Biology subjects

Gandhi, T.

Publications and source records attributed to Gandhi, T..

9 recordsLinked to original sources

bioRxiv2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis

Here we present a novel data independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for nontargeted discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation (SLIM) technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. This PAMAF mode of operation increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering method, we can avoid ion losses up to 99% while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. Additionally, by storing ions in a trapping region while the previous packet of ions is being analyzed, the PAMAF mode achieves [~]100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed approximately 6x more protein group identifications compared to a standard data-dependent acquisition (DDA) analysis without HRIM on the same QTOF instrument, and to over 100x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by DDA. Additionally, since precursor isolation in PAMAF mode is size-based rather than m/z-based, many coeluting isobars and isomers can be resolved prior to fragmentation to eliminate chimeric spectra that compromise identification accuracy. In this work we also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity. This approach, known as DIA-PAMAF mode, further reduces the frequency of chimeric fragmentation spectra, and enabled the detection of over 8,000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics that has the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/704896v1_ufig1.gif" ALT="Figure 1"> View larger version (114K): org.highwire.dtl.DTLVardef@7984c3org.highwire.dtl.DTLVardef@1fb2fe3org.highwire.dtl.DTLVardef@50d35org.highwire.dtl.DTLVardef@1a62926_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Cell type-independent timekeeping gene modules enable embryonic stage prediction in zebrafish

Gene expression changes across embryonic development reflect both differentiation and genes whose expression varies strictly with developmental time, independent of cell type. Multiple embryonic timing systems set the onset and pace of developmental events, and blocking transcription arrests many of these events. However, the genes comprising the vertebrate embryonic timing system(s) remain largely unknown. To identify genes whose expression changes with time alone, we examine naive zebrafish embryonic explants that form only two tissue types yet maintain developmental timing, thus uncoupling developmental age from most differentiation programs. By comparing longitudinal gene expression in naive explants with Nodal-induced explants that differentiate into all three germ layers, we identify "timekeeping" genes whose temporal expression patterns vary strictly with developmental age. Consensus clustering of temporally dynamic genes identified 20 gene clusters, termed "chrono-constitutive modules" (CCMs), that maintain distinct schedules of expression regardless of tissue type. These CCM trajectories are similar in intact zebrafish embryos and single embryonic cells of multiple distinct lineages. Enrichment analysis of microRNA targets and transcription factor regulons within the CCMs further reveal distinct putative regulators of several modules. Strikingly, CCM expression patterns are also largely conserved during early development of another fish species, Japanese medaka. Machine learning models trained on only zebrafish CCM transcript levels accurately predict the developmental age of embryonic explants, intact embryos, and even individual embryonic cells, demonstrating their utility in developmental timekeeping. These results support the existence of transcriptional timekeeping during early development and demonstrate its utility in embryonic stage prediction.

developmental biology↗

bioRxiv2025

Neural Oscillations during Perceptual Grouping: Insights from Alpha and Theta Bands in Visual Perception Tasks

Perceptual grouping, the brains ability to organize visual stimuli into coherent objects and patterns, is thought to rely on distinct neural oscillations across various frequency bands. However, the specific contributions of these frequency bands to perceptual grouping remain a central question in cognitive neuroscience. Our study employs spectral analysis and time-frequency analysis techniques to analyze EEG data collected during visual perception tasks. By examining the temporal dynamics and frequency-specific activity, particularly focusing on the alpha and theta bands immediately following stimulus onset, our goal is to uncover the neural processes underlying perceptual grouping. Our findings indicate significant changes in alpha and theta band activity associated with perceptual grouping in the brain. These results suggest that alpha band oscillations may play a role in directing attention and filtering relevant visual information, while theta band oscillations might facilitate the integration of visual elements into coherent percepts by engaging memory processes and enhancing neural communication across brain regions. This research contributes to a deeper understanding of how neural oscillations coordinate to support perceptual organization, potentially informing future studies and therapeutic approaches aimed at enhancing visual perception and cognitive function.

neuroscience↗

bioRxiv2025

Enhanced Identifications and Quantification through Retention Time Down-Sampling in Fast-Cycling diagonal-PASEF Methods

Data-independent acquisition mass spectrometry is essential for comprehensive quantification of proteomes, enabling deeper insights into cellular processes and disease mechanisms. On the timsTOF platform, diagonal-PASEF acquisition methods have recently been introduced to directly and continuously cover the observed diagonal shape of the peptide precursor ion distributions. Although diagonal-PASEF has shown great promise, its broad adoption as a routine workflow has been hampered by a lack of available algorithmic solutions and a paucity of demonstrated real-world applications. Here, we conducted a systematic and comprehensive optimization of diagonal-PASEF for 17-minute gradients on the timsTOF HT in conjunction to Spectronaut 19. We demonstrate that Spectronaut 19 fully supports all tested diagonal-PASEF methods independent of the number of slices or overlaps and with minimal user intervention required. Using our optimized analysis strategy we coupled diagonal-PASEF acquisitions to retention time down-sampling by summation (RTsum), thereby providing a novel mode of analysis of this data that exploits the fast-cycling nature of diagonal-PASEF methods. Through the combination of RTsum with diagonal-PASEF, we demonstrate that this strategy yields higher signal-to-noise ratios while retaining the peak shape for analytes of interest. Importantly, combining RTsum with diagonal-PASEF improved overall identifications and quantitative precision when compared to dia-PASEF with static or variable quadrupole isolation widths and across different input amounts for cell line injections. We also tested the performance of diagonal-PASEF in controlled quantitative experiments where diagonal-PASEF outperformed dia-PASEF in the overall number of retained candidates, quantitative precision and identifications on peptide level. These data indicate that RTsum demonstrates a positive use case of the high sampling rate of diagonal-PASEF. Collectively, our findings imply that diagonal-PASEF methods are superior for peptide and protein level analyses especially at lower input amounts.

