Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl.

Structural Elucidation of Intact Rough-type Lipopolysaccharides Using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.

Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging.

Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.

Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally heterogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging.

On-tissue pyrene-1-boronic acid labeling assisted MALDI imaging of catecholamines in porcine adrenal gland.

In this study, an on-tissue chemical labeling - matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method was developed for visualization of the distribution of three catecholamine (CA) compounds (dopamine, epinephrine and norepinephrine) in porcine adrenal gland. Commercially available pyrene-1-boronic acid (PBA) was employed as an effective in situ derivatizing reagent dissolved in acetonitrile containing 0.1% pyridine for the chemical labeling and the matrix coating.

Circulating Isovalerylcarnitine and Lung Cancer Risk: Evidence from Mendelian Randomization and Prediagnostic Blood Measurements.

Background: Tobacco exposure causes 8 of 10 lung cancers, and identifying additional risk factors is challenging due to confounding introduced by smoking in traditional observational studies.

Materials And Methods: We used Mendelian randomization (MR) to screen 207 metabolites for their role in lung cancer predisposition using independent genome-wide association studies (GWAS) of blood metabolite levels (n = 7,824) and lung cancer risk (n = 29,266 cases/56,450 controls). A nested case-control study (656 cases and 1,296 matched controls) was subsequently performed using prediagnostic blood samples to validate MR association with lung cancer incidence data from population-based cohorts (EPIC and NSHDS).

Targeted proteomics for evaluating risk of venous thrombosis following traumatic lower-leg injury or knee arthroscopy.

Introduction: Patients with lower-leg cast immobilization and patients undergoing knee arthroscopy have an increased risk of venous thrombosis (VT). Guidelines are ambiguous about thromboprophylaxis use, and individual risk factors for developing VT are often ignored. To assist in VT risk stratification and guide thromboprophylaxis use, various prediction models have been developed.

Structure of prion β-oligomers as determined by short-distance crosslinking constraint-guided discrete molecular dynamics simulations.

The conversion of the native monomeric cellular prion protein (PrP ) into an aggregated pathological β-oligomeric form (PrP ) and an infectious form (PrP ) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational changes involved in the conversion are still unknown. We applied mass spectrometry combined with chemical crosslinking, hydrogen/deuterium exchange, limited proteolysis, and surface modification for the differential characterization of the native and the urea+acid-converted prion β-oligomer structures to obtain insights into the mechanisms of conversion and aggregation.

Performance Assessment of a 125 Human Plasma Peptide Mixture Stored at Room Temperature for Multiple Reaction Monitoring-Mass Spectrometry.

Synthetic peptides are a critical requirement for the development and application of targeted mass spectrometry (MS)-based assays for the quantitation of proteins from biological matrices. Transporting synthetic peptides on dry ice from one laboratory to another is costly and often difficult because of country-specific import and export regulations. Therefore, in this study, we assessed the impact of leaving a lyophilized mixture consisting of 125 peptides at room temperature for up to 20 days, and we assessed the effect on the quantitative performance of multiple reaction monitoring-MS (MRM-MS) assays.

MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of > 0.

Mouse Quantitative Proteomics Knowledgebase: reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays.

Unlabelled: MotivationLaboratory mouse is the most used animal model in biological research, largely due to its high conserved synteny with human. Researchers use mice to answer various questions ranging from determining a pathological effect of knocked out/in gene to understanding drug metabolism. Our group developed >5000 quantitative targeted proteomics assays for 20 mouse tissues and determined the concentration ranges of a total of >1600 proteins using heavy labeled internal standards.

Process and Workflow for Preparation of Disparate Mouse Tissues for Proteomic Analysis.

We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets.

Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry.

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards.

Supermolecule-assisted imaging of low-molecular-weight quaternary-ammonium compounds by MALDI-MS of their non-covalent complexes with cucurbit[7]uril.

Cucurbit[7]uril was used to form non-covalent complexes with low-molecular-weight quaternary-ammonium compounds for their indirect analysis by MALDI-MS. By shifting the ion signals to a higher and interference-free mass region, the distributions of neurine, choline, and phosphocholine in rat brain were visualized by MALDI imaging with high selectivity and good sensitivity.

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g.

Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry.

Multiple reaction monitoring (MRM) is a key tool for biomarker validation and the translation of potential biomarkers into the clinic. To demonstrate the applicability of MRM towards achieving this goal, we set out to determine the concentration ranges of 267 plasma proteins, including 61 FDA-approved/LDT developed biomarkers, in 21 commercial human plasma lots, as well as to assess accuracy and precision. Each target protein was quantified by calculating the area ratio of the endogenous tryptic target peptide to its stable isotope-labelled internal standard equivalent and compared to a standard curve.

Improving Identification of Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.

An experimental and computational approach for identification of protein-protein interactions by chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale.

Multiplex quantitation of 270 plasma protein markers to identify a signature for early detection of colorectal cancer.

Blood-based protein biomarker signatures might be an alternative or supplement to existing methods for early detection of colorectal cancer (CRC) for population-based screening. The objective of this study was to derive a protein biomarker signature for early detection of CRC and its precursor advanced adenoma (AA). In a two-stage design, 270 protein markers were measured by liquid chromatography/multiple reaction monitoring/mass spectrometry in plasma samples of discovery and validation sets.

Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations.

For disordered proteins, ligand binding can be a critical event that changes their structural dynamics. The ability to characterize such changes would facilitate the development of drugs designed to stabilize disordered proteins, whose mis-folding is important for a number of pathologies, including neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. In this study, we used hydrogen/deuterium exchange, differential crosslinking, differential surface modification, and molecular dynamics (MD) simulations to characterize the structural changes in disordered proteins that result from ligand binding.

Insight into the Structure of the "Unstructured" Tau Protein.

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD), for the determination of the conformational ensemble of tau protein in solution. The predicted structures were in agreement with surface modification and long-distance crosslinking data.

MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2.

Background: MeCP2-a chromatin-binding protein associated with Rett syndrome-has two main isoforms, MeCP2-E1 and MeCP2-E2, differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest that they may also have non-overlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored.

Evaluation and Validation of Plasma Proteins Using Two Different Protein Detection Methods for Early Detection of Colorectal Cancer.

Objective: Plasma protein biomarkers could be an efficient alternative for population-based screening for early detection of colorectal cancer (CRC). The objective of this study was to evaluate and validate plasma proteins individually and as a signature for early detection of CRC.

Methods: In a three-stage design, proteins were measured firstly by liquid chromatography/multiple reaction monitoring-mass spectrometry (LC/MRM-MS) and later by proximity extension assay (PEA) in a discovery set consisting of 96 newly diagnosed CRC cases and 94 controls free of neoplasms at screening colonoscopy.

The Gut Microbiome and Metabolome of Two Riparian Communities in the Amazon.

During the last decades it has become increasingly clear that the microbes that live on and in humans are critical for health. The communities they form, termed microbiomes, are involved in fundamental processes such as the maturation and constant regulation of the immune system. Additionally, they constitute a strong defense barrier to invading pathogens, and are also intricately linked to nutrition.