Biology subjects

Shulman, N.

Publications and source records attributed to Shulman, N..

9 recordsLinked to original sources

bioRxiv2026

A quantitative proteomics dataset for assessment and prediction of low dose X-ray radiation exposure in mice.

Ionizing radiation induces molecular responses that may be used to estimate exposure when physical dosimeters are unavailable. Here we present two large-scale proteomics datasets generated from mouse dorsal skin punch samples collected following controlled X-ray exposures spanning multiple doses, dose rates, and post-exposure time points. Experiment 1 comprised 96 samples (including 16 reference samples) collected 6 days after exposure to 0-75 cGy delivered at either 30 or 300 cGy/min. Experiment 2 comprised 936 samples (including 236 reference samples) exposed to 0-100 cGy at either 3 or 28 cGy/min dose rates and harvested between 7 and 150 days post-exposure. All samples were processed using a standardized workflow involving automated bead-based digestion and data-independent acquisition mass spectrometry. The datasets include multiple pooled reference sample types, process controls, and system suitability standards ensuring high quality data. All data presented are available via ProteomeXchange at several levels of processing, from raw files through normalized peptide- and protein-level abundance matrices suitable for biomarker discovery and machine learning applications. This dataset will facilitate generation of new insights into the biological changes and molecular signatures resulting from X-ray exposure in mice and may also help inform future studies in humans.

biochemistry↗

bioRxiv2026

Carafe2 enables high quality in silico spectral library generation for timsTOF data-independent acquisition proteomics

Data-independent acquisition (DIA) proteomics enables reproducible and systematic peptide detection and quantification, and trapped ion mobility spectrometry (TIMS) on the timsTOF platform further improves DIA by synchronizing ion mobility separation with quadrupole precursor sampling. Analyzing the highly multiplexed spectra generated by DIA typically relies on spectral libraries, and fully leveraging the additional ion mobility dimension requires these libraries to include accurate retention time, fragment ion intensity, and ion mobility annotations. Existing in silico spectral library generation tools either lack ion mobility support entirely or rely on models trained on data-dependent acquisition (DDA) data, that can introduce a mismatch that may not capture unique experiment-specific biases when applied to each respective timsTOF dataset. Carafe is a software tool that uses deep learning models to generate high-quality, experiment-specific in silico libraries by training directly on DIA data. In this study, we extend Carafe to generate libraries for timsTOF DIA data, which involves fine-tuning retention time (RT), fragment ion intensity, and ion mobility prediction models using timsTOF DIA data. Carafe2 operates directly on native timsTOF raw data (Bruker .d directories) without the need for data conversion. We demonstrate the performance of Carafe2 across a wide range of DIA applications, including global proteome, phosphoproteome, and plasma proteome datasets. Comparing Carafe2 fine-tuned RT, fragment ion intensity, and ion mobility prediction models with pretrained DDA models, we find that Carafe2 models outperform pretrained models on a variety of DIA datasets. We then demonstrate the utility of in silico libraries generated by Carafe2 for peptide detection on several different types of timsTOF DIA datasets by comparing with the libraries generated with DDA-trained AlphaPeptDeep models, DIA-NN built-in models, and empirical spectral libraries generated from DDA experiments.

bioinformatics↗

bioRxiv2025

Evaluation of an Orbitrap Astral Zoom mass spectrometer prototype for quantitative proteomics - beyond identification lists

Mass spectrometry instrumentation continues to evolve rapidly yet quantifying these advances beyond conventional peptide and protein detections remain challenging. Here, we evaluate a modified Orbitrap Astral Zoom mass spectrometer (MS) prototype and compare its performance to the standard Orbitrap Astral MS. Across a range of acquisition methods and sample inputs, the prototype instrument outperformed the standard Orbitrap Astral MS in precursor and protein identifications, ion beam utilization, and quantitative precision. To enable meaningful cross-platform comparisons, we implemented an ion calibration framework that converts signal intensity from arbitrary units to ion per second. This benchmarking strategy showed that the prototype sampled 30% more ions per peptide than the original Orbitrap Astral MS. This increase in the ion beam utilization resulted in improved sensitivity and quantitative precision. To make these metrics broadly accessible, we added new metrics to the Skyline document grid to report the number of ions measured in a spectrum at the apex of the elution peak or the sum of ions between the peak integration boundaries. Taken together, our results demonstrate the prototype Orbitrap Astral Zoom as a high-performance platform for DIA proteomics and establish a generalizable framework for evaluation of mass spectrometer performance based on the number of ions detected for each analyte. Data are available on Panorama Public and on ProteomeXchange under the identifier PXD064536.

