A proteomic atlas of senescence-associated secretomes for aging biomarker development.

The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry.

Post-translational modification (PTM) of protein lysine residues by NƐ-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites

Simultaneous Quantification of the Acetylome and Succinylome by One-Pot Affinity Enrichment.

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label-free proteomic workflow-one-pot PTM enrichment-that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label-free, data-independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one-pot method are identified and quantified and peak areas are shown to be highly correlated between the one-pot and traditional single-PTM enrichments. The one-pot method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue

Flooding Induced Seismicity in the Ruhr Area – a geomechanics numerical modelling approach

The Ruhr region is characterized by centuries of coal mining at depths reaching more than 1000 meters. After the closure of the last mines, their controlled flooding started. The Floodrisk project investigates ground uplift, stress changes due to pore pressure changes and the reactivation potential of faults to explain induced seismicity. We focused on monitoring the eastern Ruhr area and are investigating in detail the relationship between mine water rise, tectonic stress and induced seismicity in the Haus Aden drainage area. In the region of the former "Bergwerk Ost", which had the highest seismicity in the Ruhr area during active mining, the RUB has installed a network of up to 30 short-period seismic stations. Continuous monitoring of seismicity and mine water levels is available for this region from the active mining phase through the post-mining phase to flooding. The temporal evolution of the mine water level after the pumps were shut down in mid-2019 shows a strong correlation with the temporal evolution of the observed microseismicity. Over 2200 induced events have been located since the beginning of flooding, showing spatial clustering. A comparison of the mine galleries, which today serve as the main underground waterways, with the localizations of the events shows that most of the events occur about 300 m below the main pillars located between the longwall panels. This study provides a compilation of the regional stress state in the eastern Ruhr area based on the mine measurements, which were re-evaluated to derive the regional stress component and compared with stress orientations from independent sources (information on stresses in deep boreholes and earthquake focal mechanisms). The spatial distribution of stress orientations in the Ruhr region shows a rather homogeneous stress pattern with only very few locations where stress orientations differ significantly from the average. Based on the geometry of the pillars, shafts and longwall panels, a generic numerical FE-model was developed using the compiled stress data for model calibration. The results indicate increased vertical stresses within and below the pillars as a result of stress arching. The horizontal stress changes are minor, thus differential stress increases in the vicinity of the event localization

Depletion-induced seismicity in NW-Germany: lessons from comprehensive investigations

Evaluating various investigations for north-German gas fields, we discuss past and actual evolutions of the rock fabric in the light of dilatant driven and spontaneous contractant critical phenomena. Features of the latter were discovered by multi-stage triaxial tests with water-saturated sandstone samples and were similarly observed around the gas fields. A Mohr–Coulomb condition with quasi-local stress components (${\hat{\sigma }}\u27_1$ and ${\hat{\sigma }}\u27_3$), and variable parameters ϕ’ and ${{\hat{c}}}\u27$, can capture successive critical states of the solid fabric. The implied driven dilatation up to a collapse with contraction is captured by a stress-dilatancy relation. Fractal patterns of shear bands (faults) dominate if the smallest principal stress ${\hat{\sigma }}\u27_3$ exceeds ${{\hat{c}}}\u27$, otherwise cracks dominate and can lead to a rockburst. Triaxial tests with X-ray attenuation, seismometry including the splitting of shear waves and/or neutron beam diffraction contribute to clarification and validation. Seismic early warning and calculation models for various geotechnical operations with dominating faults can thus be improved, but the task is more difficult for rockbursts