High-quality lipid suppression and B0 shimming for human brain 1H MRSI.

Magnetic Resonance Spectroscopic Imaging (MRSI) is a powerful technique that can map the metabolic profile in the brain non-invasively. Extracranial lipid contamination and insufficient B0 homogeneity however hampers robustness, and as a result has hindered widespread use of MRSI in clinical and research settings. Over the last six years we have developed highly effective extracranial lipid suppression methods with a second order gradient insert (ECLIPSE) utilizing inner volume selection (IVS) and outer volume suppression (OVS) methods. While ECLIPSE provides > 100-fold in lipid suppression with modest radio frequency (RF) power requirements and immunity to B1+ field variations, axial coverage is reduced for non-elliptical head shapes. In this work we detail the design, construction, and utility of MC-ECLIPSE, a pulsed second order gradient coil with Z2 and X2Y2 fields, combined with a 54-channel multi-coil (MC) array. The MC-ECLIPSE platform allows arbitrary region of interest (ROI) shaped OVS for full-axial slice coverage, in addition to MC-based B0 field shimming, for robust human brain proton MRSI. In vivo experiments demonstrate that MC-ECLIPSE allows axial brain coverage of 92-95 % is achieved following arbitrary ROI shaped OVS for various head shapes. The standard deviation (SD) of the residual B0 field following SH2 and MC shimming were 25 ± 9 Hz and 18 ± 8 Hz over a 5 cm slab, and 18 ± 5 Hz and 14 ± 6 Hz over a 1.5 cm slab, respectively. These results demonstrate that B0 magnetic field shimming with the MC array supersedes second order harmonic capabilities available on standard MRI systems for both restricted and large ROIs. Furthermore, MC based B0 shimming provides comparable shimming performance to an unrestricted SH5 shim set for both restricted, and 5-cm slab shim challenges. Phantom experiments demonstrate the high level of localization performance achievable with MC-ECLIPSE, with ROI edge chemical shift displacements ranging from 1-3 mm with a median value of 2 mm, and transition width metrics ranging from 1-2.5 mm throughout the ROI edge. Furthermore, MC based B0 shimming is comparable to performance following a full set of unrestricted spherical harmonic fields up to order 5. Short echo time MRSI and GABA-edited MRSI acquisitions in the human brain following MC-shimming and arbitrary ROI shaping demonstrate full-axial slice coverage and extracranial lipid artifact free spectra. MC-ECLIPSE allows full-axial coverage and robust MRSI acquisitions, while allowing interrogation of cortical tissue proximal to the skull, which has significant value in a wide range of neurological and psychiatric conditions.

CATEcor: An Open Science, Shaded-Truss, Externally-Occulted Coronagraph.

We present the design of a portable coronagraph, CATEcor (where CATE stands for Continental-America Telescope Eclipse), that incorporates a novel "shaded-truss" style of external occultation and serves as a proof-of-concept for that family of coronagraphs. The shaded-truss design style has the potential for broad application in various scientific settings. We conceived CATEcor itself as a simple instrument to observe the corona during the darker skies available during a partial solar eclipse, or for students or interested amateurs to detect the corona under ideal noneclipsed conditions. CATEcor is therefore optimized for simplicity and accessibility to the public. It is implemented using an existing dioptric telescope and an adapter rig that mounts in front of the objective lens, restricting the telescope aperture and providing external occultation. The adapter rig, including occulter, is fabricated using fusion deposition modeling (FDM; colloquially "3D printing"), greatly reducing cost. The structure is designed to be integrated with moderate care and may be replicated in a university or amateur setting. While CATEcor is a simple demonstration unit, the design concept, process, and trades are useful for other more sophisticated coronagraphs in the same general family, which might operate under normal daytime skies outside the annular-eclipse conditions used for CATEcor.

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits. Applied on the liquid chromatography time scale using an Orbitrap Tribrid mass spectrometer, PTCR produces easy-to-interpret mass spectra with limited ion signal overlap. We demonstrate that the accurate estimation of the number of charges submitted to the Orbitrap mass analyzer after PTCR allows for the detection of charge-reduced product ions over a wide mass-over-charge (m/z) window with low parts per million m/z accuracy. Therefore, PTCR-based MD MS analysis increases not only sequence coverage, number of uniquely identified fragments, and number of assigned complementary ion pairs, but also the general confidence in the assignment of subunit fragments. This data acquisition method can be readily applied to any class of mAbs without an apparent need for optimization, and benefits from the high resolving power of the Orbitrap mass analyzer to return sequence coverage of individual subunits exceeding 80% in a single run, and > 90% when just two experiments are combined.

