Biology subjects

Mann, M.

Publications and source records attributed to Mann, M..

15 recordsLinked to original sources

bioRxiv2018

MaxQuant.Live enables global targeting of more than 25,000 peptides

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. While achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on pre-scheduled mass-to-charge and retention time windows. Here we set out to merge these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time recalibration in mass, retention time and intensity dimensions to predict precursor identity. MaxQuant.Live is freely available (www.maxquant.live) and has a graphical user interface to specify many pre-defined data acquisition strategies. Acquisition speed is as fast as with the vendor software and the power of our approach is demonstrated with the acquisition of breakdown curves for hundreds of precursors of interest. We also uncover precursors that are not even visible in MS1 scans, using elution time prediction based on the auto-adjusted retention time alone. Finally, we successfully recognized and targeted more than 25,000 peptides in single LC-MS runs. Global targeting combines the advantages of two classical approaches in MS-based proteomics, while greatly expanding the analytical toolbox.

bioinformatics↗

bioRxiv2018

In vivo phosphoproteomics reveals pathogenic signaling changes in diabetic islets

Progressive decline of pancreatic beta cells function is key to the pathogenesis of type 2 diabetes. Protein phosphorylation is the central mechanism controlling glucose-stimulated insulin secretion in beta cells. However, if and how signaling networks are remodeled in diabetic islets in vivo remain unknowns. Here we applied high-sensitivity mass spectrometry-based proteomics and quantified the levels of about 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls. This highlighted drastic remodeling of key kinase hubs and signaling pathways. We integrated our phosphoproteomic dataset with a literature-derived signaling network, which revealed a crucial and conserved role of GSK3 kinase in the control of the beta cells-specific transcription factor PDX1 and insulin secretion, which we functionally verified. Our resource will enable the community to investigate potential mechanisms and drug targets in type 2 diabetes.

systems biology↗

bioRxiv2018

Conserved mechanism of nucleoporin regulation of the Kcnq1ot1 imprinted domain with divergence in embryonic and trophoblast stem cells

Genomic imprinting is an epigenetic phenomenon, whereby dual chromatin states lead to expression of one, and silencing of the other parental allele. Recently, we identified a nucleoporin-mediated mechanism of Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells by nucleoporins NUP107, NUP62 and NUP153. Here, we investigate their role in Kcnq1ot1 imprinted domain regulation in embryonic and trophoblast stem cells. Nucleoporin depletion in both lineages reduced Kcnq1ot1 noncoding RNA expression and volume, reduced Kcnq1ot1 paternal domain positioning at the nuclear periphery, and altered histone modifications along with histone modifier enrichment at the imprinting control region. However, while CTCF and cohesin were enriched at nucleoporin binding sites in the imprinting control region in embryonic stem cells, with reduction upon nucleoporin depletion, neither CTCF or cohesin occupied these sites in trophoblast stem cells. Finally, different subsets of silent paternal alleles were reactivated via altered histone modification upon nucleoporin depletion in embryonic and trophoblast stem cells. These results demonstrate a conserved mechanism with divergent regulation of the Kcnq1ot1 imprinted domain by NUP107, NUP62 and NUP153 in embryonic and extraembryonic lineages.\n\nSummary StatementInvestigation of nucleoporins, NUP107, NUP62, and NUP153, revealed a conserved nucleoporin-dependent mechanism that mediates Kcnq1ot1 imprinted domain regulation in ES and TS cells, although notable lineage-specific divergence was also observed.

genetics↗

bioRxiv2018

Single fiber proteomics of respiratory chain defects in mitochondrial disorders

Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. The single fiber investigation of this important diagnostic feature has been beyond the capability of large-scale technologies so far. We used laser capture microdissection (LCM) to excise thin sections of individual muscle fibers from frozen biopsies of patients suffering from chronic progressive external ophthalmoplegia. We then applied a highly sensitive mass spectrometry (MS)-based proteomics workflow to analyze healthy and defective muscle fibers within the same biopsy. We quantified more than 4000 proteins in each patient, covering 75% of all respiratory chain subunits, and compared their expression in metabolically healthy and defective muscle fibers. Our findings show that mitochondrial disease causes extensive proteomic rearrangements, affecting the OPA1-dependent cristae remodeling pathway and mitochondrial translation. We provide fiber type-specific information showing that increased expression of fatty acid oxidation enzymes occurs in defective slow but not fast muscle fibers. Our findings shed light on compensatory mechanisms in muscle fibers that struggle with energy shortage and metabolic stress.

pathology↗

bioRxiv2018

Multi-Omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency

Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remain incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre-to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of mouse embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes demarcate discrete phases of pluripotency, characterized by cell-state-specific surface marker expression. Our data provide new insights into the multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms underlying pre-to post-implantation epiblast differentiation.\n\nHIGHLIGHTSO_LIMulti-ome maps of cells transitioning from naive to primed pluripotency\nC_LIO_LIPhosphoproteome dynamics precede changes to epigenome, transcriptome, and proteome\nC_LIO_LIKinase-substrate network reconstruction uncovers signaling dynamics and crosstalk\nC_LIO_LIProteins and cell surface markers that track pluripotent state transitions\nC_LIO_LIComparative analysis of mouse and human pluripotent states\nC_LI

genomics↗

bioRxiv2018

Proteomics-based comparative mapping of the human brown and white adipocyte secretome reveals EPDR1 as a novel batokine

Secreted proteins from adipose tissue play a role in metabolic cross-talk and homeostasis. We performed high sensitivity mass spectrometry-based proteomics on the cell media of in vitro differentiated, non-immortalized brown adipocytes derived from supraclavicular adipose of adult humans and white adipocytes derived from subcutaneous adipose of adult humans. We identified 471 potentially secreted proteins covering interesting protein categories such as hormones, growth factors, growth factor binding proteins, cytokines, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated in brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, among these ependymin-related protein 1 (EPDR1). Ablation of EPDR1 impaired the induction of thermogenic transcripts in response to norepinephrine in brown adipocytes, while EPDR1-treated mice increased their energy consumption, suggesting a role in brown fat commitment and activation. Our work reveals substantial differences between the secretomes of brown and white human adipocytes and identifies novel candidate batokines.

physiology↗

bioRxiv2018

Cortical circuit alterations precede disease onset in Huntington’s disease mice

AbstractHuntingtons disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in the disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to monitor the activity of single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in neuronal activity with a clear increase in activity at the age of 8.5 weeks, preceding the onset of motor and neurological symptoms. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and HD patient samples reveal reduced inputs from parvalbumin-positive interneurons onto layer 2/3 pyramidal cells. Thus, our study provides a time-resolved description as well as mechanistic details of cortical circuit dysfunction in HD.\n\nSignificance statementFuntional alterations in the cortex are believed to play an important role in the pathogenesis of Huntingtons disease (HD). However, studies monitoring cortical activity in HD models in vivo at a single-cell resultion are still lacking. We have used chronic two-photon imaging to investigate changes in the activity of single neurons in the primary motor cortex of awake presymptomatic HD mice. We show that neuronal activity increases before the mice develop disease symptoms. Our histological analyses in mice and in human HD autopsy cases furthermore demonstrate a loss inhibitory synaptic terminals from parvalbimun-positive interneurons, revealing a potential mechanism of cortical circuit impairment in HD.

neuroscience↗

bioRxiv2018

Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

DNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.\n\nHighlightsO_LIThe proteasome, in addition to SPRTN, degrades DPCs during DNA replication\nC_LIO_LIProteasome-dependent DPC degradation requires DPC ubiquitylation\nC_LIO_LIDPC ubiquitylation is triggered by ssDNA and does not require the replisome\nC_LIO_LISPRTN-dependent DPC degradation is a post-replicative process\nC_LI

molecular biology↗

bioRxiv2018

The CMG helicase bypasses DNA protein cross-links to facilitate their repair

Covalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. Using Xenopus laevis egg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisomes response to nucleoprotein barriers.

