Development of a Multiplexed LC-MS/MS Assay for the Quantitation of Podocyte Injury Biomarkers Nephrin, Podocalyxin, and Podocin in Human Urine.

CKD is frequently diagnosed only after a significant progression. GFR is the most common indicator of kidney function but is limited in detecting early CKD cases and distinguishing glomerular, tubular, and global CKD. Aiming to provide a glomeruli specific biomarker assay, we developed a peptide immunoaffinity targeted mass spectrometry method for the quantitation of three podocyte specific proteins in human urine: nephrin, podocalyxin, and podocin.

Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.

Complement Activation by Adeno-Associated Virus-Neutralizing Antibody Complexes.

Treatment of monogenetic disorders using vectors based on adeno-associated virus (AAV) is an area of intense interest. AAV is non-pathogenic human virus, and preexisting capsid antibodies are prevalent in the population posing a challenge to the safety and efficacy of AAV-mediated gene therapies. In this study, we investigated the risk of AAV-mediated complement activation when sera from a cohort of human donors were exposed to AAV9 capsid.

Highly Multiplexed Kinase Profiling in Spleen with Targeted Mass Spectrometry Reveals Kinome Plasticity across Species.

Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences that could exist between human patients and animal models. We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog, and cynomolgus monkey).

Biomeasures and mechanistic modeling highlight PK/PD risks for a monoclonal antibody targeting Fn14 in kidney disease.

Discovery of the upregulation of fibroblast growth factor-inducible-14 (Fn14) receptor following tissue injury has prompted investigation into biotherapeutic targeting of the Fn14 receptor for the treatment of conditions such as chronic kidney diseases. In the development of monoclonal antibody (mAb) therapeutics, there is an increasing trend to use biomeasures combined with mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modeling to enable decision making in early discovery. With the aim of guiding preclinical efforts on designing an antibody with optimized properties, we developed a mechanistic site-of-action (SoA) PK/PD model for human application.

Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn's Disease.

Objective: To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn's disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody.

Methods: In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12.

Physiologically relevant binding affinity quantification of monoclonal antibody PF-00547659 to mucosal addressin cell adhesion molecule for in vitro in vivo correlation.

Background And Purpose: A monoclonal antibody (PF-00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans-membrane (mMAdCAM) target forms, showed over 30-fold difference in antibody-target K between in vitro (Biacore) and clinically derived (K ) values. Back-scattering interferometry (BSI) was applied to acquire physiologically relevant K values which were used to establish in vitro and in vivo correlation (IVIVC).

Experimental Approach: BSI was applied to obtain K values between PF-00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue.

Bioanalytical platform comparison using a generic human IgG PK assay format.

A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG₁ monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte, and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance.

Tissue bioanalysis of biotherapeutics and drug targets to support PK/PD.

Contemporary drug discovery leverages quantitative modeling and simulation with increasing emphasis, both to gain deeper knowledge of drug targets and mechanisms as well as improve predictions between preclinical models and clinical applications, such as first-in-human dose projections. Proliferation of novel biotherapeutic modalities increases the need for applied PK/PD modeling as a quantitative tool to advance new therapies. Of particular relevance is the understanding of exposure, target binding and associated pharmacology at the target site of interest.

Clinical pharmacokinetic assessment of an anti-MAdCAM monoclonal antibody therapeutic by LC-MS/MS.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be a viable tool for preclinical pharmacokinetic (PK) analysis of monoclonal antibody (mAb) therapeutics. This work describes free and total PK assays for the mAb PF-00547,659 in serum of ulcerative colitis patients in a First-In-Human study [Vermeire, S. et al.

Visualisation tool for peptide fractionation data in proteomics: application to OFFGEL isoelectric focussing.

Background: OFFGEL isoelectric focussing (IEF) has become a popular tool in proteomics to fractionate peptides or proteins. As a consequence there is a need for software solutions supporting data mining, interpretation and characterisation of experimental quality.

Results: We can assess performance characteristics of OFFGEL IEF peptide fractionation in proteomics by generating plots of the overall fractionation patterns and the pairwise comparisons of adjacent fractions.

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum.

An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection.

Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya.

The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F.

Comparison of cellular ribonucleoprotein complexes associated with the APOBEC3F and APOBEC3G antiviral proteins.

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F (APOBEC3F [A3F]) and A3G proteins are effective inhibitors of infection by various retroelements and share approximately 50% amino acid sequence identity. We therefore undertook comparative analyses of the protein and RNA compositions of A3F- and A3G-associated ribonucleoprotein complexes (RNPs). Like A3G, A3F is found associated with a complex array of cytoplasmic RNPs and can accumulate in RNA-rich cytoplasmic microdomains known as mRNA processing bodies or stress granules.

Mass spectrometric detection of CP4 EPSPS in genetically modified soya and maize.

The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS.

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides.

Protonated peptides derived from proline-rich proteins (PRP) are often difficult to sequence by standard collision-induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N-terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al.

MALDI post-source decay and LIFT-TOF/TOF investigation of alpha-cyano-4-hydroxycinnamic acid cluster interferences.

Large signals from alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix complexes with sodium and potassium ions were found to interfere with sensitive matrix-assisted laser desorption/ionization (MALDI) analysis of a hydrochloric acid digest of gelatine preparations. The nature of some selected matrix clusters was investigated by conventional post-source decay and LIFT-TOF/TOF experiments. The matrix clusters fragmented readily by neutral evaporation to give smaller sized matrix cluster species without matrix disintegration.

BSE control: detection of gelatine-derived peptides in animal feed by mass spectrometry.

The epidemic of bovine spongiform encephalopathy (BSE) is thought to have resulted from feeding scrapie-infected sheep to cattle. This has led to a ban of feeding animals with "processed animal protein"(PAP). We report a novel approach for the mass spectrometric detection of PAP contamination in animal feedstuffs by detecting gelatine, a derivative of the major animal protein collagen.