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How tissues migrate robustly through changing guidance landscapes is poorly understood. Here, quantitative imaging is combined with inducible perturbation experiments to investigate the mechanisms that ensure robust tissue migration in vivo. We show that tissues exposed to acute "chemokine floods" halt transiently before they perfectly adapt, i.e., return to the baseline migration behavior in the continued presence of elevated chemokine levels. A chemokine-triggered phosphorylation of the atypical chemokine receptor Cxcr7b reroutes it from constitutive ubiquitination-regulated degradation to plasma membrane recycling, thus coupling scavenging capacity to extracellular chemokine levels. Finally, tissues expressing phosphorylation-deficient Cxcr7b migrate normally in the presence of physiological chemokine levels but show delayed recovery when challenged with elevated chemokine concentrations. This work establishes that adaptation to chemokine fluctuations can be "outsourced" from canonical GPCR signaling to an autonomously acting scavenger receptor that both senses and dynamically buffers chemokine levels to increase the robustness of tissue migration.

Summary Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5's mode of actions, which appears to be context dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking and in P5's non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues, and organisms., Graphical abstract, Highlights • Developed four functional click-enabled analogues of pregnenolone (P5) • Chemoproteomics prioritizes 62 P5 target proteins in live cancer and immune cells • These include shared and distinct biochemical role of P5 in cancer and immune cells • P5 activity in cancer and immune cells is mediated through non-genomic pathways

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '-omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC Grant Agreement No. 232913. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012–0208. This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 634942. Funding for open access charge: European Research council (ERC) advanced Grant Agreement No. 232913, the Fundación Marcelino Botin, the Spanish Ministerio de Economía y Competitividad BIO2007-61762, the ISCIII, Subdirección General de evaluación y fomento de la investigación PI10/01702 to the ICREA researcher L.S.

Here, we describe an in-house built ultra-high pressure liquid chromatography (UHPLC) system, with little complexity in design and high separation power combined with convenience in operation. This system enables the use of long columns of 40 cm packed with 1.8 μm particles generating pressures below 1000 bar. Furthermore, the system could be operated at flow rates between 50 and 200 nL min(-1) while maintaining its separation power. Several gradients were optimized ranging from 23 to 458 minutes. With the longest gradient we identified over 4500 protein groups and more than 26,000 unique peptides from 1 μg of a human cancer cell lysate in a single run using an Orbitrap Velos - a level of performance often seen solely using multidimensional separation strategies. Further experiments using a mass spectrometer with faster sequencing speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that the combination of UHPLC with high resolution mass spectrometry at increased sequencing speeds enables extensive proteome analysis in single runs.

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.