Label-free quantification of shotgun LC–MS/MS data is the prevailing approach in quantitative proteomics but remains computationally nontrivial. The central data analysis step is the detection of ...peptide-specific signal patterns, called features. Peptide quantification is facilitated by associating signal intensities in features with peptide sequences derived from MS2 spectra; however, missing values due to imperfect feature detection are a common problem. A feature detection approach that directly targets identified peptides (minimizing missing values) but also offers robustness against false-positive features (by assigning meaningful confidence scores) would thus be highly desirable. We developed a new feature detection algorithm within the OpenMS software framework, leveraging ideas and algorithms from the OpenSWATH toolset for DIA/SRM data analysis. Our software, FeatureFinderIdentification (“FFId”), implements a targeted approach to feature detection based on information from identified peptides. This information is encoded in an MS1 assay library, based on which ion chromatogram extraction and detection of feature candidates are carried out. Significantly, when analyzing data from experiments comprising multiple samples, our approach distinguishes between “internal” and “external” (inferred) peptide identifications (IDs) for each sample. On the basis of internal IDs, two sets of positive (true) and negative (decoy) feature candidates are defined. A support vector machine (SVM) classifier is then trained to discriminate between the sets and is subsequently applied to the “uncertain” feature candidates from external IDs, facilitating selection and confidence scoring of the best feature candidate for each peptide. This approach also enables our algorithm to estimate the false discovery rate (FDR) of the feature selection step. We validated FFId based on a public benchmark data set, comprising a yeast cell lysate spiked with protein standards that provide a known ground-truth. The algorithm reached almost complete (>99%) quantification coverage for the full set of peptides identified at 1% FDR (PSM level). Compared with other software solutions for label-free quantification, this is an outstanding result, which was achieved at competitive quantification accuracy and reproducibility across replicates. The FDR for the feature selection was estimated at a low 1.5% on average per sample (3% for features inferred from external peptide IDs). The FFId software is open-source and freely available as part of OpenMS (www.openms.org).
Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which ...cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.
► Quantitative proteomic strategy reveals dynamics of cell-cycle protein interactions ► Cyclins confer biochemical specificity to cyclin/cdk interaction networks ► Cyclin A phosphorylates Sld3/c15orf42 and Wee1 to promote S phase and mitosis ► Cyclins A and B interact sequentially with proteins in G2 phase and mitosis
Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative ...palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.
Naïve CD4
T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we ...use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4
T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.
Asexual stage Plasmodium falciparum replicates and undergoes a tightly regulated developmental process in human erythrocytes. One mechanism involved in the regulation of this process is ...posttranslational modification (PTM) of parasite proteins. Palmitoylation is a PTM in which cysteine residues undergo a reversible lipid modification, which can regulate target proteins in diverse ways. Using complementary palmitoyl protein purification approaches and quantitative mass spectrometry, we examined protein palmitoylation in asexual-stage P. falciparum parasites and identified over 400 palmitoylated proteins, including those involved in cytoadherence, drug resistance, signaling, development, and invasion. Consistent with the prevalence of palmitoylated proteins, palmitoylation is essential for P. falciparum asexual development and influences erythrocyte invasion by directly regulating the stability of components of the actin-myosin invasion motor. Furthermore, P. falciparum uses palmitoylation in diverse ways, stably modifying some proteins while dynamically palmitoylating others. Palmitoylation therefore plays a central role in regulating P. falciparum blood stage development.
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► A global approach identified >400 palmitoylated proteins in Plasmodium falciparum ► Palmitoyl proteins are central to invasion and other virulence-associated processes ► Palmitoylation is required for completion of the P. falciparum asexual life cycle ► P. falciparum uses palmitoylation dynamically for diverse regulatory purposes
Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution ...of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.
The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are ...used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.
Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 ...remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.
Genes encoding the core cell cycle machinery are transcriptionally regulated by the MuvB family of protein complexes in a cell cycle-specific manner. Complexes of MuvB with the transcription factors ...B-MYB and FOXM1 activate mitotic genes during cell proliferation. The mechanisms of transcriptional regulation by these complexes are still poorly characterised. Here, we combine biochemical analysis and in vitro reconstitution, with structural analysis by cryo-electron microscopy and cross-linking mass spectrometry, to functionally examine these complexes. We find that the MuvB:B-MYB complex binds and remodels nucleosomes, thereby exposing nucleosomal DNA. This remodelling activity is supported by B-MYB which directly binds the remodelled DNA. Given the remodelling activity on the nucleosome, we propose that the MuvB:B-MYB complex functions as a pioneer transcription factor complex. In this work, we rationalise prior biochemical and cellular studies and provide a molecular framework of interactions on a protein complex that is key for cell cycle regulation.
Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of ...pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.
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