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At least 55 records · Page 3

Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography–Mass Spectrometry for High-Throughput Single-Cell Proteomics

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. Here, we have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ~77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Abstract Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

59 BASIC BIOLOGICAL SCIENCES↗

Robust Anthropogenic Signal Identified in the Seasonal Cycle of Tropospheric Temperature

Previous work identified an anthropogenic fingerprint pattern in T AC (x, t), the amplitude of the seasonal cycle of mid- to upper-tropospheric temperature (TMT), but did not explicitly consider whether fingerprint identification in satellite T AC (x, t) data could have been influenced by real-world multidecadal internal variability (MIV). Here we address this question here using large ensembles (LEs) performed with five climate models. LEs provide many different sequences of internal variability noise superimposed on an underlying forced signal. Despite differences in historical external forcings, climate sensitivity, and MIV properties of the five models, their T AC (x, t) fingerprints are similar and statistically identifiable in 239 of the 240 LE realizations of historical climate change. Comparing simulated and observed variability spectra reveals that consistent fingerprint identification is unlikely to be biased by model underestimates of observed MIV. Even in the presence of large (factor of 3–4) intermodel and inter-realization differences in the amplitude of MIV, the anthropogenic fingerprints of seasonal cycle changes are robustly identifiable in models and satellite data. This is primarily due to the fact that the distinctive, global-scale fingerprint patterns are spatially dissimilar to the smaller-scale patterns of internal T AC (x, t) variability associated with the Atlantic multidecadal oscillation and El Niño–Southern Oscillation. The robustness of the seasonal cycle detection and attribution results shown here, taken together with the evidence from idealized aquaplanet simulations, suggest that basic physical processes are dictating a common pattern of forced T AC (x, t) changes in observations and in the five LEs. The key processes involved include GHG-induced expansion of the tropics, lapse-rate changes, land surface drying, and sea ice decrease.

54 ENVIRONMENTAL SCIENCES↗

Chlamydomonas reinhardtii responses to Fe-excess, Fe-deficiency, and Fe-limitation in either photoautotrophic or mixotrophic growth

A systems level analysis of Chlamydomonas reinhardtii grown photoautotrophically or mixotrophically with a reduced carbon source, acetate, under four different defined Fe stages of Fe-replete, Fe-deficient, Fe-limited, or Fe-excess. Samples were digested with trypsin, labeled with TMT 10-Plex, then analyzed by LC-MS/MS. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline. [doi:10.25345/C5707X12X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

59 BASIC BIOLOGICAL SCIENCES↗

Rhodotorula toruloides Nitrogen Limitation PTM Profiling Multi-Omics (TZ-DP1)

The purpose of this experiment was to evaluate the regulatory stress response of Oleaginous yeast species Rhodotorula toruloides NBRC 0880 (JGI strain IFO0880 v4.0) under nitrogen-rich and nitrogen-limited conditions over time. Time course experimental samples (0, 24, 48, and 72 hours after inoculation) were prepared using a semi-automated multi-PTM proteomic approach, using tandem mass tag 18-plex (TMT18), and lipidome remodeling for downstream multi-omics analysis. Processed datasets are openly accessible from PNNL DataHub and contain secondary processed proteomic (redox, phospho, and global TMT) and lipidomic (positive and negative ion mode) results files and experimental design metadata.

59 BASIC BIOLOGICAL SCIENCES↗

Microbial Co-Culture Control Proteomics (MC-DP1)

Co-cultured S. elongatus PCC 7942 CscB/SPS and R. toruloides IFO0880 presented as an effective photosynthesis-driven biofuel production platform. The goal of this experiment was to understand the molecular mechanism of this co-culture system at redox post-translational modification level. The redox proteome of the co-cultured strains was compared to a mono-cultured S. elongatus in light or dark conditions. Samples were processed using a resin-assisted capture (RAC) workflow with TMT labeling to enrich and quantify modified cysteines at proteome level. The datasets were generated by a Q Exactive Plus Orbitrap Mass Spectrometer coupled with a Waters nanoAcquity UPLC, then searched by MSGF+ for downstream redox PTM analysis.

Source record↗

S. elongatus PCC 7942 Carbon Metabolism Proteomics (MC-DP2)

The purpose of this experiment was to examine the redox proteome of S. elongatus PCC 7942 CscB/SPS under different light conditions (light and dark) and culture conditions (dense and dilute optical density). Samples were processed using a resin-assisted capture (RAC) workflow with TMT labeling to enrich and quantify protein cysteines which were differentially oxidized under four conditions. The datasets were generated by a Q Exactive Plus Orbitrap Mass Spectrometer coupled with a Waters nanoAcquity UPLC, then searched by MSGF+ for downstream redox PTM analysis.

59 BASIC BIOLOGICAL SCIENCES↗

PPI DataHub Project Data Package: S. elongatus PCC 7942 Limited Proteolysis and Thermal Proteome Profiling Structural Proteomics (JM-PB-DP3)

The purpose of this experiment was to investigate structural alterations in proteins involved in central carbon metabolism and photosynthetic electron transfer pathways in Synechococcus elongatus PCC 7942. Sample data was obtained from S. elongatus cell lysates using three complementary mass spectrometry (MS) techniques using limited proteolysis (LiP-MS), thermal proteome profiling (TPP-MS), and redox enrichment (Redox-MS) in evaluating alterations solvent accessibility and structural stability caused by light perturbation at the molecular level. Experimentally processed sample data for LiP and TPP proteomic datasets were derived from the same cell culture stock, prepared simultaneously in parallel, and acquired by mass spectrometry. Processed datasets are openly accessible from the download button and contain secondary processed proteomic results files, computed outputs, and supporting metadata materials. Experimental samples processed for LiP-MS label-free quantification (LFQ) or TPP-MS tandem mass tag (TMT) 10-plex were acquired using a Q-Exactive HF-X mass spectrometer and processed/compiled using either MSGF+ (v2024.03.26) or ​​​​PlexedPiper for proteome evaluation. Additional software supporting downstream proteomic analysis include FragPipe (v.4.0), MSFragger (v.22.1), and an adapted Microbial Isolate LiP Analysis Workflow (located at Zenodo). Processed proteomic data downloads include a sample naming key, normalized quantification results files, and processed protein annotated abundance files.

59 BASIC BIOLOGICAL SCIENCES↗

Segment alignment control system

The segmented primary mirror for the LDR will require a special segment alignment control system to precisely control the orientation of each of the segments so that the resulting composite reflector behaves like a monolith. The W.M. Keck Ten Meter Telescope will utilize a primary mirror made up of 36 actively controlled segments. Thus the primary mirror and its segment alignment control system are directly analogous to the LDR. The problems of controlling the segments in the face of disturbances and control/structures interaction, as analyzed for the TMT, are virtually identical to those for the LDR. The two systems are briefly compared.

Aubrun, JEAN-N.↗

On magnetodynamic effects initiated by a high-speed impact of a large cosmic body upon the Earth's surface

The impact of a large cosmic body with typical size R approximately = 1 km (mass M approximately = 4-10 Gt for a stony or icy body) moving with velocity V approximately = 50-70 km/s (kinetic energy of the order of 10 exp 21 J or 10 exp 6 Mt of TMT) on the Earth's surface leads to a full vaporization of a body and of a significant part of substance of the upper layers of the Earth and even to the ionization of this vapor cloud. As a result, a hypersonic jet of air and erosion plasma is formed. The kinetic energy E sub J is far above the total energy of the geomagnetic field of the Earth (approximately equivalent to the energy of 100 Mt) and the total mass of a fast-moving part of the jet M sub j approximately = 10 exp 12 kg is far above the mass of atmosphere in the jet expansion cone. Thus, the jet will propagate practically inertially with the constant mean velocity U approximately = 10-20 km/s and even higher. The interaction of this plasma jet with the Earth's magnetic field causes magnetodynamic effects similar to those which are produced by cosmic nuclear explosions but of a larger scale. The preliminary results of experimental and numerical modeling of the plasma jet-magnetosphere interaction are presented.

Nemchinov, I. V.↗

High Contrast Imaging with the JWST NIRCAM Coronagraph

Relative to ground-based telescopes, the James Webb Space Telescope (JWST) will have a substantial sensitivity advantage in the 2.2-5pm wavelength range where brown dwarfs and hot Jupiters are thought to have significant brightness enhancements. To facilitate high contrast imaging within this band, the Near-Infrared Camera (NIRCAM) will employ a Lyot coronagraph with an array of band-limited image-plane occulting spots. In this paper, we provide the science motivation for high contrast imaging with NIRCAM, comparing its expected performance to that of the Keck, Gemini and 30 m (TMT) telescopes equipped with Adaptive Optics systems of different capabilities. We then describe our design for the NIRCAM coronagraph that enables imaging over the entire sensitivity range of the instrument while providing significant operational flexibility. We describe the various design tradeoffs that were made in consideration of alignment and aberration sensitivities and present contrast performance in the presence of JWST's expected optical aberrations. Finally we show an example of a that can provide 10-5 companion sensitivity at sub-arcsecond separations.