Validation of Acuros for total body irradiation at extended distance.

PURPOSE: Standardized and accurately reported doses are essential in conventional total body irradiation (TBI), especially lung doses. This study evaluates the accuracy of the Acuros algorithm in predicting doses for extended-distance TBI. METHODS: Measurements and calculations were done with both 6 and 18 MV. Tissue Maximum Ratio (TMR), output and off axis ratios (OAR) were measured at 200 and 500 cm source to detector distance and compared to Acuros calculated values. Two end-to-end tests were carried out, one with an in-house phantom (solid water and Styrofoam) with inserted ion chambers and the other was with the Imaging and Radiation Oncology Core (IROC) TBI anthropomorphic phantom equipped with TLDs. The end-to-end test was done for 6 and 18 MV both with and without lung blocks. The source to midplane distance for both phantoms were at 518 and 508 cm respectively. Lung blocks were placed at the phantom surface and a beam spoiler was positioned 30 cm from the surface of the phantoms as per our clinical set up. RESULTS: The agreement between measured and calculated TMR, output and off axis ratios for both 6 and 18 MV were within 2%. Ion chamber measurements in both the Styrofoam and solid water for both energies carried out with and without lung blocks were within 2% of calculated values. TLD measured doses for both 6 and 18 MV in the IROC phantom were within 5% of calculated doses which is within the uncertainty of the TLD measurement. CONCLUSIONS: The results indicate that the clinical beam model for Acuros 16.1 commissioned at standard clinical distances is capable of calculating doses accurately at extended distances up to 500 cm.

Commissioning and initial validation of Eclipse eMC algorithm for the electron FLASH research extension (FLEX) system for pre-clinical studies.

PURPOSE: To investigate the feasibility of commissioning the 16 MeV electron FLASH Extension (FLEX) in the commercial treatment planning system (TPS) for biomedical research with cell and mouse models, and in silico treatment planning studies. METHODS: To commission the FLEX system with the electron Monte Carlo (eMC) algorithm in the commercial TPS, radiochromic film was used to measure the vendor-recommended beam data. Once the beam model was generated for the eMC algorithm, supplemental measurements were collected for validation purposes and compared against the TPS-calculated results. Additionally, the newly commissioned 16 MeV FLASH beam was compared to the corresponding 16 MeV conventional electron beam. RESULTS: The eMC algorithm effectively modeled the FLEX system. The eMC-calculated PDDs and profiles for the 16 MeV electron FLASH beam agreed with measured values within 1%, on average, for 6 × 6 cm2 and 10 × 10 cm2 applicators. Flatness and symmetry deviated by less than 1%, while FWHM and penumbra agreed within 1 mm for both eMC-calculated and measured profiles. Additionally, the small field (i.e., 2-cm diameter cutout) that was measured for validation purposes agreed with TPS-calculated results within 1%, on average, for both the PDD and profiles. The FLASH and conventional dose rate 16 MeV electron beam were in agreement in regard to energy, but the profiles for larger field sizes began to deviate (>10 × 10 cm2) due to the forward-peaked nature of the FLASH beam. For cell irradiation experiments, the measured and eMC-calculated in-plane and cross-plane absolute dose profiles agreed within 1%, on average. CONCLUSIONS: The FLEX system was successfully commissioned in the commercial TPS using the eMC algorithm, which accurately modeled the forward-peaked nature of the FLASH beam. A commissioned TPS for FLASH will be useful for pre-clinical cell and animal studies, as well as in silico FLASH treatment planning studies for future clinical implementation.

Recommendations for using analytical anisotropic algorithm and AcurosXB for epidermal dose calculations in breast radiotherapy from an in vivo Gafchromic film study.