biochemistry↗

bioRxiv2024

A Comprehensive and Robust Multiplex-DIA Workflow Profiles Protein Turnover Regulations Associated with Cisplatin Resistance

Measuring protein turnover is essential for understanding cellular biological processes and advancing drug discovery. The multiplex DIA mass spectrometry (DIA-MS) approach, combined with dynamic SILAC labeling (pulse-SILAC, or pSILAC), has proven to be a reliable method for analyzing protein turnover and degradation kinetics. Previous multiplex DIA-MS workflows have employed various strategies, including leveraging the highest isotopic labeling channels of peptides to enhance the detection of isotopic MS signal pairs or clusters. In this study, we introduce an improved and robust workflow that integrates a novel machine learning strategy and channel-specific statistical filtering, enabling dynamic adaptation to systematic or temporal variations in channel ratios. This allows comprehensive profiling of protein turnover throughout the pSILAC experiment without relying solely on the highest channel signals. Additionally, we developed KdeggeR, a data processing and analysis package optimized for pSILAC-DIA experiments, which estimates and visualizes peptide and protein degradation rates and dynamic profiles. Our integrative workflow was benchmarked on both 2-channel and 3-channel standard DIA datasets and workflow to an aneuploid cancer cell model before and after cisplatin resistance development demonstrated a strong negative correlation between transcript regulation and protein degradation for major protein complex subunits. We also identified specific protein turnover signatures associated with cisplatin resistance.

systems biology↗

bioRxiv2023

Substantial Downregulation of Mitochondrial and Peroxisomal Proteins during Acute Kidney Injury revealed by Data-Independent Acquisition Proteomics

Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A fast-acquisition rate ZenoTOF 7600 mass spectrometer was introduced for data-independent acquisition (DIA) for comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3,945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured mice exhibited severely declined health. The comprehensive and sensitive kidney-specific DIA assays highlighted here feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome and will serve as useful tools for developing novel therapeutics to remediate kidney function.

molecular biology↗

bioRxiv2022

MicroRNA-mRNA networks are dysregulated in opioid use disorder postmortem brain: further evidence for opioid-induced neurovascular alterations

To understand mechanisms and identify potential targets for intervention in the current crisis of opioid use disorder (OUD), postmortem brains represent an under-utilized resource. To refine previously reported gene signatures of neurobiological alterations in OUD from the dorsolateral prefrontal cortex (Brodmann Area 9, BA9), we explored the role of microRNAs (miRNA) as powerful epigenetic regulators of gene function. Building on the growing appreciation that miRNAs can cross the blood-brain barrier, we carried out miRNA profiling in same-subject postmortem samples from BA9 and blood tissues. miRNA-mRNA network analysis showed that even though miRNAs identified in BA9 and blood were fairly distinct, their target genes and corresponding enriched pathways were highly overlapping, with tube development and morphogenesis, and pathways related to endothelial cell function and vascular organization, among the dominant enriched biological processes. These findings point to robust, redundant, and systemic opioid-induced miRNA dysregulation with potential functional impact on transcriptomic changes. Further, using correlation network analysis we identified cell-type specific miRNA targets, specifically in astrocytes, neurons, and endothelial cells, associated with OUD transcriptomic dysregulation. Our refined miRNA-mRNA networks enabled identification of novel pharmaco-chemical interventions for OUD, particularly targeting the TGF beta-p38MAPK signaling pathway. Finally, leveraging a collection of control brain transcriptomes from the Genotype-Tissue Expression (GTEx) project, we identified correlation of OUD miRNA targets with TGF beta, hypoxia, angiogenesis, coagulation, immune system and inflammatory pathways. These findings support previous reports of neurovascular and immune system alterations as a consequence of opioid abuse.

bioinformatics↗

bioRxiv2019

Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries

Quantitative phosphoproteomics has in recent years revolutionized understanding of cell signaling, but it remains a challenge to scale the technology for high-throughput analyses. Here we present a rapid and reproducible phosphoproteomics approach to systematically analyze hundreds of samples by fast liquid chromatography tandem mass spectrometry using data independent acquisition (DIA). To overcome the inherent issue of positional phosphopeptide isomers in DIA-based phosphoproteomics, we developed and employed an accurate site localization scoring algorithm, which is incorporated into the Spectronaut software tool. Using a library of synthetic phosphopeptides spiked-in to a yeast phosphoproteome in different ratios we show that it is on par with the top site localization score for data-dependent acquisition (DDA) based phosphoproteomics. Single-shot DIA-based phosphoproteomics achieved an order of magnitude broader dynamic range, higher reproducibility of identification and improved sensitivity and accuracy of quantification compared to state-of-the-art DDA-based phosphoproteomics. Importantly, direct DIA without the need of spectral libraries performed almost on par with analyses using specific project-specific libraries. Moreover, we implemented and benchmarked an algorithm for globally determining phosphorylation site stoichiometry in DIA. Finally, we demonstrate the scalability of the DIA approach by systematically analyzing the effects of thirty different kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that a large proportion of EGF-dependent phospho-regulation is mediated by a specific set of protein kinases.

biochemistry↗

bioRxiv2018

Quantifying dynamic protein acetylation using quantitative stoichiometry

Protein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell, however identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation are important factors to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under pro-growth conditions. Lastly, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well-described.

biochemistry↗