coronagraph↗

The Advanced Technology Large Aperture Space Telescope (ATLAST): Science Drivers, Technology Developments, and Synergies with Other Future Facilities

The Advanced Technology Large-Aperture Space Telescope (ATLAST) is a concept for an 8-meter to 16-meter UVOIR space observatory for launch in the 2025-2030 era. ATLAST will allow astronomers to answer fundamental questions at the forefront of modern astrophysics, including "Is there life elsewhere in the Galaxy?" We present a range of science drivers that define the main performance requirements for ATLAST (8 to 16 milliarcsec angular resolution, diffraction limited imaging at 0.5 m wavelength, minimum collecting area of 45 square meters, high sensitivity to light wavelengths from 0.1 m to 2.4 m, high stability in wavefront sensing and control). We will also discuss the synergy between ATLAST and other anticipated future facilities (e.g., TMT, EELT, ALMA) and the priorities for technology development that will enable the construction for a cost that is comparable to current generation observatory-class space missions.

Postman, Marc↗

Control System Modeling for the Thirty Meter Telescope Primary Mirror

The Thirty Meter Telescope primary mirror is composed of 492 segments that are controlled to high precision in the presence of wind and vibration disturbances, despite the interaction with structural dynamics. The higher bandwidth and larger number of segments compared with the Keck telescopes requires greater attention to modeling to ensure success. We focus here on the development and validation of a suite of quasi-static and dynamic modeling tools required to support the design process, including robustness verification, performance estimation, and requirements flowdown. Models are used to predict the dynamic response due to wind and vibration disturbances, estimate achievable bandwidth in the presence of control-structure-interaction (CSI) and uncertainty in the interaction matrix, and simulate and analyze control algorithms and strategies, e.g. for control of focus-mode, and sensor calibration. Representative results illustrate TMT performance scaling with parameters, but the emphasis is on the modeling framework itself.

uncetainty↗

Titan Science with the James Webb Space Telescope

The James Webb Space Telescope (JWST), scheduled for launch in 2018, is the successor to the Hubble Space Telescope (HST) but with a signicantly larger aperture (6.5 m) and advanced instrumentation focusing on infrared science (0.6-28.0 microns). In this paper, we examine the potential for scientic investigation of Titan using JWST, primarily with three of the four instruments: NIRSpec, NIRCam, and MIRI, noting that science with NIRISS will be complementary. Five core scientic themes are identied: (1) surface (2) tropospheric clouds (3) tropospheric gases (4) stratospheric composition, and (5) stratospheric hazes. We discuss each theme in depth, including the scientic purpose, capabilities, and limitations of the instrument suite and suggested observing schemes. We pay particular attention to saturation, which is a problem for all three instruments, but may be alleviated for NIRCam through use of selecting small sub-arrays of the detectorssufcient to encompass Titan, but with signicantly faster readout times. We nd that JWST has very signicant potential for advancing Titan science, with a spectral resolution exceeding the Cassini instrument suite at near-infrared wavelengths and a spatial resolution exceeding HST at the same wavelengths. In particular, JWST will be valuable for time-domain monitoring of Titan, given a ve- to ten-year expected lifetime for the observatory, for example, monitoring the seasonal appearance of clouds. JWST observations in the post-Cassini period will complement those of other large facilities such as HST, ALMA, SOFIA, and next-generation ground-based telescopes (TMT, GMT, EELT).

Nixon, Conor A.↗

Synergies of Subaru and CGI

Given the limited observing time and demanding scenarios of the WFIRST coronagraph instrument (CGI), it is critical to consider how Subaru observations can benefit its observing program. Subaru telescope has a suite of instruments with their adaptive optics (AO) and extreme adaptive optics modules (SCExAO). With SCExAO, the Subaru telescope is capable of detection and spectral characterization of binaries and bright (greater than 5(exp -6) contrast) companions in the near-infrared. This will enable the vetting of targets, disk detection and characterization, and potentially some additional science should CGI identify interesting targets during its technology demonstration and potential guest observer program. Additionally, large companions that are within the inner working angle of the coronagraph can be identified using the VAMPIRES aperture masking interferometer. With highly complementary target brightness and significantly overlapping fields of view, there is a great deal of potential for combined observations with Subaru and CGI. This will represent the first time single observations spanning the visible to near-infrared will be possible for high contrast imaging. We will discuss the overlap of instrumentation over time, the implication of instrument evolution as TMT comes online, and how this can be used to improve both science and technology demonstrations for CGI.

adaptive optics (AO↗

Synergies Between the Kepler, K2 and TESS Missions with the PLATO Mission (Revised)

