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Capturing proteins on anion-exchange discs facilitates concentration of diluted samples and removal of contaminants, allowing more efficient sample pretreatment for bottom-up/cross-linking mass spectrometry than in-solution and in-gel.

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. We developed an easy-to-implement and inexpensive procedure combining the earlier established Passively Eluting Proteins from Polyacrylamide gels as Intact species (PEPPI) protocol with an optimized Anion-Exchange disk-assisted Sequential sample Preparation (AnExSP) method for the removal of stains and SDS. The protocol was compared with a methanol-chloroform-water (MCW)-based protein precipitation protocol. The results show that the PEPPI-AnExSP procedure is better suited for the identification of low-molecular-weight proteoforms, whereas the MCW-based protocol showed advantages for higher-molecular-weight proteoforms. Moreover, complementary results were observed with the two methods in terms of hydrophobicity and isoelectric points of the identified proteoforms. In total, 8500 proteoforms could be identified in a human proteome standard, showing the effectiveness of the gel-based sample fractionation approaches in combination with LC-FAIMS-MS.

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.

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. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. Conclusions We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short also allows a second cycle of assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. We refer to this approach as the ALACAT strategy as it permits a la carte design of quantification standards.

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 N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.

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. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

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). Results In the RemIT-JAV RPGN study, serum levels of TIMP-1 were significantly higher in mildly active AAV patients with MPA and GPA 6 months after the initiation of remission-induction therapy than in patients in remission. Regarding maintenance therapy, elevated levels of TIMP-1 in patients in remission were associated with relapse and/or difficulty reducing the glucocorticoid dosage after 6 to 12 months. In the MAAV-EU study, serum levels of TIMP-1 were elevated in relapsed patients 6 months before relapse, earlier than the increase in serum levels of CRP. Analyses of both studies revealed that approximately 30% of patients in remission with a serum TIMP-1 level ≥ 150 ng/mL relapsed after 6 to 12 months, while the majority of patients with a TIMP-1 level Conclusion We herein demonstrated that TIMP-1 is more useful as a predictive biomarker of sustained remission than as a predictor of relapse in maintenance therapy for AAV. TIMP-1 levels

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism., T cells can alter their metabolism during activation and differentiation. Here the authors show that the tumor suppressor menin regulates CD8 T-cell fate via the modulation of central carbon metabolism.

Polyacrylamide gel electrophoresis (PAGE) is a powerful technique for separating proteins from complex biological samples. However, the difficulty in recovering proteins with high yields from polyacrylamide matrices often precludes further analyses of intact proteins. Here, we propose a novel experimental workflow named Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows intact mass spectrometry (MS) of PAGE separated proteins. We discovered that staining proteins with certain Coomassie brilliant blue formulations immediately after PAGE improves the efficiency of extraction in a medium with pH 7–11. Post-staining, proteins spanning a broad range of molecular weights were recovered efficiently in a 10-minute procedure. High recovery yields were also obtained from dried and archived gels. This workflow is effective for top-down proteomics analysis of the target molecular region in the gel. An alternative procedure was developed for the extraction of protein complexes exceeding 400 kDa, which were separated using native PAGE, from unstained gels. Non-covalent hemoglobin tetramer, purified from cell lysate with two-dimensional native PAGE and extracted with the mild detergent octyl-β-Dglucopyranoside, was amenable for native MS analysis. We anticipate that the established workflow will facilitate the purification, storage, and transport of proteins destined for detailed characterization by MS.

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. Results Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins. Conclusions We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes. Electronic supplementary material The online version of this article (10.1186/s13007-019-0427-7) contains supplementary material, which is available to authorized users.

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.

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 H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model. CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a fourfold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by fivefold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood–Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

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 H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model. CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a fourfold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by fivefold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood–Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses. Electronic supplementary material The online version of this article (10.1186/s13068-018-1052-9) contains supplementary material, which is available to authorized users.

