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Bone marrow mesenchymal stromal cells (BMSCs) can differentiate into adipocytes and osteoblasts, and are important regulators of the haematopoietic system. Ageing associates with an increased ratio of bone marrow adipocytes to osteoblasts and immune dysregulation. Here, we carried out an integrative multiomics analysis of ATAC-Seq, RNA-Seq and proteomics data from primary human BMSCs in a healthy cohort age between 20 - 60. We identified age-sensitive elements uniquely affecting each molecular level where transcription is mostly spared, and characterised the underlying biological pathways, revealing the interplay of age-related gene expression mechanism changes spanning multiple gene regulatory layers. Through data integration with enhancer-mediated gene regulatory network analysis, we discovered that enhancers and transcription factors influence cell differentiation potential in the ageing BMSCs. By combining our results with genome-wide association study data, we found that age-specific changes could contribute to common traits related to BMSC-derived tissues such as bone and adipose tissue, and to immune-related traits on a systemic level such as asthma. We demonstrate here that a multiomics approach is crucial for unravelling complex information, providing new insights on how ageing contributes to bone marrow- and immune-related disorders.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

Introduction: RNA modifications are emerging as important determinants of cell identity and cell fate. Small nucleolar RNAs (snoRNA) guide pseudouridylation and 2'-O-methylation of RNA species. C/D box snoRNAs are essential for AML1/ETO-induced leukemia (Zhou et al. Nat Cell Biol 2017). Dynamics and relevance of these modifications in hematopoiesis are unknown. Here, we aimed to determine the plasticity of ribosomal 2'-O-methylation (Ribomethylome) patterns in hematopoietic cell populations and the interdependence with snoRNA expression, transcriptomics and proteomics. Methods: Healthy donors (19-86yrs) donated bone marrow and six cell populations were sorted or prepared: Hematopoietic stem/progenitor cell (HPC), Monocyte/macrophage precursor (MON), Granulocytic precursor (GRA), Erythroid precursor (ERY), Lymphocyte progenitor (LYM), and Mesenchymal stem/stromal cell (MSC). Small RNA sequencing and Ribometh-seq data were obtained for 65 and 55 samples, respectively. Data were analyzed together with accompanying RNA-seq and Mass-spec proteomics data which were available for all the specimens. Bioinformatics analyses were based on PCA, tSNE, spearman correlation, paired t-test, GSEA and ANOVA. Results: The analyses of 2'-O-methylation (Ribomethylome) in six bone marrow cell types from healthy donors revealed that ribosomal modifications occurred different during the process of hematopoietic differentiation. Among these sides, HPC and Myeloid lineage showed significant variability between different cell populations. Ribomethylome patterns differed between cell types and PCA analyses indicated that cellular identity was matched with a specific Ribomethylome pattern. Plasticity in Ribomethylomes were most evident for HPC, LYM, GRA and MON which showed high levels of 2'-O-methylation (almost 100% of rRNA methylated) whereas methylation levels in MSC cells were much lower (Spearman correlation Conclusion: Our finding based on multi-omics analyses identifies cell type specific Ribomethylomes. Myeloid differentiation is associated with specific Ribomethylome changes. Distinct Ribomethylomes may contribute to cellular identity by directing translation of specific sets of mRNAs. Figure 1: The effects of ribomethylome and protein translation were evident and separated by different cell populations(tSNE). Figure 1 Disclosures Müller-Tidow: Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau.

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

12 p.-5 fig.-2 tab., Saccharomyces cerevisiae τ55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (τ55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of τ55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify τ55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.

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.

Over the past decade peptide sequencing by collision induced dissociation (CID) has become the method of choice in mass spectrometry-based proteomics. The development of alternative fragmentation techniques such as electron transfer dissociation (ETD) has extended the possibilities within tandem mass spectrometry. Recent advances in instrumentation allow peptide fragment ions to be detected with high speed and sensitivity (e.g., in a 2D or 3D ion trap) or at high resolution and high mass accuracy (e.g., an Orbitrap or a ToF). Here, we describe a comprehensive experimental comparison of using ETD, ion-trap CID, and beam type CID (HCD) in combination with either linear ion trap or Orbitrap readout for the large-scale analysis of tryptic peptides. We investigate which combination of fragmentation technique and mass analyzer provides the best performance for the analysis of distinct peptide populations such as N-acetylated, phosphorylated, and tryptic peptides with up to two missed cleavages. We found that HCD provides more peptide identifications than CID and ETD for doubly charged peptides. In terms of Mascot score, ETD FT outperforms the other techniques for peptides with charge states higher than 2. Our data shows that there is a trade-off between spectral quality and speed when using the Orbitrap for fragment ion detection. We conclude that a decision-tree regulated combination of higher-energy collisional dissociation (HCD) and ETD can improve the average Mascot score.

Background: Diminishing potential to replace damaged tissues is a hallmark for aging of somatic stem cells, but the mechanisms leading to aging remain elusive. Applying comprehensive proteomics studies on human hematopoietic stem and progenitor cells (HPCs) as well as 5 other cell populations that constitute the bone marrow niche, we have acquired a systems understanding of the mechanisms involved in aging of HPCs. Methods and materials: We present a proteome-wide atlas of age-associated alterations in HPCs along with 5 other cell populations including mesenchymal stromal cells (MSC) that comprise the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified was assessed in a cohort of 59 human subjects from different age groups. In selected samples, transcriptomics, metabolomics, and single cell RNA-Sequencing studies were simultaneously performed. Results: As the HPCs become older, one of the most prominent changes is the increase in abundance of proteins involved in the pathways for central carbon metabolism. This change is found only in HPCs and is reminiscent of the Warburg effect where glycolytic intermediates are rerouted towards pentose phosphate shunt and anabolism. Transcriptomic data confirm the increase in abundance of mRNA for the respective glycolytic enzymes in older HPCs. Metabolomics analyses provide further proofs demonstrating the trend towards anabolism upon aging. Altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation at the expense of lymphoid development. Simultaneously, significant and complementary alterations in the bone marrow niche are observed. Whereas key factors responsible for homing, egress and adhesion of HPCs, e.g. SDF1/CXCL12, VCAM1, FN1, integrins α4, αL, β1 andβ2 decrease in abundance in MSCs, soluble factors responsible for myeloid differentiation, e.g.TGF-beta1, increase in abundance in the cellular niche with age. Transcriptomic analyses of single-sorted HPCs have demonstrated unequivocally that the mRNA levels of age-dependent increase in glycolytic enzymes are expressed at significantly higher levels in myeloid-biased versus than those in lymphoid-biased HPCs, whereas age-unaffected enzymes have similar mRNA levels in both subsets. The increase in abundance of glycolytic enzymes is hence linked with skewing of myeloid-biased HPCs in older human subjects. Conclusion: We have generated a comprehensive atlas of alterations in proteome landscapes of human HPCs and niche cells in bone marrow upon aging. In addition to findings that recapitulate the results derived from murine studies, the major novelties are (a) the alterations in central carbon metabolism in aging human HPCs, (b) the complementary decrease in levels of adhesive molecules and respective ligands in MSCs and HPCs. Single-cell studies have demonstrated that the age-related increase in abundance of the glycolytic enzymes is linked to the myeloid-biased HPCs. These data represent a valuable resource and serve as a basis for development of strategies targeting metabolic changes to enhance HPC regeneration. Disclosures No relevant conflicts of interest to declare.

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.

Disruption of epithelial architecture is a fundamental event during epithelial tumorigenesis. We show that the expression of the cancer-promoting phosphatase PRL-3 (PTP4A3), which is overexpressed in several epithelial cancers, in polarized epithelial MDCK and Caco2 cells leads to invasion and the formation of multiple ectopic, fully polarized lumens in cysts. Both processes disrupt epithelial architecture and are hallmarks of cancer. The pathological relevance of these findings is supported by the knockdown of endogenous PRL-3 in MCF-7 breast cancer cells grown in three-dimensional branched structures, showing the rescue from multiple-lumen- to single-lumen-containing branch ends. Mechanistically, it has been previously shown that ectopic lumens can arise from midbodies that have been mislocalized through the loss of mitotic spindle orientation or through the loss of asymmetric abscission. Here, we show that PRL-3 triggers ectopic lumen formation through midbody mispositioning without altering the spindle orientation or asymmetric abscission, instead, PRL-3 accelerates cytokinesis, suggesting that this process is an alternative new mechanism for ectopic lumen formation in MDCK cysts. The disruption of epithelial architecture by PRL-3 revealed here is a newly recognized mechanism for PRL-3-promoted cancer progression.

Posttranslational modifications (PTMs) of proteins represent an important level of cellular control. They participate in the efficient transduction of signals and form the basis of long-term cellular memory, allowing cells to adapt to a rapidly changing environment. More than 200 different PTMs have been described that affect many aspects of protein functions, and the importance of these modifications is evident from the number of diseases that arise from their deregulation. The proteome-wide analysis of certain PTMs, such as phosphorylation, acetylation, glycosylation, methylation, ubiquitination, and sumoylation, has become a standard procedure in many laboratories. We highlight and discuss some important aspects of systems-wide PTM analyses using mass spectrometry-based methods.

Background The multifunctional Ca 2+ ‐ and calmodulin‐dependent protein kinase II (Ca MKII ) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of Ca MKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining Ca MKII 's role therein is large‐scale analysis of phosphorylation events by mass spectrometry. However, current large‐scale phosphoproteomics approaches have proved inadequate for high‐fidelity identification of kinase‐specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify Ca MKII 's downstream effects in cardiac tissue. Methods and Results To identify putative downstream Ca MKII targets in cardiac tissue, animals with myocardial‐delimited expression of the specific peptide inhibitor of Ca MKII ( AC 3‐I) or an inactive control ( AC 3‐C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce Ca MKII activation downstream of β‐adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC 3‐I and AC 3‐C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high‐resolution LC ‐ MS / MS . Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC 3‐I‐mediated Ca MKII inhibition. Conclusions Our data set included known Ca MKII substrates, as well as several new candidate proteins involved in functions not previously implicated in Ca MKII signaling.

Background: Whereas proteomics analyses at a cellular level have been performed in murine HPC, most proteomics studies on human material have thus far focused on examining human tissues with a mixture of cell types and not at a specific cellular level. Whole proteomics analyses might reveal genuine molecular alterations und mechanisms of aging on human hematopoietic cells. Aim: To identify the age-related molecular changes in human hematopoietic progenitor cells (HPC) as well as in the cellular elements of the human bone marrow niche, we have applied mass spectrometry-based platform for proteomics analysis of this network of bone marrow (BM) cells at a cellular level. The overarching goal is to acquire a systems understanding of the molecular mechanisms involved in aging of human HPC as well as other cellular constituents of the marrow niche, and to combine the cell biological, proteomic, and genetic studies for a better understanding of age-related diseases such as MDS. Methods: In this study, we have simultaneously characterized at the proteomics level HPC as well as the cellular microenvironment in BM from each individual human subject. Cells were harvested from the BM of 69 healthy human subjects [Age Group (AG) 20-29 years: n=19; AG 30-39: n=16; AG 40-49: n=9; AG 50+: N=15]. BM cells were separated for nucleated cells using Ficoll, followed by sorting with specific marker constellations, i.e. HPC (CD34+), lymphoid cells (CD34-/CD45+/SSClow), myeloid precursors (CD34-/CD45med/SSChigh), monocyte-macrophages (CD34-/CD45+/CD14+), erythroid precursors (CD34-/CD45-), and mesenchymal stromal cells (MSC) based on plastic adherence upon culture. Results: In 60 of these 69 healthy subjects we were able to harvest an adequate amount of material from 5 to 6 the aforementioned subsets from the respective individual, yielding altogether 342 samples for proteomics analysis. After digestion (trypsinization), isobaric labelling of peptides (TMT) of appropriate quality was successfully performed in 270 of these 342 samples derived from 69 healthy human subjects. These 270 samples underwent tandem mass-spectrometry analyses. The number of proteins identified were >12,000, covering 65% of the estimated human proteome (HPC: >7,500; Lymphoid cells: >8,500; Myeloid Precursors: >7,500; Mono/Macro: >8,500; Erythroid Precursors: >6,500; and in MSC: >9,000 proteins). We have then focused on the molecular mechanisms, specifically proteomic alterations involved in aging of these 6 cell types. Gene Set Enrichment Analysis (GSEA) using previously defined gene sets specific for common lymphoid progenitors (CLP), bi-potent granulocyte/macrophage progenitors (GMP), and megakaryocyte progenitors (MKP) revealed significant down-regulation of CLP related proteins and significant up-regulation of MKP related proteins in HPC upon aging. The same down-regulation of CLP proteins and up-regulation of MKP proteins were also found in the BM lymphoid cells. Further significant alterations of note are a decrease in cell cycle related proteins upon aging in HPC, lymphoid cells, as well as in MSC, and an increase in GMP related proteins upon aging in lymphoid cells, erythroid precursors and in MSC. Extensive pathway and network analyses are underway. Conclusions: We have for the first time accomplished a multiplex and comprehensive analysis of a community of cells in human BM, comprising HPC and 5 other cell types that constitute the niche and have identified >12,000 proteins in this cellular network. Proteomics analyses have demonstrated a significant down-regulation of CLP related proteins in human HPC and in marrow lymphoid cells, as well as an up-regulation of MKP in all the cell types tested upon aging. Of note is also a decrease in cell cycle related proteins with age. This study has thus provided evidence that lineage skewing towards megakaryocytic and decrease in lymphoid development upon aging occur in human HPC as well as in other human BM cells. Disclosures No relevant conflicts of interest to declare.

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.