Biology subjects

Pichler, P.

Publications and source records attributed to Pichler, P..

7 recordsLinked to original sources

bioRxiv2024

Challenging the Astral mass analyzer - going beyond 5200 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.

A detailed proteome map is crucial for understanding molecular pathways and protein functions. Despite significant advancements in sample preparation, instrumentation, and data analysis, single-cell proteomics is currently limited by proteomic depth and quantitative performance. We combine a zero dead-end volume chromatographic column running at high throughput with the Thermo Scientific Orbitrap Astral mass spectrometer running in DIA mode. We demonstrate unprecedented depth of proteome coverage as well as accuracy and precision for quantification of ultra-low input amounts. Using a tailored library, we identify up to 7400 protein groups from as little as 250 pg HeLa at a throughput of 50 samples per day (SPD). We benchmark multiple data analysis strategies, estimate their influence on FDR and show that FDR on protein level can easily be maintained at 1 %. Using a two-proteome mix, we check for optimal parameters of quantification and show that fold change differences of 2 can still be successfully determined at single-cell level inputs. Eventually, we apply our workflow to A549 cells yielding a proteome coverage of up to 5300 protein groups from a single cell, which allows the observation of heterogeneity in a cellular population and studying dependencies between cell size and cell-cycle phase. Additionally, our work-flow enables us to distinguish between in vitro analogs of two human blastocyst lineages: naive human pluripotent stem cells (epiblast) and trophectoderm (TE)-like cells. Gene Ontology analysis of enriched proteins in TE-like cells harmoniously aligns with transcriptomic data, indicating that single-cell proteomics possesses the capability to identify biologically relevant differences between these two lineages within the blastocyst.

biochemistry↗

bioRxiv2023

A method for deep and quantitative protein profiling of urine sediment, soluble and exosome fractions for biomarker research

Urine collection is painless and offers the potential for kidney liquid-biopsy(1), which appears particularly appealing with regard to the diagnosis of kidney disease (2) and patient follow-up after renal transplantation (3). From a nephrological point of view, urinary sediment and the soluble and exosome fractions of urine constitute different biological entities. We here describe a method that allows deep profiling of the protein content of the above-mentioned three fractions of urine by quantitative data-independent label-free proteomics. The method was evaluated using 19 urine samples from the Nephrology outpatient clinic at Vienna General Hospital, comprising a diverse set of chronic kidney disease (CKD) as well as patients after kidney transplantation (NTX). Peptide separation was accomplished through 60 min active gradients. A timsTOF Pro2 mass spectrometer was operated in DIA mode. The total analysis time per urine sample (three fractions) was around four hours. We demonstrate adequate technical and experimental reproducibility. Our data suggest that the protein information content of these three fractions is diverse, strengthening the importance of separate analysis. The depth of our quantitative proteomics approach permitted a detection of proteins characteristic for different parts of the nephron, such as Podocin, CD2-AP and Podocalyxin (Podocytes), SLC22A8 and SLC22A13 (proximal tubule) and Aquaporin-2 (collecting duct), suggesting that our method is sensitive enough to detect and quantify biologically relevant proteins that might serve as potential biomarkers. To the best of our knowledge, the ability to quantify up to 4000 protein groups per urine sample and more than 6000 protein groups in total makes our strategy the deepest proteome profiling study of urine to date. In conclusion, we established a method with promising figures of merit that we consider broadly applicable and useful for future clinical biomarker research studies in urine.

biochemistry↗

bioRxiv2022

Robust and easy-to-use one pot workflow for label free single cell proteomics

The analysis of ultra-low input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a comprehensive workflow that includes optimized strategies for all steps from cell lysis to data analysis. Thanks to convenient to handle 1 L sample volume and standardized 384 well plates the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using the CellenONE(R), which allows for highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced -pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA) and data-independent acquisition (DIA), and commonly used advanced data-analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single cell level input in a 20-min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.