BACKGROUND AND PURPOSE: This study recommends clinical epidermal dose calculation methods based on in-vivo film measurements and registered skin dose distributions with the Eclipse (Varian Medical Systems) treatment planning system's Analytical Anisotropic Algorithm (AAA) and Acuros XB (AXB) dose calculation algorithms. MATERIALS AND METHODS: Eighteen AAA V13.6 breast plans were recalculated using AXB (dose to medium) V13.5 with the same beam parameters and monitor units as in the original plans. These are compared against in-vivo Gafchromic film measurements from the lateral and inferior breast regions. Three skin structures in the treatment planning system are evaluated: a surface layer of voxels of the body contour, a 0.2 cm internal skin rind, and a 0.5 cm internal skin rind. RESULTS: Systematic shifts are demonstrated between the film measurements of skin dose and the Eclipse dose calculations. On average, the dose to the surface layer of pixels is underestimated by AAA by 8% and overestimated by AXB by 3%. A 5 mm skin rind extended into the body can increase epidermal dose calculations on average by 8% for AAA and 4% for AXB. CONCLUSION: This is the first study to register in-vivo skin dose distributions in the breast to the treatment planning system for comparison. Based on the results from this study it is recommended that epidermal dose is calculated with a 0.5 cm skin rind for the AAA algorithm and with rind thickness up to 0.2 cm for the AXB algorithm.

Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for Sample Multiplexing-Based Proteomics.

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three data sets, one using only BPRP fractionation and two others of each SAX-partition followed by BPRP. The three data sets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition data set contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines, as well as two additional experiments using ovarian and pancreatic cancer cell lines.

Enriching Cysteine-Containing Peptides Using a Sulfhydryl-Reactive Alkylating Reagent with a Phosphonic Acid Group and Immobilized Metal Affinity Chromatography.

The reduction of disulfide bonds and their subsequent alkylation are commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of the SH-SY5Y human cell line following 24 h treatments with two proteasome inhibitors (bortezomib and MG-132) in a tandem mass tag (TMT)pro9-plex experiment. We acquire three datasets─(1) Cys-peptide enriched, (2) the unbound complement, and (3) the non-depleted control─and compare the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-Cys phosphonate adaptable tag (6C-CysPAT) can quantify over 38,000 Cys-containing peptides in 5 h with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9900 protein abundance profiles exhibiting the effects of two different proteasome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this "mini-Cys" dataset can be used to preview and assess the quality of a deep, fractionated dataset.

Management of a broken guiding catheter tip: Cut and fix technique.

A 49-year-old male presented with class III exertional angina, 1 year after angioplasty of the left anterior descending artery (LAD) and right coronary artery. Coronary angiogram revealed 90% in-stent restenosis (ISR) in mid-LAD with angiographic impression of stent fracture. Optical coherence tomographic evaluation of mid-LAD ISR showed a distinct 3 mm long "eclipse sign" indicating embolized, broken guiding catheter tip as a cause of ISR, which was confirmed on reviewing 1-year-old angiographic images. This was managed with "cut and fix technique" using cutting balloon and another drug-eluting stent. Optical coherence tomographic at 9 months showed well endothelialized stent with a thin layer of neo-intimal hyperplasia over the sandwiched broken guiding tip.

Multiple cohort study of hospitalized SARS-CoV-2 in-host infection dynamics: Parameter estimates, identifiability, sensitivity and the eclipse phase profile.

Within-host SARS-CoV-2 modelling studies have been published throughout the COVID-19 pandemic. These studies contain highly variable numbers of individuals and capture varying timescales of pathogen dynamics; some studies capture the time of disease onset, the peak viral load and subsequent heterogeneity in clearance dynamics across individuals, while others capture late-time post-peak dynamics. In this study, we curate multiple previously published SARS-CoV-2 viral load data sets, fit these data with a consistent modelling approach, and estimate the variability of in-host parameters including the basic reproduction number, R0, as well as the best-fit eclipse phase profile. We find that fitted dynamics can be highly variable across data sets, and highly variable within data sets, particularly when key components of the dynamic trajectories (e.g. peak viral load) are not represented in the data. Further, we investigated the role of the eclipse phase time distribution in fitting SARS-CoV-2 viral load data. By varying the shape parameter of an Erlang distribution, we demonstrate that models with either no eclipse phase, or with an exponentially-distributed eclipse phase, offer significantly worse fits to these data, whereas models with less dispersion around the mean eclipse time (shape parameter two or more) offered the best fits to the available data across all data sets used in this work. This manuscript was submitted as part of a theme issue on "Modelling COVID-19 and Preparedness for Future Pandemics".

A comprehensive evaluation of advanced dose calculation algorithms for brain stereotactic radiosurgery.