Two transit survey missions will have been flown by NASA prior to the launch of ESA's PLATO Mission in 2026, laying the groundwork for exoplanet discovery via the transit method. The Kepler Mission, which launched in 2009, collected data on its 100+ square degree field of view for four years before failure of a reaction wheel ended its primary mission. The results from Kepler include 2300+ confirmed or validated exoplanets, 2200+ planetary candidates, 2100+ eclipsing binaries. Kepler also revolutionized the field of asteroseismology by measuring the pressure mode oscillations of over 15000 solar-like stars spanning the lifecycle of such stars from hydrogen-burning dwarfs to helium-burning red giants. The re-purposed Kepler Mission, dubbed K2, continues to observe fields of view in and near the ecliptic plane for 80 days each, significantly broadening the scope of the astrophysical investigations as well as discovering an additional 156 exoplanets to date. The TESS mission will launch in 2017 to conduct an all-sky survey for small exoplanets orbiting stars 10X closer and 100X brighter than Kepler exoplanet host stars, allowing for far greater follow-up and characterization of their masses as well as their sizes for at least 50 small planets. Future assets such as James Webb Space Telescope, and ground-based assets such as ESOs Very Large Telescope (VLT) array, the Exremely Large Telescope (ELT), and the Thirty Meter Telescope (TMT) will be able to characterize the atmospheric composition and properties of these small planets. TESS will observe each 24 X 96 field of view for 30 days and thereby cover first the southern and then the northern hemisphere over 13 pointings during each year of the primary mission. The pole-most camera will observe the James Webb continuous viewing zone for one year in each hemisphere, permitting much longer period planets to be detected in this region. The PLATO mission will seek to detect habitable Earth-like planets with an instrument composed of 26 small telescopes in several 2232 square deg FOVs with a range of observation durations over a mission lifetime of up to eight years. This paper summarizes the findings of the KeplerK2 missions, previews the likely results from the TESS mission, and explores the lessons learned and to be learned from these prior missions that can be incorporated into the observation and data reduction strategy for the PLATO Mission so as to maximize the science return.

exoplanet detection↗

The Alignment and Phasing System for the Thirty Meter Telescope: Risk Mitigation and Status Update

Alignment and Phasing System (APS) is responsible for the optical alignment via starlight of the approximately 12,000 degrees of freedom of the primary, secondary and tertiary mirrors of Thirty Meter Telescope (TMT). APS is based on the successful Phasing Camera System (PCS) used to align the Keck Telescopes. Since the successful APS conceptual design in 2007, work has concentrated on risk mitigation, use case generation, and alignment algorithm development and improvement. Much of the risk mitigation effort has centered around development and testing of prototype APS software which will replace the current PCS software used at Keck. We present an updated APS design, example use cases and discuss, in detail, the risk mitigation efforts.

Optical Alignment↗

TRAPPIST-1 Habitable Atmosphere Intercomparison (THAI): motivations and protocol version 1.0

Upcoming telescopes such as the James Webb Space Telescope (JWST), the European Extremely Large Telescope (E-ELT), the Thirty Meter Telescope (TMT) or the Giant Magellan Telescope (GMT) may soon be able to characterize, through transmission, emission or reflection spectroscopy, the atmospheres of rocky exoplanets orbiting nearby M dwarfs. One of the most promising candidates is the late M-dwarf system TRAPPIST-1, which has seven known transiting planets for which transit timing variation (TTV) measurements suggest that they are terrestrial in nature, with a possible enrichment in volatiles. Among these seven planets, TRAPPIST-1e seems to be the most promising candidate to have habitable surface conditions, receiving ∼66 % of the Earth's incident radiation and thus needing only modest greenhouse gas inventories to raise surface temperatures to allow surface liquid water to exist. TRAPPIST-1e is, therefore, one of the prime targets for the JWST atmospheric characterization. In this context, the modeling of its potential atmosphere is an essential step prior to observation. Global climate models (GCMs) offer the most detailed way to simulate planetary atmospheres. However, intrinsic differences exist between GCMs which can lead to different climate prediction and thus observability of gas and/or cloud features in transmission and thermal emission spectra. Such differences should preferably be known prior to observations. In this paper we present a protocol to intercompare planetary GCMs. Four testing cases are considered for TRAPPIST-1e, but the methodology is applicable to other rocky exoplanets in the habitable zone. The four test cases included two land planets composed of modern-Earth and pure-CO2 atmospheres and two aqua planets with the same atmospheric compositions. Currently, there are four participating models (LMDG, ROCKE-3D, ExoCAM, UM); however, this protocol is intended to let other teams participate as well.

Thomas Jean Daniel Fauchez↗