System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap

Yeast remains an important model for systems biology and for evaluating proteomics strategies. In-depth shotgun proteomics studies have reached nearly comprehensive coverage, and rapid, targeted approaches have been developed for this organism. Recently, we demonstrated that single LC-MS/MS analysis using long columns and gradients coupled to a linear ion trap Orbitrap instrument had an unexpectedly large dynamic range of protein identification (Thakur, S. S., Geiger, T., Chatterjee, B., Bandilla, P., Frohlich, F., Cox, J., and Mann, M. (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol. Cell Proteomics 10, 10.1074/mcp.M110.003699). Here we couple an ultra high pressure liquid chromatography system to a novel bench top Orbitrap mass spectrometer (Q Exactive) with the goal of nearly complete, rapid, and robust analysis of the yeast proteome. Single runs of filter-aided sample preparation (FASP)-prepared and LysC-digested yeast cell lysates identified an average of 3923 proteins. Combined analysis of six single runs improved these values to more than 4000 identified proteins/run, close to the total number of proteins expressed under standard conditions, with median sequence coverage of 23%. Because of the absence of fractionation steps, only minuscule amounts of sample are required. Thus the yeast model proteome can now largely be covered within a few hours of measurement time and at high sensitivity. Median coverage of proteins in Kyoto Encyclopedia of Genes and Genomes pathways with at least 10 members was 88%, and pathways not covered were not expected to be active under the conditions used. To study perturbations of the yeast proteome, we developed an external, heavy lysine-labeled SILAC yeast standard representing different proteome states. This spike-in standard was employed to measure the heat shock response of the yeast proteome. Bioinformatic analysis of the heat shock response revealed that translation-related functions were down-regulated prominently, including nucleolar processes. Conversely, stress-related pathways were up-regulated. The proteomic technology described here is straightforward, rapid, and robust, potentially enabling widespread use in the yeast and other biological research communities

The BINGO Project IX: Search for Fast Radio Bursts -- A Forecast for the BINGO Interferometry System

The Baryon Acoustic Oscillations (BAO) from Integrated Neutral Gas Observations (BINGO) radio telescope will use the neutral Hydrogen emission line to map the Universe in the redshift range $0.127 \le z \le 0.449$, with the main goal of probing BAO. In addition, the instrument optical design and hardware configuration support the search for Fast Radio Bursts (FRBs). In this work, we propose the use of a BINGO Interferometry System (BIS) including new auxiliary, smaller, radio telescopes (hereafter \emph{outriggers}). The interferometric approach makes it possible to pinpoint the FRB sources in the sky. We present here the results of several BIS configurations combining BINGO horns with and without mirrors ($4$ m, $5$ m, and $6$ m) and 5, 7, 9, or 10 for single horns. We developed a new {\tt Python} package, the {\tt FRBlip}, which generates synthetic FRB mock catalogs and computes, based on a telescope model, the observed signal-to-noise ratio (S/N) that we used to compute numerically the detection rates of the telescopes and how many interferometry pairs of telescopes (\emph{baselines}) can observe an FRB. FRBs observed by more than one baseline are the ones whose location can be determined. We thus evaluate the performance of BIS regarding FRB localization. We found that BIS will be able to localize 23 FRBs yearly with single horn outriggers in the best configuration (using 10 outriggers of 6 m mirrors), with redshift $z \leq 0.96$; the full localization capability depends on the number and the type of the outriggers. Wider beams are best to pinpoint FRB sources because potential candidates will be observed by more baselines, while narrow beams look deep in redshift. The BIS can be a powerful extension of the regular BINGO telescope, dedicated to observe hundreds of FRBs during Phase 1. Many of them will be well localized with a single horn + 6 m dish as outriggers.(Abridged)Comment: 12 pages, 9 figures, 5 tables, submitted to A&

Cosmoglobe DR1 results. I. Improved Wilkinson Microwave Anisotropy Probe maps through Bayesian end-to-end analysis

We present Cosmoglobe Data Release 1, which implements the first joint analysis of WMAP and Planck LFI time-ordered data, processed within a single Bayesian end-to-end framework. This framework builds directly on a similar analysis of the LFI measurements by the BeyondPlanck collaboration, and approaches the CMB analysis challenge through Gibbs sampling of a global posterior distribution, simultaneously accounting for calibration, mapmaking, and component separation. The computational cost of producing one complete WMAP+LFI Gibbs sample is 812 CPU-hr, of which 603 CPU-hrs are spent on WMAP low-level processing; this demonstrates that end-to-end Bayesian analysis of the WMAP data is computationally feasible. We find that our WMAP posterior mean temperature sky maps and CMB temperature power spectrum are largely consistent with the official WMAP9 results. Perhaps the most notable difference is that our CMB dipole amplitude is $3366.2 \pm 1.4\ \mathrm{\mu K}$, which is $11\ \mathrm{\mu K}$higher than the WMAP9 estimate and$2.5\ {\sigma}$ higher than BeyondPlanck; however, it is in perfect agreement with the HFI-dominated Planck PR4 result. In contrast, our WMAP polarization maps differ more notably from the WMAP9 results, and in general exhibit significantly lower large-scale residuals. We attribute this to a better constrained gain and transmission imbalance model. It is particularly noteworthy that the W-band polarization sky map, which was excluded from the official WMAP cosmological analysis, for the first time appears visually consistent with the V-band sky map. Similarly, the long standing discrepancy between the WMAP K-band and LFI 30 GHz maps is finally resolved, and the difference between the two maps appears consistent with instrumental noise at high Galactic latitudes. All maps and the associated code are made publicly available through the Cosmoglobe web page.Comment: 65 pages, 61 figures. Data available at cosmoglobe.uio.no. Submitted to A&

BeyondPlanck IV. On end-to-end simulations in CMB analysis -- Bayesian versus frequentist statistics

End-to-end simulations play a key role in the analysis of any high-sensitivity CMB experiment, providing high-fidelity systematic error propagation capabilities unmatched by any other means. In this paper, we address an important issue regarding such simulations, namely how to define the inputs in terms of sky model and instrument parameters. These may either be taken as a constrained realization derived from the data, or as a random realization independent from the data. We refer to these as Bayesian and frequentist simulations, respectively. We show that the two options lead to significantly different correlation structures, as frequentist simulations, contrary to Bayesian simulations, effectively include cosmic variance, but exclude realization-specific correlations from non-linear degeneracies. Consequently, they quantify fundamentally different types of uncertainties, and we argue that they therefore also have different and complementary scientific uses, even if this dichotomy is not absolute. Before BeyondPlanck, most pipelines have used a mix of constrained and random inputs, and used the same hybrid simulations for all applications, even though the statistical justification for this is not always evident. BeyondPlanck represents the first end-to-end CMB simulation framework that is able to generate both types of simulations, and these new capabilities have brought this topic to the forefront. The Bayesian BeyondPlanck simulations and their uses are described extensively in a suite of companion papers. In this paper we consider one important applications of the corresponding frequentist simulations, namely code validation. That is, we generate a set of 1-year LFI 30 GHz frequentist simulations with known inputs, and use these to validate the core low-level BeyondPlanck algorithms; gain estimation, correlated noise estimation, and mapmaking

A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage

Online parallel accumulation - serial fragmentation (PASEF) with a novel trapped on mobility mass spectrometer

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et aL, PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, Max-Quant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease