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Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification. SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.

Regions of hypoxia occur in most if not all solid cancers. Although the presence of tumor hypoxia is a common occurrence, the levels of hypoxia and proportion of the tumor that are hypoxic vary significantly. Importantly, even within tumors, oxygen levels fluctuate due to changes in red blood cell flux, vascular remodeling and thermoregulation. Together, this leads to cyclic or intermittent hypoxia. Tumor hypoxia predicts for poor patient outcome, in part due to increased resistance to all standard therapies. However, it is less clear how cyclic hypoxia impacts therapy response. Here, we discuss the causes of cyclic hypoxia and, importantly, which imaging modalities are best suited to detecting cyclic vs. chronic hypoxia. In addition, we provide a comparison of the biological response to chronic and cyclic hypoxia, including how the levels of reactive oxygen species and HIF-1 are likely impacted. Together, we highlight the importance of remembering that tumor hypoxia is not a static condition and that the fluctuations in oxygen levels have significant biological consequences.

Nanomagnetic materials offer exciting avenues for probing cell mechanics and activating mechanosensitive ion channels, as well as for advancing cancer therapies. Most experimental works so far have used superparamagnetic materials. This report describes a first approach based on interfacing cells with lithographically defined microdiscs that possess a spin-vortex ground state. When an alternating magnetic field is applied the microdisc vortices shift, creating an oscillation, which transmits a mechanical force to the cell. Because reduced sensitivity of cancer cells toward apoptosis leads to inappropriate cell survival and malignant progression, selective induction of apoptosis is of great importance for the anticancer therapeutic strategies. We show that the spin-vortex-mediated stimulus creates two dramatic effects: compromised integrity of the cellular membrane, and initiation of programmed cell death. A low-frequency field of a few tens of hertz applied for only ten minutes was sufficient to achieve ∼90% cancer-cell destruction in vitro . Biocompatible, lithographically defined, ferromagnetic microdiscs that have a spin-vortex ground state oscillate when activated by an alternating magnetic field. This oscillation compromises the integrity of the cell membrane and initiates programmed cell death in ∼90% of cancer cells in vitro , even with a low-frequency field applied for only ten minutes.

Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35). Design Type(s) reference design • replicate design • protein expression profiling • quality control testing design Measurement Type(s) protein expression profiling Technology Type(s) mass spectrometry assay Factor Type(s) purification • Proteolysis Sample Characteristic(s) Homo sapiens • Monocyte • Neutrophil • Kidney • Muscle • Lung • Blood platelet • Gut • Blood plasma • 293 cell • THP-1 cell • U2 OS cell • HeLa cell • U2OS & HeLa • NCI60 • LNCAP cell • CAL-51 cell Machine-accessible metadata file describing the reported data (ISA-Tab format)

Data-independent acquisition (DIA) mass spectrometry, also known as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH), is a popular label-free proteomics strategy to comprehensively quantify peptides/proteins utilizing mass spectral libraries to decipher inherently multiplexed spectra collected linearly across a mass range. Although there are many spectral libraries produced worldwide, the quality control of these libraries is lacking. We present the DIALib-QC (DIA library quality control) software tool for the systematic evaluation of a library’s characteristics, completeness and correctness across 62 parameters of compliance, and further provide the option to improve its quality. We demonstrate its utility in assessing and repairing spectral libraries for correctness, accuracy and sensitivity. Most data-independent acquisition (DIA) methods depend on mass spectral libraries for peptide identification but tools to assess library quality are lacking. Here, the authors develop DIALib- QC for the systematic evaluation and correction of spectral libraries.

A high-conductivity two-dimensional (2D) hole gas, analogous to the ubiquitous 2D electron gas, is desirable in nitride semiconductors for wide-bandgap p-channel transistors. We report the observation of a polarization-induced high-density 2D hole gas in epitaxially grown gallium nitride on aluminium nitride and show that such hole gases can form without acceptor dopants. The measured high 2D hole gas densities of about 5 × 1013 per square centimeters remain unchanged down to cryogenic temperatures and allow some of the lowest p-type sheet resistances among all wide-bandgap semiconductors. The observed results provide a probe for studying the valence band structure and transport properties of wide-bandgap nitride interfaces.

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). Measurement(s) Proteome • database type spectral library Technology Type(s) SWATH MS protein profiling assay • mass spectrometry • Data-Independent Acquisition Sample Characteristic - Organism Escherichia coli Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.13078733

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400  S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus . From approximately 2.15 × 10 5   S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.