biochemistry↗

bioRxiv2018

An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics

Aging promotes lung function decline and susceptibility to chronic lung diseases, which are the third leading cause of death worldwide. We used single cell transcriptomics and mass spectrometry to quantify changes in cellular activity states of 30 cell types and the tissue proteome from lungs of young and old mice. Aging led to increased transcriptional noise, indicating deregulated epigenetic control. We observed highly distinct effects of aging on cell type level, uncovering increased cholesterol biosynthesis in type-2 pneumocytes and lipofibroblasts as a novel hallmark of lung aging. Proteomic profiling revealed extracellular matrix remodeling in old mice, including increased collagen IV and XVI and decreased Fraser syndrome complex proteins and Collagen XIV. Computational integration of the aging proteome and single cell transcriptomes predicted the cellular source of regulated proteins and created a first unbiased reference of the aging lung. The lung aging atlas can be accessed via an interactive user-friendly webtool at: https://theislab.github.io/LungAgingAtlas

systems biology↗

bioRxiv2018

Online parallel accumulation - serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, while mass spectra are acquired in about 100 microseconds with time-of-fight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive due to its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 800,000 fragmentation spectra in standard 120 min LC runs are easily achievable, which can be used for near exhaustive precursor selection in complex mixtures or re-sequencing weak precursors. MaxQuant identified more than 6,400 proteins in single run HeLa analyses without matching to a library, and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,900 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

systems biology↗

bioRxiv2018

A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system-termed Evosep One-analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

biochemistry↗

bioRxiv2017

EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification

We developed EASI-tag (Easily Abstractable Sulfoxide-based Isobaric tag), a new generation of amine-derivatizing and sulfoxide-containing isobaric labelling reagents, which dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level enable accurate determination of quantitative differences between multiplexed samples. EASI-tag makes isobaric labeling applicable to any bottom up proteomics workflow and to benchtop mass spectrometers.

biochemistry↗

bioRxiv2017

Skeletal muscle fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH-dependent regulation of adipogenesis

Fibro adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fat infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. Nonetheless, factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition. An unbiased mass spectrometry-based proteomic analysis of FAPs from muscles of wild type and mdx mice, revealed that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. These results offer a basis for rationalizing the pathological outcomes of fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fatty depositions in muscles of dystrophic patients.\n\nHighlightsO_LISingle-cell mass cytometry reveals that wt and mdx FAPs are in different cell states.\nC_LIO_LIActivation of the NOTCH signaling pathway negatively regulates adipogenesis of wt but not mdx FAPs.\nC_LIO_LIDeep proteomics suggests a mechanism explaining the different sensitivity of mdx- FAPs to NOTCH.\nC_LIO_LITNF-a stimulation restores the anti-adipogenic effect of NOTCH in mdx FAPs.\nC_LI

cell biology↗

bioRxiv2017

Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli are Altered with Age

Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal's lifespan. With age, however, they produce a myeloid-biased output that may lead to poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA-seq, we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is much more common amongst aged LT-HSCs and is uniquely primed to respond to acute inflammatory challenge. We predict several transcription factors to regulate differentially expressed genes between mLT-HSCs and other LT-HSC subsets. Among these, we show that Klf5, Ikzf1 and Stat3 play important roles in age-related inflammatory myeloid bias. These factors may regulate myeloid versus lymphoid balance with age, and can potentially mitigate the long-term deleterious effects of inflammation that lead to hematopoietic pathologies.\n\nHighlightsO_LILT-HSCs from young and aged mice have differential responses to acute inflammatory challenge.\nC_LIO_LIHSPCs directly sense inflammatory stimuli in vitro and have a robust transcriptional response.\nC_LIO_LIAged LT-HSCs demonstrate a cell-intrinsic myeloid bias during inflammatory challenge.\nC_LIO_LISingle-cell RNA-seq unmasked the existence of two subsets within the LT-HSC population that was apparent upon stimulation but not steady-state. One of the LT-HSC subsets is more prevalent in young and the other in aged mice.\nC_LIO_LIKlf5, Ikzf1 and Stat3 regulate age- and inflammation-related LT-HSC myeloid-bias.\nC_LI\n\nOne sentence summaryMurine hematopoietic stem cells display transcriptional heterogeneity that is quantitatively altered with age and leads to the age-dependent myeloid bias evident after inflammatory challenge.

immunology↗