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. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. One of the major challenges in proteomics is absolute quantification of individual proteins. The predominant technology in large scale protein quantification is MS of (usually tryptic) peptides derived from proteolysis of the proteome in vitro and it is well understood that although mass spectrometers can deliver linearity of response over many orders of magnitude, the response factor (signal intensity per mol of peptide) varies considerably among individual peptides (1, 2). One outcome is that commonly used “label-free” methods that sum the precursor ion intensities for the peptides derived from a single protein, are excellent for relative quantification, but are less satisfactory for absolute quantification. MS-based absolute quantification of proteins can be supported by external standards that are analyzed before and/or after the analyte or by stable-isotope labeled internal standards that are coanalyzed and which define the response factor for each peptide (3). These peptides can be individually synthesized and quantified (4) and there have been some remarkable large-scale studies. However, large numbers of accurately quantified peptides are costly. Further, a commercially produced, accurately quantified standard peptide is a finite resource and is hence best focused on low numbers of assays of a small number of target proteins. Intact protein standards (5⇓–7), or large fragments (8) provide multiple potential peptides for quantification of the targets. In 2005, a novel approach to the creation of standard peptides by biosynthesis was proposed in the form of QconCATs (9⇓⇓⇓–13). QconCATs are artificial proteins that are concatenations of standard peptides from multiple natural proteins, sometimes interspersed by short peptides to recapitulate the primary sequence context of the natural counterpart (14, 15). Peptides suitable for quantification are referred to as Q-peptides, and are not synonymous with proteotypic peptides, as the latter term refers to peptides, unique to one protein, that drive protein identification, not quantification. QconCATs genes are synthesized de novo and are routinely expressed in E. coli cultured in media supplemented with appropriate stable isotope labeled amino acids, such that peptides derived from QconCATs are discriminable from natural peptides within the mass spectrometer. The purified QconCATs are mixed with the biological analyte sample and coproteolyzed to generate a mixture of labeled (standard) and unlabeled (analyte) peptide pairs that can be analyzed by liquid chromatography coupled to MS to yield absolute quantification of the analyte proteins. QconCATs have the added advantage that with appropriate control of proteolysis (11) all standards are, by definition, in a 1:1 ratio, rendering independent quantification of each standard unnecessary; a single common peptide can function to quantify the QconCAT (13). However, successful expression of novel QconCATs in E. coli is not always guaranteed. In a large-scale quantification project that used over 100 independently designed and expressed QconCATs, we discovered that ∼1 in 10 of the concatamers would fail to express, even when a range of expression conditions were explored. Further, at a low frequency, some QconCATs were prone to proteolysis in the bacterial cell or during purification, rendering them of reduced value for quantification. Effective QconCAT deployment across large scale proteome quantification studies would require a high level of confidence in expression of every new construct. In addition, living-cell based synthesis systems are not ideal for high-throughput preparation of multiple QconCATs and many mass spectrometry laboratories are not equipped for the basic molecular biology that would be needed to subclone and express recombinant proteins. To enhance the potential of QconCAT technology for large-scale proteome quantification, we here focus on a wheat germ cell-free protein synthesis system (WGCFS)1 as a major enhancement to the workflow of high throughput QconCAT synthesis. WGCFS, which uses the powerful translation system for germination stored in wheat germ, realizes the highest yield of translation among commercially available eukaryotic derived cell-free systems (16⇓⇓⇓–20). Using WGCFS, we previously demonstrated the feasibility of synthesis of single, small QconCATs, typically 25 kDa (21). In the present study, we first assessed whether WGCFS could be used to express more typical QconCATs at approx. 60 kDa (for quantification of ∼25 proteins at two peptides per target protein), whether WGCFS would rescue “failed” QconCATs and whether this cell free system was able to reduce the risk of proteolytic degradation. Further, we established whether an additional step in efficiency could be derived from coexpression of multiple QconCATs in a single WGCFS reaction.

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.

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. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

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. After enzymatic protein digestion in BAC gel, we completely dissolved the gel by reductive treatment with tris-(2-carboxyethyl) phosphine to release the digested peptides from the gel. Our analysis revealed that the reductive dissolution of the BAC gel enhances the peptide recovery, which has a significantly higher protein identification capability than the conventional method, using an insoluble polyacrylamide gel. In addition, protein samples trapped in dehydrated BAC gel were stable at room temperature and reproducible sample recovery was obtained after storage for one week. These results indicate that the proposed method could be an effective tool for conducting sample pretreatment for mass spectrometry-based protein analysis.

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. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2–Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf–Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2–Batf interactions are required to prevent an excessive Th2 response., Bach2 limits T cell effector functions. Here the authors show that Bach2–Batf complex antagonizes the recruitment of the Batf–Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production, and describe mechanisms of negative feedback by which Bach2 restricts Baft-mediated Th2 response.