PURPOSE: Accurate dose calculation is important in both target and low dose normal tissue regions for brain stereotactic radiosurgery (SRS). In this study, we aim to evaluate the dosimetric accuracy of the two advanced dose calculation algorithms for brain SRS. METHODS: Retrospective clinical case study and phantom study were performed. For the clinical study, 138 SRS patient plans (443 targets) were generated using BrainLab Elements Voxel Monte Carlo (VMC). To evaluate the dose calculation accuracy, the plans were exported into Eclipse and recalculated with Acuros XB (AXB) algorithm with identical beam parameters. The calculated dose at the target center (Dref), dose to 95% target volume (D95), and the average dose to target (Dmean) were compared. Also, the distance from the skull was analyzed. For the phantom study, a cylindrical phantom and a head phantom were used, and the delivered dose was measured by an ion chamber and EBT3 film, respectively, at various locations. The measurement was compared with the calculated doses from VMC and AXB. RESULTS: In clinical cases, VMC dose calculations tended to be higher than AXB. It was found that the difference in Dref showed > 5% in some cases for smaller volumes < 0.3 cm3 . Dmean and D95 differences were also higher for small targets. No obvious trend was found between the dose difference and the distance from the skull. In phantom studies, VMC dose was also higher than AXB for smaller targets, and VMC showed better agreement with the measurements than AXB for both point dose and high dose spread. CONCLUSION: The two advanced calculation algorithms were extensively compared. For brain SRS, AXB sometimes calculates a noticeable lower target dose for small targets than VMC, and VMC tends to have a slightly closer agreement with measurements than AXB.

Extending the EclipseTM AcurosXB output factor table for small field radiosurgery.

PURPOSE: To investigate the necessity of extending the output factor table (OF Table) of the Varian EclipseTM Treatment Planning System for small field stereotactic radiosurgery (SRS) and stereotactic body radiosurgery (SBRT) treatments. METHODS: A new AcurosXB 15.6 beam model was created in the Eclipse Beam Configuration, which is identical to the one that has been used in the clinic with a default 3 × 3 cm to 40 × 40 cm OF Table, except the OF Table in the new model was extended to cover the range from 1 × 1 cm to 40 × 40 cm. 80 small square and rectangular output factors were measured on a Varian TrueBeam utilizing a Standard Imaging Exradin W2-1×1 scintillator detector, inside a PTW BeamScan water tank with 95 cm SSD at 5 cm depth. Cerenkov contamination was corrected using a rectangular field method (2 cm × 15 cm field). Nine Radiosurgery plans with primary jaw setting ranging from 0.7 cm to 2.0 cm were evaluated by both beam models. The monitor unit (MU) differences between the two beam models were calculated for identical 3-dimensional (3D) absolute dose distributions. Output factors, measured versus Eclipse calculated, were compared down to 0.5 × 0.5 cm primary jaw setting. RESULTS: For the 6FFF beam, the difference between the two beam models was ∼ 6% for 1 × 1 cm jaw settings and 4% at 1.5 × 1.5 cm, with the extended OF Table requiring higher MUs for the same dose prescription and same 3-dimensional isodose distribution. For the 6MV beam, the corresponding difference is ∼7.5% for 1 × 1 cm, 5% for 1.5 × 1.5 cm, and 3% for 2 × 2 cm jaw settings, with the extended OF Table requiring higher MUs. For jaw settings smaller than 1 × 1 cm, measured dose can be considerably smaller than Eclipse predicted dose, even with the OF Table extension. This is reflected by the fact that the output factor for 0.5 × 0.5 cm, calculated via Eclipse external beam, was more than 30% greater than that measured for both 6FFF and 6MV beams. CONCLUSIONS: Eclipse does a satisfactory job for primary jaw sizes down to 2 cm. For jaw settings smaller than 1.5 cm, the OF Table in Eclipse should be extended to improve the dose calculation accuracy.

Assessing interference in isobaric tag-based sample multiplexing using an 18-plex interference standard.

Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO viewing tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that are acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.

Comparison of Eclipse Smart Segmentation and MIM Atlas Segment for liver delineation for yttrium-90 selective internal radiation therapy.