biochemistry↗

bioRxiv2022

Control algorithms underlying the translational optomotor response

The optomotor response (OMR) is central to the locomotory behavior in diverse animal species including insects, fish and mammals. Furthermore, the study of the OMR in larval zebrafish has become a key model system for investigating the neural basis of sensorimotor control. However, a comprehensive understanding of the underlying control algorithms is still outstanding. In fish it is often assumed that the OMR, by reducing average optic flow across the retina, serves to stabilize position with respect to the ground. Yet the degree to which this assumption is true, and how this could emerge from the intermittent burst dynamics of larval zebrafish swimming is unclear. Here, we combine detailed computational modeling with a new approach to free-swimming experiments, in which feedback gain - i.e., the degree of sensory feedback produced by swimming - is manipulated by varying the height of larval zebrafish above a moving stimulus. We develop an account of underlying feedback control mechanisms that describes not only bout initiation but also the control of swim speed during bouts. We observe that the degree to which fish stabilize their position is only partial and gain-dependent, suggesting that the OMR may not primarily function in fish to prevent drift. We find the speed profile during bouts follows a fixed temporal pattern independent of bout intensity, suggesting that bout termination and bout duration are not explicitly controlled. We also find that the reverse optic flow, experienced when the fish is swimming faster than the stimulus, plays a minimal role in control of the OMR despite carrying most of the sensory information about self-movement. These results shed new light on the underlying dynamics of the OMR in larval zebrafish and will be crucial for future work aimed at identifying the neural basis of this behavior. Author SummaryIn many animals vision is central to the control of locomotory behaviors. In particular, innate motor responses to optic flow allow flying animals to react to gusts of air and fish to changes of current. As fish are washed downstream, for example, movement of the riverbed image across the retina evokes forward movement in bouts (short periods of swimming followed by rest periods). It is typically assumed that this translational optomotor response stabilizes the fishs position to prevent it drifting downstream. In larval zebrafish, this response has become a key model system for investigating the neural basis of sensorimotor behaviors in a vertebrate. Here, we combine behavioral experiments and computational modeling to elucidate the underlying control algorithms. We unpick the detailed relationship between visual stimuli and the initiation, termination and intensity of swim bouts. We find initiation and intensity are controlled separately, and that termination is not explicitly controlled. Surprisingly we also found that the degree of stabilization is only partial and varies systematically with height above ground, raising questions of the function of this response. These findings shed new light on the underlying dynamics which will be crucial for future work to identify the neural basis of this behavior.

animal behavior and cognition↗

bioRxiv2020

μSPIM: A Software Platform for Selective Plane Illumination Microscopy

Selective Plane Illumination Microscopy (SPIM) is a fluorescence imaging technique that allows volumetric imaging at high spatio-temporal resolution to monitor neural activity in live organisms such as larval zebrafish. A major challenge in the construction of a custom SPIM microscope is the control and synchronization of the various hardware components. Here we present a control toolset, SPIM, built around the open-source MicroManager platform that has already been widely adopted for the control of microscopy hardware. Installation of SPIM is relatively straightforward, involving a single C++ executable and a Java-based extension to Micro-Manager. Imaging protocols are defined through the SPIM extension to Micro-Manager. The extension then synchronizes the camera shutter with the galvanometer mirrors to create a light-sheet that is scanned in the z-dimension, in synchrony with the imaging objective, to produce volumetric recordings. A key advantage of SPIM is that a series of calibration procedures optimizes acquisition for a given set-up making it relatively independent of the optical design of the microscope, or the hardware used to build it. Two laser illumination arms can be used while also allowing for the introduction of illumination masks. SPIM allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution as well as slower imaging to reconstruct cell populations, for example, in the cleared brains of mice.

neuroscience↗

bioRxiv2019

Motor behaviour selectively inhibits hair cells activated by forward motion in the lateral line of Zebrafish

How do sensory systems disambiguate events in the external world from signals generated by the motor behaviour of the animal? One strategy is to suppress the sensory input whenever the motor system is active, but the cellular mechanisms remain unclear. We investigated how motor behaviour modulates signals transmitted by the lateral line of zebrafish, which senses pressure changes around the body of the animal. Activation of motor neurons during fictive swimming caused co-activation of efferent fibers and suppression of synaptic transmission from the primary mechanoreceptors, the hair cells. In some hair cells, a single motor spike inhibited glutamate release by about 50% and block was often complete within 50-100 ms of the start of swimming. All hair cells polarized to be activated by posterior deflections, as would occur during forward swimming, were suppressed by >90%, while only half of those polarized in the anterior direction were inhibited and by an average of just 45%. The selective inhibition of hair cells activated during motor behaviour provides a mechanism for the suppression of self-generated signals while maintaining sensitivity to stimuli originating in the external world.

neuroscience↗

bioRxiv2018

Hair cells with heterogeneous transfer characteristics encode mechanical stimuli in the lateral line of zebrafish

Ribbon synapses of hair cells transmit mechanical information but the transfer characteristics relating deflection of the hair bundle to glutamate release have not been assessed directly. Here we have imaged glutamate to investigate how hair cells encode information in the lateral line of zebrafish. Half the hair cells signalled cupula motion in either direction from rest, achieving maximum sensitivity for deflections of ~40 nm in the preferred direction. The remainder rectified completely and were less sensitive, extending the operating range of the neuromast beyond 1m. Adaptation was also heterogeneous, with some hair cells generating sustained synaptic outputs and others transient. A unique signal encoded a return to rest: a transient burst from hair cells unresponsive to the initial stimulus. A mixed population of hair cells with these various transfer characteristics will allow a neuromast to encode weak stimuli as well as the amplitude and duration of stronger deflections.

neuroscience↗