biochemistry↗

bioRxiv2024

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the StellarTM mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

biochemistry↗

bioRxiv2024

Hybrid Quadrupole Mass Filter Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics

Targeted mass spectrometry (MS) methods are powerful tools for selective and sensitive analysis of peptides identified by global discovery experiments. Selected reaction monitoring (SRM) is currently the most widely accepted MS method in the clinic, due to its reliability and analytical performance. However, due to limited throughput and the difficulty in setting up and analyzing large scale assays, SRM and parallel reaction monitoring (PRM) are typically used only for very refined assays of on the order of 100 targets or less. Here we introduce a new MS platform with a quadrupole mass filter, collision cell, linear ion trap architecture that has increased acquisition rates compared to the analogous hardware found in the Orbitrap Tribrid series instruments. The platform can target more analytes than existing SRM and PRM instruments - in the range of 5000 to 8000 peptides per hour. This capability for high multiplexing is enabled by acquisition rates of 70-100 Hz for peptide applications, and the incorporation of real-time chromatogram alignment that adjusts for retention time drift and enables narrow time scheduled acquisition windows. Finally, we describe a Skyline external software tool that implements the building of targeted methods based on data independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. We show that the platform delivers ~10x lower LOQs than traditional SRM analysis for a highly multiplex assay and also demonstrate how analytical figures of merit change while varying method duration with a constant number of analytes, or by keeping a constant time duration while varying the number of analytes.

biochemistry↗

bioRxiv2024

A Workflow for Improved Analysis of Cross-linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent analysis (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

bioinformatics↗

bioRxiv2023

Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data independent acquisition

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

biochemistry↗

bioRxiv2021

Development and Application of Multidimensional Lipid Libraries to Investigate Lipidomic Dysregulation Related to Smoke Inhalation Injury Severity

Lipids play many biological roles including membrane formation, protection, insulation, energy storage, and cell division. These functions have brought great interest to lipidomic studies for understanding their dysregulation in toxic exposure, inflammation, and diseases. However, lipids have shown to be analytically challenging due to their highly isomeric nature and vast concentration ranges in biological matrices. Therefore, powerful multidimensional techniques such as those integrating liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC-IMS-CID-MS) have recently been implemented to separate lipid isomers as well as provide structural information and increased feature identification confidence. These multidimensional datasets are however extremely large and highly complex, resulting in challenges in data processing and annotation. Here, we have overcome these challenges by developing sample-specific multidimensional libraries using the freely available software Skyline. Specifically, the human plasma library developed for this work contains over 500 unique, experimentally validated lipids, which is combined with adapted Skyline functions for highly confident lipid annotations such as indexed retention time (iRT) for retention time prediction and IMS drift time filtering for increased sensitivity and selectivity. For broad comparison with other lipidomic studies, this human plasma database was initially used to annotate LC-IMS-CID-MS data from a NIST SRM 1950 extract, giving comparable results to previous studies. This workflow was then utilized to assess matched plasma and bronchoalveolar lavage fluid (BALF) samples from patients with varying degrees of smoke inhalation injury to identify potential lipid-based patient prognostic and diagnostic markers. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2019

Avant-garde: An automated data-driven DIA data curation tool.

Data-Independent Acquisition (DIA) is a technique that promises to comprehensively detect and quantify all peptides above an instruments limit of detection. Several software tools to analyze DIA data have been developed in recent years. However, several challenges still remain, like confidently identifying peptides, defining integration boundaries, dealing with interference for selected transitions, and scoring and filtering of peptide signals in order to control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. Avant-garde is a new tool to refine DIA (and PRM) by removing interfered transitions, adjusting integration boundaries and scoring peaks to control the FDR. Unlike other tools where MS runs are scored independently from each other, Avant-garde uses a novel data-driven scoring strategy. DIA signals are refined by learning from the data itself, using all measurements in all samples together to achieve the best optimization. We evaluated the performances of Avant-garde with a calibrated sample using spiked-in standards in a complex background, a phospho-enriched dataset (Abelin et al, 2016), and two complex hybrid proteome samples for benchmarking DIA software tools. The results clearly showed that Avant-garde is capable of improving the selectivity, accuracy, and reproducibility of the quantification results in very complex biological matrices. We have further shown that it can evaluate the suitability of a peak to be used for quantification reaching the same levels of selectivity, accuracy, and reproducibility obtained with manual validation.

bioinformatics↗