PURPOSE: The aim was to compare Smart Segmentation of Eclipse treatment planning system and Atlas Segment of MIM software for liver delineation for resin yttrium-90 (Y-90) procedures. MATERIALS AND METHODS: CT images of 20 patients treated with resin Y-90 selective internal radiation therapy (SIRT) were tested. Liver contours generated with Smart Segmentation and Atlas Segment were compared with physician manually delineated contours. Dice similarity coefficient (DSC), mean distance to agreement (MDA), and ratio of volume (RV) were calculated. The contours were evaluated with activity calculations and ratio of activity (RA) was calculated. RESULTS: Mean DSCs were 0.77 and 0.83, MDAs were 0.88 and 0.71 cm, mean RVs were 0.95 and 1.02, and mean RAs were 1.00 and 1.00, for Eclipse and MIM results, respectively. CONCLUSION: MIM outperformed Eclipse in both DSC and MDA, whereas the differences in liver volumes and calculated activities were statistically insignificant between the Eclipse and MIM results. Both auto-segmentation tools can be used to generate initial liver contours for resin Y-90 SIRT, which need to be reviewed and edited by physicians.

Technical note: Institutional solution of clinical cine MRI for tumor motion evaluation in radiotherapy.

PURPOSE: Since 4D-MRI is inadequate to capture dynamic respiratory variations, real-time cinematographic (cine) MRI is actively used in MR-guided radiotherapy (MRgRT) for tumor motion evaluation, delineation, and tracking. However, most radiotherapy imaging platforms do not support the format of cine MRI from clinical MRI systems. This study developed an institutional solution of clinical cine MRI for tumor motion evaluation in radiotherapy applications. METHODS: Cine MRI manipulation software (called Cine Viewer) was developed within a commercial Treatment Planning System (TPS). It consists of (1) single/orthogonal viewers, (2) display controllers, (3) measurement grids/markers, and (4) manual contouring tools. RESULTS: The institutional solution of clinical cine MRI incorporated with radiotherapy application was assessed through case presentations (liver cancer). Cine Viewer loaded cine MRIs from 1.5T Philips Ingenia MRI, handling MRI DICOM format. The measurement grids and markers were used to quantify the displacement of anatomical structures in addition to the tumor. The contouring tool was utilized to localize the tumor and surrogates on the designated frame. The stacks of the contours were exhibited to present the ranges of tumor and surrogate motions. For example, the stacks of the tumor contours from case-1 were used to determine the ranges of tumor motions (∼8.17 mm on the x-direction [AP-direction] and ∼14 mm on the y-direction [SI-direction]). In addition, the patterns of the displacement of the contours over frames were analyzed and reported using in-house software. In the case-1 review, the tumor was displaced from +146.0 mm on the x-direction and +125.0 mm on the y-direction from the ROI of the abdominal surface. CONCLUSION: We demonstrated the institutional solution of clinical cine MRI in radiotherapy. The proposed tools can streamline the utilization of cine MRI for tumor motion evaluation using Eclipse for treatment planning.

Comparison of PRIMO Monte Carlo code and Eclipse treatment planning system in calculation of dosimetric parameters in brain cancer radiotherapy.

Background: It is important to evaluate the dose calculated by treatment planning systems (TPSs) and dose distribution in tumor and organs at risk (OARs). The aim of this study is to compare dose calculated by the PRIMO Monte Carlo code and Eclipse TPS in radiotherapy of brain cancer patients. Materials and methods: PRIMO simulation code was used to simulate a Varian Clinac 600C linac. The simulations were validated for the linac by comparison of the simulation and measured results. In the case of brain cancer patients, the dosimetric parameters obtained by the PRIMO code were compared with those calculated by Eclipse TPS. Gamma function analysis with 3%, 3 mm criteria was utilized to compare the dose distributions. The evaluations were based on the dosimetric parameters for the planning target volume (PTV) and OAR including D min, D mean, and D max, homogeneity index (HI), and conformity index (CI). Results: The gamma function analysis showed a 98% agreement between the results obtained by the PRIMO code and measurement for the percent depth dose (PDD) and dose profiles. The corresponding value in comparing the dosimetric parameters from PRIMO code and Eclipse TPS for the brain patients was 94%, on average. The results of the PRIMO simulation were in good agreement with the measured data and Eclipse TPS calculations. Conclusions: Based on the results of this study, the PRIMO code can be utilized to simulate a medical linac with good accuracy and to evaluate the accuracy of treatment plans for patients with brain cancer.

In the Shadow of the 1919 Total Solar Eclipse: The Two British Expeditions and the Politics of Invisibility.

