Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry.

Targeting and Crossing the Blood-Brain Barrier with Extracellular Vesicles.

The blood-brain barrier (BBB) is one of the most complex and selective barriers in the human organism. Its role is to protect the brain and preserve the homeostasis of the central nervous system (CNS). The central elements of this physical and physiological barrier are the endothelial cells that form a monolayer of tightly joined cells covering the brain capillaries.

Discovery of a putative blood-based protein signature associated with response to ALK tyrosine kinase inhibition.

Background: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib.

An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression.

Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson's disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression.

Mass-Spectrometry-Based Method To Quantify in Parallel Tau and Amyloid β 1-42 in CSF for the Diagnosis of Alzheimer's Disease.

Alzheimer's disease (AD), the most common form of dementia, afflicts about 50 million people worldwide. Currently, AD diagnosis is primarily based on psychological evaluation and can only be confirmed post-mortem. Reliable and objective biomarkers for prognosis and diagnosis have been sought for years.

(I) Pharmacological profiling of a novel modulator of the α7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains.

The primary cause of Alzheimer's disease is unlikely to be the much studied markers amyloid beta or tau. Their widespread distribution throughout the brain does not account for the specific identity and deep subcortical location of the primarily vulnerable neurons. Moreover an unusual and intriguing feature of these neurons is that, despite their diverse transmitters, they all contain acetylcholinesterase.

Proteomics and the blood-brain barrier: how recent findings help drug development.

The drug discovery and development processes are divided into different stages separated by milestones to indicate that significant progress has been made and that certain criteria for target validation, hits, leads and candidate drugs have been met. Proteomics is a promising approach for the identification of protein targets and biochemical pathways involved in disease process and thus plays an important role in several stages of the drug development. The blood-brain barrier is considered as a major bottleneck when trying to target new compounds to treat neurodegenerative diseases.

Glial-cell-mediated re-induction of the blood-brain barrier phenotype in brain capillary endothelial cells: a differential gel electrophoresis study.

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood-brain barrier (BBB) models. Hence, the re-induction of barrier properties is crucial for the relevance of BBB models.

TNAP and EHD1 are over-expressed in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties.

Although the physiological properties of the blood-brain barrier (BBB) are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs) has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation.

Comparison of 4-plex to 8-plex iTRAQ quantitative measurements of proteins in human plasma samples.

Methods for isobaric tagging of peptides, iTRAQ or TMT, are commonly used platforms in mass spectrometry based quantitative proteomics. These two methods are very often used to quantitate proteins in complex samples, e.g.

Plasma proteomic profiling in HIV-1 infected methamphetamine abusers.

We wanted to determine whether methamphetamine use affects a subset of plasma proteins in HIV-infected persons. Plasma samples from two visits were identified for subjects from four groups: HIV+, ongoing, persistent METH use; HIV+, short-term METH abstinent; HIV+, long term METH abstinence; HIV negative, no history of METH use. Among 390 proteins identified, 28 showed significant changes in expression in the HIV+/persistent METH+ group over the two visits, which were not attributable to HIV itself.

Elucidating protein inter- and intramolecular interacting domains using chemical cross-linking and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry.

Among many methods used to investigate protein/protein interactions, chemical cross-linking combined with mass spectrometry remains a vital experimental approach. Mapping peptides modified by cross-linker provides clues about proteins' interacting domains. One complication is that such modification may result from intra- but not intermolecular interactions.

A differential proteomic approach identifies structural and functional components that contribute to the differentiation of brain capillary endothelial cells.

When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells.

MALDI-TOF MS profiling as the first-tier screen for sickle cell disease in neonates: matching throughput to objectives.

Purpose: Universal newborn screening for sickle cell diseases (SCDs) is not currently performed in many countries concerned by this public health problem. Owing to the technical and financial limitations of standard profiling methods (IEF coupled to subsequent HPLC), ethnically targeted neonatal screening is often preferred. Here, we demonstrate that MALDI-MS-based SCD newborn screening could be considered as a potential method for a strategy to universal screening because of its high throughput, cost-effectiveness, sensitivity and ability to automatically discriminate sickle haemoglobin.

Comparative proteomic analysis of blood eosinophils reveals redox signaling modifications in patients with FIP1L1-PDGFRA-associated chronic eosinophilic leukemia.

The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos).

Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation.

Background: Proteomic-based discovery of biomarkers for disease has recently come under scrutiny for a variety of issues; one prominent issue is the lack of orthogonal validation for biomarkers following discovery. Validation by ELISA or Western blot requires the use of antibodies, which for many potential biomarkers are under-characterized and may lead to misleading or inconclusive results. Gelsolin is one such biomarker candidate in HIV-associated neurocognitive disorders.

A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties.

Background: Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g.

Understanding the blood-brain barrier using gene and protein expression profiling technologies.

The blood-brain barrier (BBB) contributes to the brain homeostasis by regulating the passage of endogenous and exogenous compounds. This function is in part due to well-known proteins such as tight junction proteins, plasma membrane transporters and metabolic barrier proteins. Over the last decade, genomics and proteomics have emerged as supplementary tools for BBB research.

Actin, gelsolin and filamin-A are dynamic actors in the cytoskeleton remodelling contributing to the blood brain barrier phenotype.

The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co-culture with astrocytes compared with endothelial cells cultured alone.

Proteomic analysis of left ventricular remodeling in an experimental model of heart failure.

The development of chronic heart failure (CHF) following myocardial infarction is characterized by progressive alterations of left ventricle (LV) structure and function called left ventricular remodeling (LVR), but the mechanism of LVR remains still unclear. Moreover, information concerning the global alteration protein pattern during the LVR will be helpful for a better understanding of the process. We performed differential proteomic analysis of whole LV proteins using an experimental model of CHF in which myocardial infarction was induced in adult male rats by left coronary ligation.