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A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes(1)) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure–function analyses of the superbinding mechanisms of sFes(1) and superSrc-SH2 (sSrc(1)), another SH2 superbinder. We grafted the superbinder motifs from sFes(1) and sSrc(1) into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

By far the largest contribution to ion detectability in liquid chromatography-driven mass spectrometry-based proteomics is the efficient generation of peptide ions by the electrospray source. To maximize the transfer of peptides from liquid to a gaseous phase to allow molecular ions to enter the mass spectrometer at micro-spray flow rates, an efficient electrospray process is required. Here we describe superior performance of new Vacuum-Insulated-Probe-Heated-ElectroSpray-Ionization source (VIP-HESI) coupled with micro-spray flow rate chromatography and Bruker timsTOF PRO mass spectrometer. VIP-HESI significantly improves chromatography signals in comparison to nano-spray ionization using the CaptiveSpray source and provides increased protein detection with higher quantitative precision, enhancing reproducibility of sample injection amounts. Protein quantitation of human K562 lymphoblast samples displayed excellent chromatographic retention time reproducibility (

The analysis of protein phosphorylation site identification by mass spectrometry-based methods continues to improve with increased efficiency at multiple points of the pipeline including affinity isolation, sample handling with automated protein digestion and phosphopeptide extraction routines, and more sensitive phosphopeptide detection capabilities. The role mass spectrometry parameters play in the quantity and quality of results is understudied, with some reports presenting improvements without sufficient details of how these parameters were derived, nor how they were fully optimized. Here, we systematically scrutinize and optimize parameters of two Orbitrap Tribrid mass spectrometers by varying instrument parameters at both the MS1 and MS2 levels using Titanium IMAC-enriched phosphopeptide samples to illustrate how the recovery of phosphopeptides, and the confidence of phosphosite localizations, vary accordingly. These optimized values are then used to compare different IMAC beads for phosphopeptide-enrichment, and optimal bead combinations used to further improve phosphopeptide detection. These results have implications for planning detailed phosphoprotein identification and quantitation experiments to provide confidence in detection, site localization and reproducibility.

Aging manifests as progressive deterioration in cellular and systemic homeostasis, requiring systems-level perspectives to understand the gradual molecular dysregulation of underlying biological processes. Here, we report systems-level changes in the molecular regulation of biological processes under multiple lifespan-extending interventions in mice and across age in humans. In mouse cohorts, Differential Rank Conservation (DIRAC) analyses of liver proteomics and transcriptomics show that mechanistically distinct prolongevity interventions tighten the regulation of aging-related biological modules, including fatty acid metabolism and inflammation processes. An integrated analysis of liver transcriptomics with mouse genome-scale metabolic model supports the shifts in fatty acid metabolism. Additionally, the difference in DIRAC patterns between proteins and transcripts suggests biological modules which may be tightly regulated via cap-independent translation. In a human cohort spanning the majority of the adult lifespan, DIRAC analyses of blood proteomics and metabolomics demonstrate that regulation of biological modules does not monotonically loosen with age; instead, the regulatory patterns shift according to both chronological and biological ages. Our findings highlight the power of systems-level approaches to identifying and characterizing the biological processes involved in aging and longevity.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4− late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4− late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector.IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

Summary We subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe., Graphical abstract, Highlights • Gut microbiome and diet of European salt miners determined using paleofeces • Until the Baroque, the microbiome resembled that of modern non-Westernized people • Food-fermenting fungi in Iron Age feces indicates blue cheese and beer consumption, Maixner et al. describe the gut microbiome and diet of European salt miners using paleofeces dating from the Bronze Age to the Baroque period. This analysis provides evidence for recent changes in the gut microbiome due to industrialization and for the consumption of fermented food and beverages in Iron Age Europe.

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion., Dengue (DENV) and Zika (ZIKV) viruses normally display as smooth spherical particles, while DENV can also become bumpy-surfaced, resulting in immune evasion. Here, Morrone et al. report DENV and ZIKV infectious club-shaped particles (clubSP) that display distinct antibody binding properties.