This paper addresses the legendary total solar eclipse of 29 May 1919. Two British teams confirmed the light bending prediction by Albert Einstein: Charles R. Davidson and Andrew C. C. Crommelin in Sobral, Brazil and Arthur S. Eddington and Edwin T. Cottingham on the African island of Príncipe, then part of the Portuguese empire. By jointly analyzing the two astronomical expeditions supported by written and visual sources, I show how, despite extensive scholarship on this famous historical episode and the historiographical emphasis on the plural dimensions of knowledge construction, many human and non-human actors have been kept in the shadow of the eclipse. I do so by focusing on what I call knowledge from the periphery together with knowledge from below, grounded literally on how localities (sites) affect choices and events, and growing outward to encompass a wide range of participants. I show how the geopolitical status of the two nations where the observational sites were located, and specifically Portugal's condition of colonial power, affected main decisions and events, while highlighting the active role of participants, ranging from experts from the peripheries and those involved in the travels to local elites and anonymous peoples, some of whom contributed to the observation of totality.

Isobaric Tag-Based Protein Profiling across Eight Human Cell Lines Using High-Field Asymmetric Ion Mobility Spectrometry and Real-Time Database Searching.

A vast number of human cell lines are available for cell culture model-based studies, and as such the potential exists for discrepancies in findings due to cell line selection. To investigate this concept, the authors determine the relative protein abundance profiles of a panel of eight diverse, but commonly studied human cell lines. This panel includes HAP1, HEK293T, HeLa, HepG2, Jurkat, Panc1, SH-SY5Y, and SVGp12. A mass spectrometry-based proteomics workflow designed to enhance quantitative accuracy while maintaining analytical depth is used. To this end, this strategy leverages TMTpro16-based sample multiplexing, high-field asymmetric ion mobility spectrometry, and real-time database searching. The data show that the differences in the relative protein abundance profiles reflect cell line diversity. The authors also determine several hundred proteins to be highly enriched for a given cell line, and perform gene ontology and pathway analysis on these cell line-enriched proteins. An R Shiny application is designed to query protein abundance profiles and retrieve proteins with similar patterns. The workflows used herein can be applied to additional cell lines to aid cell line selection for addressing a given scientific inquiry or for improving an experimental design.

A Compendium of Murine (Phospho)Peptides Encompassing Different Isobaric Labeling and Data Acquisition Strategies.

Targeted mass spectrometry-based assays typically rely on previously acquired large data sets for peptide target selection. Such repositories are widely available for unlabeled peptides. However, they are less common for isobaric tagged peptides. Here we have assembled two series of six data sets originating from a mouse embryonic fibroblast cell line (NIH/3T3). One series is of peptides derived from a tryptic digest of a whole cell proteome and a second from enriched phosphopeptides. These data sets encompass three labeling approaches (unlabeled, TMT11-labeled, and TMTpro16-labeled) and two data acquisition strategies (ion trap MS2 with and without FAIMS-based gas phase separation). We identified a total of 1 509 526 peptide-spectrum matches which covered 11 482 proteins from the whole cell proteome tryptic digest, and 188 849 phosphopeptides from the phosphopeptide enrichment. The data sets were of similar depth, and while overlap across data sets was modest, protein overlap was high, thus reinforcing the comprehensiveness of these data sets. The data also supported FAIMS as a means to increase data set depth. These data sets provide a rich resource of peptides that may be used as starting points for targeted assays. Future data sets may be compiled for any genome-sequenced organism using the technologies and strategies highlighted herein. The data have been deposited in the ProteomeXchange Consortium with data set identifier PXD024298.

Temporal Proteomic Profiling of SH-SY5Y Differentiation with Retinoic Acid Using FAIMS and Real-Time Searching.

The SH-SY5Y cell line is often used as a surrogate for neurons in cell-based studies. This cell line is frequently differentiated with all-trans retinoic acid (ATRA) over a 7-day period, which confers neuron-like properties to the cells. However, no analysis of proteome remodeling has followed the progress of this transition. Here, we quantitatively profiled over 9400 proteins across a 7-day treatment with retinoic acid using state-of-the-art mass spectrometry-based proteomics technologies, including FAIMS, real-time database searching, and TMTpro16 sample multiplexing. Gene ontology analysis revealed that categories with the highest increases in protein abundance were related to the plasma membrane/extracellular space. To showcase our data set, we surveyed the protein abundance profiles linked to neurofilament bundle assembly, neuron projections, and neuronal cell body formation. These proteins exhibited increases in abundance level, yet we observed multiple patterns among the queried proteins. The data presented represent a rich resource for investigating temporal protein abundance changes in SH-SY5Y cells differentiated with retinoic acid. Moreover, the sample preparation and data acquisition strategies used here can be readily applied to any analogous cell line differentiation analysis.