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.

Deep proteome coverage advances knowledge of Treponema pallidum protein expression profiles during infection.

Comprehensive proteome-wide analysis of the syphilis spirochete, Treponema pallidum ssp. pallidum, is technically challenging due to high sample complexity, difficulties with obtaining sufficient quantities of bacteria for analysis, and the inherent fragility of the T. pallidum cell envelope which further complicates proteomic identification of rare T.

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.

Sample Preparation Methods for Targeted Single-Cell Proteomics.

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution.

Chemoproteomic identification of CO-dependent lysine carboxylation in proteins.

Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO are, accordingly, of great interest. Because CO is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues.

Profiling of antimicrobial metabolites of plant growth promoting spp. isolated from different plant hosts.

Unlabelled: In this study, nine strains of and , and two isolates of sp: At1RP4 and RS-1, were characterized for the in-vitro production of secondary metabolites in LB, DMB, and King's B media, and of the genes responsible for the production of antagonistic metabolites. Based on 16S rRNA gene sequence, isolates At1RP4 and RS-1 were identified as strains of and . Five phenazine derivatives comprising phenazine, phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine-1-carboxylic acid (2-OH-Phz-1-COOH), phenazine-1,6-dicarboxylic acid (Phz-1,6-di-COOH), and 2-hydroxyphenazine (2-OH-Phz) were produced by all strains in all three culture media including DMB, King's B and LB.

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated.

Biosecurity risks posed by a large sea-going passenger vessel: challenges of terrestrial arthropod species detection and eradication.

Large sea-going passenger vessels can pose a high biosecurity risk. The risk posed by marine species is well documented, but rarely the risk posed by terrestrial arthropods. We conducted the longest running, most extensive monitoring program of terrestrial arthropods undertaken on board a passenger vessel.

Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays.

Mouse is the predominant experimental model for the study of human disease due, in part, to phylogenetic relationship, ease of breeding, and the availability of molecular tools for genetic manipulation. Advances in genome-editing methodologies, such as CRISPR-Cas9, enable the rapid production of new transgenic mouse strains, necessitating complementary high-throughput and systematic phenotyping technologies. In contrast to traditional protein phenotyping techniques, multiple reaction monitoring (MRM) mass spectrometry can be highly multiplexed without forgoing specificity or quantitative precision.

Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles.

To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches.

Zero-tolerance biosecurity protects high-conservation-value island nature reserve.

Barrow Island, north-west coast of Australia, is one of the world's significant conservation areas, harboring marsupials that have become extinct or threatened on mainland Australia as well as a rich diversity of plants and animals, some endemic. Access to construct a Liquefied Natural Gas (LNG) plant, Australia's largest infrastructure development, on the island was conditional on no non-indigenous species (NIS) becoming established. We developed a comprehensive biosecurity system to protect the island's biodiversity.

Metabolomic profiling of prostate cancer by matrix assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry imaging using Matrix Coating Assisted by an Electric Field (MCAEF).

In this work, we combined the use of two MALDI matrices (quercetin and 9-aminoacridine), a recently developed new matrix coating technique - matrix coating assisted by an electric field (MCAEF), and matrix-assisted laser desorption/ionization - Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect and image endogenous compounds in the cancerous and non-cancerous regions of three human prostate cancer (stage II) tissue specimens. After three rounds of imaging data acquisitions (i.e.

Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by , although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by identified ∼80 parasite proteins, with the majority being -sialidases.

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment.

An increasingly popular "absolute" quantitative technique involves the SRM or MRM approach with stable isotope-labeled standards (SIS). Using this approach, many proteins in human plasma/serum have been quantified for biomarker assessment and disease stratification. Due to the complexity of plasma and the invasive nature of its collection, alternative biosamples are currently being explored.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards.

Insights from the pollination drop proteome and the ovule transcriptome of Cephalotaxus at the time of pollination drop production.

Background And Aims: Many gymnosperms produce an ovular secretion, the pollination drop, during reproduction. The drops serve as a landing site for pollen, but also contain a suite of ions and organic compounds, including proteins, that suggests diverse roles for the drop during pollination. Proteins in the drops of species of Chamaecyparis, Juniperus, Taxus, Pseudotsuga, Ephedra and Welwitschia are thought to function in the conversion of sugars, defence against pathogens, and pollen growth and development.

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers.

In this work, we combined a newly developed matrix coating technique - matrix coating assisted by an electric field (MCAEF) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal-to-noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500-37500 mass range on a time-of-flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100-A9, S100-A10, and S100-A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2, a fragment of cAMP-regulated phosphoprotein 19, 3 apolipoproteins (C-I, A-I, and A-II), 2 S100 proteins (A6 and A8), β-microseminoprotein, tumor protein D52, α-1-acid glycoprotein 1, heat shock protein β-1, prostate-specific antigen, and 2 unidentified large peptides at m/z 5002.

Fast Comparative Structural Characterization of Intact Therapeutic Antibodies Using Hydrogen-Deuterium Exchange and Electron Transfer Dissociation.

Higher-order structural characterization plays an important role in many stages of therapeutic antibody production. Herein, we report a new top-down mass spectrometry approach for characterizing the higher-order structure of intact antibodies, by combining hydrogen/deuterium exchange (HDX), subzero temperature chromatography, and electron transfer dissociation on the Orbitrap mass spectrometer. Individual IgG domain-level deuteration information was obtained for 6 IgG domains on Herceptin (HER), which included the antigen binding sites.

Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.

Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date.

Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.

Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF.

Reducing insecticide use in broad-acre grains production: an Australian study.

Prophylactic use of broad-spectrum insecticides is a common feature of broad-acre grains production systems around the world. Efforts to reduce pesticide use in these systems have the potential to deliver environmental benefits to large areas of agricultural land. However, research and extension initiatives aimed at decoupling pest management decisions from the simple act of applying a cheap insecticide have languished.

Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS.

Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples.