Lysosomal targetomics of ghr KO mice shows chaperone-mediated autophagy degrades nucleocytosolic acetyl-coA enzymes.

Mice deficient in GHR (growth hormone receptor; ghr KO) have a dramatic lifespan extension and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in ghr KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice.Abbreviations: CMA: chaperone-mediated autophagy; DIA: data-independent acquisition; ghr KO: growth hormone receptor knockout; GO: gene ontology; I-WAT: inguinal white adipose tissue; KFERQ: a consensus sequence resembling Lys-Phe-Glu-Arg-Gln; LAMP2A: lysosomal-associated membrane protein 2A; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; PBS: phosphate-buffered saline; PI3K: phosphoinositide 3-kinase.

efam: an expanded, metaproteome-supported HMM profile database of viral protein families.

MOTIVATION: Viruses infect, reprogram and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases. RESULTS: Here, we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240 311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, pan-ecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from 'conservative' to 'eXtremely Conservative' resulted in 37 841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by ∼24% on average (up to ∼42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem. AVAILABILITY AND IMPLEMENTATION: The resources are available on the iVirus platform at (doi.org/10.25739/9vze-4143). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Calibration Using a Single-Point External Reference Material Harmonizes Quantitative Mass Spectrometry Proteomics Data between Platforms and Laboratories.

Mass spectrometry (MS) measurements are not inherently calibrated. Researchers use various calibration methods to assign meaning to arbitrary signal intensities and improve precision. Internal calibration (IC) methods use internal standards (IS) such as synthesized or recombinant proteins or peptides to calibrate MS measurements by comparing endogenous analyte signal to the signal from known IS concentrations spiked into the same sample. However, recent work suggests that using IS as IC introduces quantitative biases that affect comparison across studies because of the inability of IS to capture all sources of variation present throughout an MS workflow. Here, we describe a single-point external calibration strategy to calibrate signal intensity measurements to a common reference material, placing MS measurements on the same scale and harmonizing signal intensities between instruments, acquisition methods, and sites. We demonstrate data harmonization between laboratories and methodologies using this generalizable approach.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples.

Laser capture microdissection (LCM)-enabled region-specific tissue analyses are critical to better understand complex multicellular processes. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, impacting measurement robustness, quantification and throughput. Here, we coupled LCM with a proteomics workflow that provides fully automated analysis of proteomes from microdissected tissues. Benchmarking against the current state-of-the-art in ultrasensitive global proteomics (FASP workflow), our approach demonstrated significant improvements in quantification (~2-fold lower variance) and throughput (>5 times faster). Using our approach we for the first time characterized, to a depth of >3,400 proteins, the ontogeny of protein changes during normal lung development in microdissected alveolar tissue containing only 4,000 cells. Our analysis revealed seven defined modules of coordinated transcription factor-signaling molecule expression patterns, suggesting a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates.

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and ß-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores.

SNaPP: Simplified Nanoproteomics Platform for Reproducible Global Proteomic Analysis of Nanogram Protein Quantities.

Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious approach to achieve in-depth protein coverage and quantitative reproducibility. Biological samples are often severely mass limited and can preclude the application of more robust bulk sample processing workflows. In this study, we present a system that minimizes sample handling by using online immobilized trypsin digestion and solid phase extraction to create a simple, sensitive, robust, and reproducible platform for the analysis of nanogram-size proteomic samples. To demonstrate the effectiveness of our simplified nanoproteomics platform, we used the system to analyze preimplantation blastocysts collected on day 4 of pregnancy by flushing the uterine horns with saline. For each of our three sample groups, blastocysts were pooled from three mice resulting in 22, 22, and 25 blastocysts, respectively. The resulting proteomic data provide novel insight into mouse blastocyst protein expression on day 4 of normal pregnancy because we characterized 348 proteins that were identified in at least two sample groups, including 59 enzymes and blastocyst specific proteins (eg, zona pellucida proteins). This technology represents an important advance in which future studies could perform global proteomic analyses of blastocysts obtained from an individual mouse, thereby enabling researchers to investigate interindividual variation as well as increase the statistical power without increasing animal numbers. This approach is also easily adaptable to other mass-limited sample types.

