) in clinical samples. With Zhang et al.''s Z'' factor ... More
Development and mechanism of ?-secretase modulators for Alzheimer's disease.
AuthorsCrump CJ, Johnson DS, Li YM
JournalBiochemistry
PubMed ID23614767
'?-Secretase is an aspartyl intramembranal protease composed of presenilin, Nicastrin, Aph1, and Pen2 with 19 transmembrane domains. ?-Secretase cleaves the amyloid precursor proteins (APP) to release Aß peptides that likely play a causative role in the pathogenesis of Alzheimer's disease (AD). In addition, ?-secretase cleaves Notch and other type I ... More
Recommended mass spectrometry-based strategies to identify botulinum neurotoxin-containing samples.
AuthorsKalb SR, Baudys J, Wang D, Barr JR
Journal
PubMed ID25996606
'Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between ... More
Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry.
AuthorsSchieltz DM, McWilliams LG, Kuklenyik Z, Prezioso SM, Carter AJ, Williamson YM, McGrath SC, Morse SA, Barr JR
Journal
PubMed ID25576235
'The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) ... More
Using phage and yeast display to select hundreds of monoclonal antibodies: application to antigen 85, a tuberculosis biomarker.
'Current diagnostic methods for tuberculosis (TB), a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen ... More
Pyroglutamate amyloid ß (Aß) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease.
AuthorsWittnam JL, Portelius E, Zetterberg H, Gustavsson MK, Schilling S, Koch B, Demuth HU, Blennow K, Wirths O, Bayer TA
JournalJ Biol Chem
PubMed ID22267726
'Pyroglutamate-modified Aß peptides at amino acid position three (Aß(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aß(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the ... More
Minimalistic sample preparation strategies for LC-MS quantification of large molecule biopharmaceuticals: a case study highlighting alpha-1 antitrypsin protein.
AuthorsWright K, Dufield D
Journal
PubMed ID25157487
Large molecule biotherapeutics pose a distinctive bioanalytical challenge for LC-MS assay development, particularly when optimizing sample enrichment steps. Alpha-1 antitrypsin (AAT) is used as an example for highlighting large-molecule assay-development strategies. ... More
Cerebrospinal fluid biomarkers of ß-amyloid metabolism in multiple sclerosis.
AuthorsAugutis K, Axelsson M, Portelius E, Brinkmalm G, Andreasson U, Gustavsson MK, Malmeström C, Lycke J, Blennow K, Zetterberg H, Mattsson N
JournalMult Scler
PubMed ID23069872
Amyloid precursor protein (APP) and amyloid ß (Aß) peptides are intensely studied in neuroscience and their cerebrospinal fluid (CSF) measurements may be used to track the metabolic pathways of APP in vivo. Reduced CSF levels of Aß and soluble APP (sAPP) fragments are reported in inflammatory diseases, including multiple sclerosis ... More
PK assays for antibody-drug conjugates: case study with ado-trastuzumab emtansine.
AuthorsDere R, Yi JH, Lei C, Saad OM, Huang C, Li Y, Baudys J, Kaur S
JournalBioanalysis
PubMed ID23641694
Antibody-drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory ... More
Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC-MS/MS.
AuthorsPalandra J, Finelli A, Zhu M, Masferrer J, Neubert H
JournalAnal Chem
PubMed ID23638938
A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides ... More
AFFIRM--a multiplexed immunoaffinity platform that combines recombinant antibody fragments and LC-SRM analysis.
AuthorsSäll A, Carlsson F, Olsson N, Wingren C, Ohlin M, Persson H, Waldemarson S
Journal
PubMed ID25337893
Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex ... More
An enhanced butyrylcholinesterase method to measure organophosphorus nerve agent exposure in humans.
AuthorsPantazides BG, Watson CM, Carter MD, Crow BS, Perez JW, Blake TA, Thomas JD, Johnson RC
Journal
PubMed ID24604326
Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G- and V-series OPNA adducts to BChE in 75 µL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass ... More
A quantitative high-throughput in vitro splicing assay identifies inhibitors of spliceosome catalysis.
AuthorsBerg MG, Wan L, Younis I, Diem MD, Soo M, Wang C, Dreyfuss G
JournalMol Cell Biol
PubMed ID22252314
Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, ... More
SorLA complement-type repeat domains protect the amyloid precursor protein against processing.
