SPIN enables high throughput species identification of archaeological bone by proteomics.

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce "Species by Proteome INvestigation" (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Multifunctional optrode for opsin delivery, optical stimulation, and electrophysiological recordings in freely moving rats.

Objective. Optogenetics involves delivery of light-sensitive opsins to the target brain region, as well as introduction of optical and electrical devices to manipulate and record neural activity, respectively, from the targeted neural population. Combining these functionalities in a single implantable device is of great importance for a precise investigation of neural networks while minimizing tissue damage.Approach. We report on the development, characterization, andin vivovalidation of a multifunctional optrode that combines a silicon-based neural probe with an integrated microfluidic channel, and an optical glass fiber in a compact assembly. The silicon probe comprises an 11-µm-wide fluidic channel and 32 recording electrodes (diameter 30µm) on a tapered probe shank with a length, thickness, and maximum width of 7.5 mm, 50µm, and 150µm, respectively. The size and position of fluidic channels, electrodes, and optical fiber can be precisely tuned according to thein vivoapplication.Main results.With a total system weight of 0.97 g, our multifunctional optrode is suitable for chronicin vivoexperiments requiring simultaneous drug delivery, optical stimulation, and neural recording. We demonstrate the utility of our device in optogenetics by injecting a viral vector carrying a ChR2-construct in the prefrontal cortex and subsequent photostimulation of the transduced neurons while recording neural activity from both the target and adjacent regions in a freely moving rat for up to 9 weeks post-implantation. Additionally, we demonstrate a pharmacological application of our device by injecting GABA antagonist bicuculline in an anesthetized rat brain and simultaneously recording the electrophysiological response.Significance. Our triple-modality device enables a single-step optogenetic surgery. In comparison to conventional multi-step surgeries, our approach achieves higher spatial specificity while minimizing tissue damage.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients.

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation.

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.

Molecular basis of Tousled-Like Kinase 2 activation.

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.

Active Control of Dye Release for Neuronal Tracing Using PEDOT-PSS Coated Electrodes.

Penetrating neural probes comprising arrays of microelectrodes are commonly used to monitor local field potentials and multi-unit activity in animal brain over time frames of weeks. To correlate these recorded signals to specific tissue areas, histological analysis is performed after the experimental endpoint. Even if the lesion of the penetrating probe shaft can be observed, a precise reconstruction of the exact electrode positions is still challenging. To overcome these experimental difficulties, we developed a new concept, whereupon recording electrodes are coated with a poly (3, 4-ethylenedioxythiophene/ polystyrenesulfonate) (PEDOT/PSS)-based film. The conducting polymer acts as dye reservoir over several weeks and afterwards provides controlled delivery of neurotracers. This paper presents a recording electrode based on a PEDOT/PSS bilayer optimized for dye delivery and with reduced impedance. Controlled exchange of neurotracer dye is successfully demonstrated in vitro using spectrofluorometry and in neuroblastoma cell cultures. A second PEDOT/PSS capping layer on top of the dye reservoir lowers the passive leakage of dye by a factor of 6.4 and prevents a direct contact of the dye filled layer with the cells. Stability tests over four weeks demonstrate the electrochemical stability of the PEDOT coating, as well as retained functionality of the dye delivery system.

Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity.

Here, we report on the first systematic long-term study of fibroblast therapy in a mouse model for recessive dystrophic epidermolysis bullosa (RDEB), a severe skin-blistering disorder caused by loss-of-function of collagen VII. Intradermal injection of wild-type (WT) fibroblasts in >50 mice increased the collagen VII content at the dermal-epidermal junction 3.5- to 4.7-fold. Although the active biosynthesis lasted <28 days, collagen VII remained stable and dramatically improved skin integrity and resistance to mechanical forces for at least 100 days, as measured with a digital 3D-skin sensor for shear forces. Experiments using species-specific antibodies, collagen VII-deficient fibroblasts, gene expression analyses, and cytokine arrays demonstrated that the injected fibroblasts are the major source of newly deposited collagen VII. Apart from transitory mild inflammation, no adverse effects were observed. The cells remained within an area