Comparative Shotgun Proteomic Analysis of Wastewater-Cultured Microalgae: Nitrogen Sensing and Carbon Fixation for Growth and Nutrient Removal in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii was batch-cultured for 12 days under continuous illumination to investigate nitrogen uptake and metabolic responses to wastewater processing. Our approach compared two conditions: (1) artificial wastewater containing nitrate and ammonia and (2) nutrient-sufficient control containing nitrate as sole form of nitrogen. Treatments did not differ in final biomass; however, comparison of group proteomes revealed significant differences. Label-free shotgun proteomic analysis identified 2358 proteins, of which 92 were significantly differentially abundant. Wastewater cells showed higher relative abundances of photosynthetic antenna proteins, enzymes related to carbon fixation, and biosynthesis of amino acids and secondary metabolites. Control cells showed higher abundances of enzymes and proteins related to nitrogen metabolism and assimilation, synthesis and utilization of starch, amino acid recycling, evidence of oxidative stress, and little lipid biosynthesis. This study of the eukaryotic microalgal proteome response to nitrogen source, availability, and switching highlights tightly controlled pathways essential to the maintenance of culture health and productivity in concert with light absorption and carbon assimilation. Enriched pathways in artificial wastewater, notably, photosynthetic carbon fixation and biosynthesis of plant hormones, and those in nitrate only control, most notably, nitrogen, amino acid, and starch metabolism, represent potential targets for genetic improvement requiring targeted elucidation.

The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation.

Leaf-cutter ants are dominant herbivores in ecosystems throughout the Neotropics that feed on fungus gardens cultivated on fresh foliar biomass. Although recent investigations have shed light on how plant biomass is degraded in fungus gardens, the cycling of nutrients that takes place in these specialized microbial ecosystems is still not well understood. Here, using metabolomic and metaproteomic techniques, we examine the dynamics of nutrient turnover in these gardens. Our results reveal that numerous free amino acids and sugars are depleted throughout the process of biomass degradation, indicating that easily accessible nutrients from plant material are readily consumed by microbes in these ecosystems. Accumulation of cellobiose and lignin derivatives near the end of the degradation process is consistent with previous characterization of lignocellulases produced by the fungal cultivar of the ants. Our results also suggest that ureides may be an important source of nitrogen in fungus gardens, especially during nitrogen-limiting conditions. No free arginine was detected in our metabolomic experiments despite evidence that the host ants cannot produce this amino acid, suggesting that biosynthesis of this metabolite may be tightly regulated in fungus gardens. These results provide new insights into microbial community-level processes that underlie this important ant-fungus symbiosis.

The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique.

System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. The high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins.

Temporal analysis of xylose fermentation by Scheffersomyces stipitis using shotgun proteomics.

Proteomics and fermentation technology have begun to integrate to investigate fermentation organisms in bioprocess development. This is the first shotgun proteomics study employed to monitor the proteomes of Scheffersomyces stipitis during xylose fermentation under oxygen limitation. We identified 958 nonredundant proteins and observed highly similar proteomes from exponential to early stationary phases. In analyzing the temporal proteome, we identified unique expression patterns in biological processes and metabolic pathways, including alternative respiration salicylhydroxamic acid (SHAM) pathway, activation of glyoxylate cycle, expression of galactose enzymes, and secondary zinc-containing alcohol dehydrogenase and O-glycosyl hydrolases. We identified the expression of a putative, high-affinity xylose sugar transporter Xut1p, but low-affinity xylose transporters were absent. Throughout cell growth, housekeeping processes included oxidative phosphorylation, glycolysis, nonoxidative branch of the pentose phosphate pathway, gluconeogenesis, biosynthesis of amino acids and aminoacyl total RNA (tRNA), protein synthesis and proteolysis, fatty acid metabolism, and cell division. This study emphasized qualitative analysis and demonstrated that shotgun proteomics is capable of monitoring S. stipitis fermentation and identifying physiological states, such as nutrient deficiency.