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  1. Abstract

    Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requiresa priorigenerated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish,E. coliand few other organisms. However, a spectral assay library for the extreme halophilic archaeonHalobacterium salinarumNRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotatedH. salinarumNRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. TheH. salinarumNRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

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  2. Medema, Marnix (Ed.)
    ABSTRACT The scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation in environmental acclimation are underexplored for organisms of the domain Archaea . Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating the transcriptome-wide locations of transcript processing sites (TPSs) and SmAP1 binding, the genome-wide locations of antisense RNAs (asRNAs), and the consequences of RNase_2099C knockout on the differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely being regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (transcriptome sequencing [RNA-Seq]) and protein levels (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry [SWATH-MS]) for 2,579 genes over four phases of batch culture growth in complex medium generated additional evidence for the conditional post-transcriptional regulation of 7% of all protein-coding genes. We demonstrate that post-transcriptional regulation may act to fine-tune specialized and rapid acclimation to stressful environments, e.g., as a switch to turn on gas vesicle biogenesis to promote vertical relocation under anoxic conditions and modulate the frequency of transposition by insertion sequence (IS) elements of the IS 200 /IS 605 , IS 4 , and IS H3 families. Findings from this study are provided as an atlas in a public Web resource ( ). IMPORTANCE While the transcriptional regulation landscape of archaea has been extensively investigated, we currently have limited knowledge about post-transcriptional regulation and its driving mechanisms in this domain of life. In this study, we collected and integrated omics data from multiple sources and technologies to infer post-transcriptionally regulated genes and the putative mechanisms modulating their expression at the protein level in Halobacterium salinarum NRC-1. The results suggest that post-transcriptional regulation may drive environmental acclimation by regulating hallmark biological processes. To foster discoveries by other research groups interested in the topic, we extended our integrated data to the public in the form of an interactive atlas ( ). 
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  3. Weiss, Louis M. (Ed.)
    ABSTRACT 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. 
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  4. Abstract Data-Independent Acquisition (DIA) is a method to improve consistent identification and precise quantitation of peptides and proteins by mass spectrometry (MS). The targeted data analysis strategy in DIA relies on spectral assay libraries that are generally derived from a priori measurements of peptides for each species. Although Escherichia coli ( E. coli ) is among the best studied model organisms, so far there is no spectral assay library for the bacterium publicly available. Here, we generated a spectral assay library for 4,014 of the 4,389 annotated E. coli proteins using one- and two-dimensional fractionated samples, and ion mobility separation enabling deep proteome coverage. We demonstrate the utility of this high-quality library with robustness in quantitation of the E. coli proteome and with rapid-chromatography to enhance throughput by targeted DIA-MS. The spectral assay library supports the detection and quantification of 91.5% of all E. coli proteins at high-confidence with 56,182 proteotypic peptides, making it a valuable resource for the scientific community. Data and spectral libraries are available via ProteomeXchange (PXD020761, PXD020785) and SWATHAtlas (SAL00222-28). 
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  5. Abstract The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP’s tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases. 
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