GeLC-FAIMS-MS workflow for in-depth middle-down proteomics.

Middle-down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu-C to generate large peptides (>3 kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS MS, for deep MDP.

Sample preparation for structural mass spectrometry via polyacrylamide gel electrophoresis.

Mass spectrometry is an analytical technique that can detect protein molecules with high sensitivity. Its use is not limited to the mere identification of protein components in biological samples, but is recently being utilized for large-scale analysis of protein structures in vivo as well. Top-down mass spectrometry with an ultra-high resolution mass spectrometer, for example, ionizes proteins in their intact state and allows rapid analysis of their chemical structure, which is used to determine proteoform profiles.

Size-Based Proteome Fractionation through Polyacrylamide Gel Electrophoresis Combined with LC-FAIMS-MS for In-Depth Top-Down Proteomics.

The combination of liquid chromatography (LC) and gas-phase separation by field-asymmetric ion mobility spectrometry (FAIMS) is a powerful proteoform separation system for top-down proteomics. Here, we present an in-depth top-down proteomics workflow, GeLC-FAIMS-MS, in which a molecular-weight-based proteome fractionation approach using SDS-polyacrylamide gel electrophoresis is performed prior to LC-FAIMS-MS. Since individual bands and their corresponding mass ranges require different compensating voltages (CVs), the MS parameters for each gel band and CV were optimized to increase the number and reliability of proteoform identifications further.

Absolute Proteome Quantification in the Gas-Fermenting Acetogen .

Microbes that can recycle one-carbon (C) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness.

Bottom-up/cross-linking mass spectrometry simplified sample processing on anion-exchange solid-phase extraction spin column.

We introduce a simple single-column protein digestion method for low-microgram-level samples containing sodium dodecyl sulfate and Coomassie dye that can be completed within a few hours.

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

Background: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process.

Results: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks.

De novo reconstitution of chromatin using wheat germ cell-free protein synthesis.

DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro. Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell-free protein synthesis.

Usefulness of tissue inhibitor of metalloproteinase 1 as a predictor of sustained remission in patients with antineutrophil cytoplasmic antibody-associated vasculitis.

Background: We previously identified tissue inhibitor of metalloproteinase 1 (TIMP-1) as a biomarker of disease activity that distinguished mildly or highly active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) from remission 6 months after the initiation of remission-induction therapy. In the present study, we investigated whether TIMP-1 is clinically useful as a predictor of relapse and sustained remission in AAV patients with microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) during maintenance therapy.

Methods: The relationship between serum TIMP-1 levels and clinical outcomes in AAV patients receiving maintenance therapy was assessed using the follow-up data of a Japanese large-cohort study (the RemIT-JAV-RPGN study) and data collected from AAV patients on maintenance therapy in our hospital (the MAAV-EU study).

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using ,'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment.

T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses.

Although the important roles of glycolysis in T cells have been demonstrated, the regulatory mechanism of glycolysis in activated T cells has not been fully elucidated. Furthermore, the influences of glycolytic failure on the T cell-dependent immune response in vivo remain unclear. We therefore assessed the role of glycolysis in the T cell-dependent immune response using T cell-specific Pgam1-deficient mice.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation.

Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy.

Background: Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins.

Identification of Proteins on Archived 2D Gels.

Separation technology of proteins from a complex mixture by two-dimensional gel (2D gel) was invented more than 40 years ago. With a good laboratory practice, the 2D gels are likely to be dried and stored at ambient temperature as archived record. Up until the beginning of this century, it had been difficult to identify the protein spots isolated on 2D gels.

H drives metabolic rearrangements in gas-fermenting .

Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H should diminish substrate loss to CO and facilitate production of reduced and energy-intensive products.

MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification.

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system.

Targeted proteomics reveals promising biomarkers of disease activity and organ involvement in antineutrophil cytoplasmic antibody-associated vasculitis.

Background: Targeted proteomics, which involves quantitative analysis of targeted proteins using selected reaction monitoring (SRM) mass spectrometry, has emerged as a new methodology for discovery of clinical biomarkers. In this study, we used targeted serum proteomics to identify circulating biomarkers for prediction of disease activity and organ involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

Methods: A large-scale SRM assay targeting 135 biomarker candidates was established using a triple-quadrupole mass spectrometer coupled with nanoflow liquid chromatography.

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry.

Bach2-Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop.

Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the Batf-Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production.

High-throughput production of a stable isotope-labeled peptide library for targeted proteomics using a wheat germ cell-free synthesis system.

Quantitative proteomic approaches using selected reaction monitoring (SRM) are currently limited by the difficulty in the preparation of reference standards. In this study, we demonstrat the high-throughput production of a reference peptide library using a wheat germ cell-free synthesis system to develop a large-scale SRM assay for targeted proteomics.

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues.

Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown.

High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.

Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

Enzymatic protein digestion using a dissolvable polyacrylamide gel and its application to mass spectrometry-based proteomics.

Enzymatic protein digestion in polyacrylamide gel has been used for sample pretreatment in mass spectrometry-based proteomics due to its effectiveness in removing contaminants that interfere with sample ionization. However, the difficulty of recovering the digested peptides from the solid gel matrix has been a drawback of this method. Here we have developed a novel in-gel digestion method to enhance peptide recovery using a dissolvable, bis-acrylylcystamine (BAC)-crosslinked polyacrylamide gel.