AuthorsMehmedbasic A, Christensen SK, Nilsson J, Rüetschi U, Gustafsen C, Poulsen AS, Rasmussen RW, Fjorback AN, Larson G, Andersen OM
Journal
PubMed ID25525276
SorLA is a neuronal sorting receptor that is genetically associated with Alzheimer disease. SorLA interacts directly with the amyloid precursor protein (APP) and affects the processing of the precursor, leading to a decreased generation of the amyloid-ß peptide. The SorLA complement-type repeat (CR) domains associate in vitro with APP, but ... More
Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum.
AuthorsBjörnstad K, Tevell Åberg A, Kalb SR, Wang D, Barr JR, Bondesson U, Hedeland M
Journal
PubMed ID25228079
Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an ... More
Detection of Botulinum Toxins A, B, E, and F in Foods by Endopep-MS.
AuthorsKalb SR, Krilich JC, Dykes JK, Lúquez C, Maslanka SE, Barr JR
Journal
PubMed ID25578960
Botulism is caused by exposure to botulinum neurotoxins (BoNTs). BoNTs are proteins secreted by some species of clostridia; these neurotoxins are known to interfere with nerve impulse transmission, thus causing paralysis. Botulism may be contracted through consumption of food either naturally or intentionally contaminated with BoNT. The human lethal dose ... More
Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.
Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically ... More
High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.
AuthorsWhiteaker JR, Zhao L, Frisch C, Ylera F, Harth S, Knappik A, Paulovich AG
Journal
PubMed ID24568200
High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an ... More
Enhanced stability of blood matrices using a dried sample spot assay to measure human butyrylcholinesterase activity and nerve agent adducts.
AuthorsPerez JW, Pantazides BG, Watson CM, Thomas JD, Blake TA, Johnson RC
Journal
PubMed ID25955132
Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intraspot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) ... More
Performance of a novel high throughput method for the determination of VX in drinking water samples.
AuthorsKnaack JS, Zhou Y, Magnuson M, Silvestri E, Johnson RC
JournalAnal Chem
PubMed ID23402290
VX (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothioate) is a highly toxic organophosphorus nerve agent, and even low levels of contamination in water can be harmful. Measurement of low concentrations of VX in aqueous matrixes is possible using an immunomagnetic scavenging technique and detection using liquid chromatography/tandem-mass spectrometry. Performance of the method was characterized in ... More
Functional characterization of botulinum neurotoxin serotype H as a hybrid of known serotypes F and A (BoNT F/A).
AuthorsKalb SR, Baudys J, Raphael BH, Dykes JK, Lúquez C, Maslanka SE, Barr JR
Journal
PubMed ID25731972
A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known ... More
Anti-idiotypic monobodies derived from a fibronectin scaffold.
AuthorsSullivan MA, Brooks LR, Weidenborner P, Domm W, Mattiacio J, Xu Q, Tiberio M, Wentworth T, Kobie J, Bryk P, Zheng B, Murphy M, Sanz I, Dewhurst S
JournalBiochemistry
PubMed ID23394681
Mimetics of conformational protein epitopes have broad applications but have been difficult to identify using conventional peptide phage display. The 10th type III domain of human fibronectin (FNfn10) has two extended, randomizable surface-exposed loops and might be more amenable to the identification of such mimetics. We therefore selected a library ... More
Multiplexed, Quantitative Workflow for Sensitive Biomarker Discovery in Plasma Yields Novel Candidates for Early Myocardial Injury.
AuthorsKeshishian H, Burgess MW, Gillette MA, Mertins P, Clauser KR, Mani DR, Kuhn EW, Farrell LA, Gerszten RE, Carr SA
Journal
PubMed ID25724909
We have developed a novel plasma protein analysis platform with optimized sample preparation, chromatography, and MS analysis protocols. The workflow, which utilizes chemical isobaric mass tag labeling for relative quantification of plasma proteins, achieves far greater depth of proteome detection and quantification while simultaneously having increased sample throughput than prior ... More
Anti-peptide monoclonal antibodies generated for immuno-multiple reaction monitoring-mass spectrometry assays have a high probability of supporting Western blot and ELISA.
AuthorsSchoenherr RM, Saul RG, Whiteaker JR, Yan P, Whiteley GR, Paulovich AG
Journal
PubMed ID25512614
Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, ... More
Quantitation of ortho-cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic-UHPLC-MS/MS.
AuthorsJohnson D, Carter MD, Crow BS, Isenberg SL, Graham LA, Erol HA, Watson CM, Pantazides BG, van der Schans MJ, Langenberg JP, Noort D, Blake TA, Thomas JD, Johnson RC
Journal
PubMed ID26149113
Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged ... More
Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS.
AuthorsCarter MD, Crow BS, Pantazides BG, Watson CM, Thomas JD, Blake TA, Johnson RC
Journal
PubMed ID24205842
Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of "aging" and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has ... More
Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.
AuthorsKuhn E, Whiteaker JR, Mani DR, Jackson AM, Zhao L, Pope ME, Smith D, Rivera KD, Anderson NL, Skates SJ, Pearson TW, Paulovich AG, Carr SA
JournalMol Cell Proteomics
PubMed ID22199228
The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can ... More
High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.
AuthorsBoyer AE, Gallegos-Candela M, Quinn CP, Woolfitt AR, Brumlow JO, Isbell K, Hoffmaster AR, Lins RC, Barr JR
Journal
PubMed ID25673244
Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, ... More
Novel dual-mode immunomagnetic method for studying reactivation of nerve agent-inhibited butyrylcholinesterase.
AuthorsAbney CW, Knaack JL, Ali AA, Johnson RC
JournalChem Res Toxicol
PubMed ID23656164
A novel immunomagnetic method has been developed for the simultaneous measurement of organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BuChE) and free OPNAs in serum. This new approach, deemed dual-mode immunomagnetic analysis (Dual-Mode IMA), combines immunomagnetic separation (IMS) and immunomagnetic scavenging (IMSc) and has been used to measure the effectiveness ... More
Targeted MS Assay Predicting Tamoxifen Resistance in Estrogen-Receptor-Positive Breast Cancer Tissues and Sera.
AuthorsDe Marchi T, Kuhn E, Dekker LJ, Stingl C, Braakman RB, Opdam M, Linn SC, Sweep FC, Span PN, Luider TM, Foekens JA, Martens JW, Carr SA, Umar A
JournalJ Proteome Res
PubMed ID26958999
'We recently reported on the development of a 4-protein-based classifier (PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen treatment in recurrent, estrogen-receptor-positive breast cancer based on high-resolution MS data. A precise and high-throughput assay to measure these proteins in a multiplexed, targeted fashion would be favorable to ... More
Cerebrospinal Fluid Amyloid Beta and Tau Concentrations Are Not Modulated by 16 Weeks of Moderate- to High-Intensity Physical Exercise in Patients with Alzheimer Disease.
Physical exercise may have some effect on cognition in patients with Alzheimer disease (AD). However, the underlying biochemical effects are unclear. Animal studies have shown that amyloid beta (Aß), one of the pathological hallmarks of AD, can be altered with high levels of physical activity. ... More
A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood.
AuthorsCartwright M, Rottman M, Shapiro NI, Seiler B, Lombardo P, Gamini N, Tomolonis J, Watters AL, Waterhouse A, Leslie D, Bolgen D, Graveline A, Kang JH, Didar T, Dimitrakakis N, Cartwright D, Super M, Ingber DE
JournalEBioMedicine
PubMed ID27333027
Blood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis. ... More
A high-throughput UHPLC-MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents.
AuthorsGraham LA, Johnson D, Carter MD, Stout EG, Erol HA, Isenberg SL, Mathews TP, Thomas JD, Johnson RC
JournalBiomed Chromatogr
PubMed ID27572107
Organophosphorus nerve agents (OPNAs) are toxic compounds that are classified as prohibited Schedule 1 chemical weapons. In the body, OPNAs bind to butyrylcholinesterase (BChE) to form nerve agent adducts (OPNA-BChE). OPNA-BChE adducts can provide a reliable, long-term protein biomarker for assessing human exposure. A major challenge facing OPNA-BChE detection is ... More
Generation and Characterization of an IgG4 Monomeric Fc Platform.
AuthorsShan L, Colazet M, Rosenthal KL, Yu XQ, Bee JS, Ferguson A, Damschroder MM, Wu H, Dall'Acqua WF, Tsui P, Oganesyan V
JournalPLoS One
PubMed ID27479095
The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of ... More
A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange.
AuthorsSo BR, Wan L, Zhang Z, Li P, Babiash E, Duan J, Younis I, Dreyfuss G
JournalNat Struct Mol Biol
PubMed ID26828962
Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins. ... More
Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage.
AuthorsXu L, Packer LE, Qian MG, Wu JT
JournalJ Pharm Biomed Anal
PubMed ID27281578
Antibody-drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for ... More