Explore the vast range of titles available.

Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation. Localizing phosphorylation sites by data-independent acquisition (DIA)-based proteomics is still challenging. Here, the authors develop algorithms for phosphosite localization and stoichiometry determination, and incorporate them into single-shot DIA-phosphoproteomics workflows.

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS 2 - and MS 3 -measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS 2 -based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS 3 -based TMT can partly rescue. However, MS 2 -based TMT outperforms MS 3 -based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly regulated phosphopeptides. Finally, we utilize the TMT multiplexing capabilities to develop an algorithm for determining phosphorylation site stoichiometry, showing that such applications benefit from the high accuracy of MS 3 -based TMT. Quantitative phosphoproteomics has become a standard method in molecular and cell biology. Here, the authors compare performance and parameters of phosphoproteome quantification by LFQ, SILAC, and MS 2 -/MS 3 -based TMT and introduce a TMT-adapted algorithm for calculating phosphorylation site stoichiometry.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and de phosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the de phosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types. Traditional proteomics methods are complex and resource-intensive. Here, the authors develop One-Tip, a highly simplified approach that enables efficient, sensitive, and comprehensive analysis across various sample types, from blood plasma to single cells.

Background Glucocorticoids are corticosteroid hormones that are commonly used for treating systemic inflammatory diseases and acute infections. Immunosuppressive effects of glucocorticoids have been studied in many cell types, particularly macrophages and T cells. Despite the importance and abundance of neutrophils in the human immune system, glucocorticoid responses remain understudied in neutrophils. Results Here, we perform quantitative mass spectrometry-based proteomics of primary neutrophils and neutrophil-like cells differentiated from human HL-60 promyelocyte cells. Primary neutrophils exhibited CK2 kinase activation and increase phosphorylation of HSP90 following 2-h incubation, highlighting potential effects of short-term ex vivo handling. Proteome and flow cytometry analysis show that neutrophil-like cells share features of neutrophils. Quantitative proteomics and phosphoproteomics of neutrophil-like cells treated with two synthetic glucocorticoid compounds, the clinical drugs dexamethasone and prednisolone, identify higher numbers of significantly regulated proteins and phosphosites compared to parental HL-60 cells. Glucocorticoid treatments modulated toll-like receptor signaling and CXCR4 serine phosphorylation. In addition, we identify RIPOR2 as a glucocorticoid-regulated protein associated with Rho GTPase signaling networks and actin cytoskeletal remodeling in neutrophils and neutrophil-like cells, though its exact functional role requires further investigation. Conclusions Our results not only reveal unconventional regulatory mechanisms of glucocorticoids in the human immune system but also provide valuable resources for discovering novel glucocorticoid-responsive protein targets in neutrophils.

Genetic and fragmented palaeoanthropological data suggest that Denisovans were once widely distributed across eastern Eurasia 1 – 3 . Despite limited archaeological evidence, this indicates that Denisovans were capable of adapting to a highly diverse range of environments. Here we integrate zooarchaeological and proteomic analyses of the late Middle to Late Pleistocene faunal assemblage from Baishiya Karst Cave on the Tibetan Plateau, where a Denisovan mandible and Denisovan sedimentary mitochondrial DNA were found 3 , 4 . Using zooarchaeology by mass spectrometry, we identify a new hominin rib specimen that dates to approximately 48–32 thousand years ago (layer 3). Shotgun proteomic analysis taxonomically assigns this specimen to the Denisovan lineage, extending their presence at Baishiya Karst Cave well into the Late Pleistocene. Throughout the stratigraphic sequence, the faunal assemblage is dominated by Caprinae, together with megaherbivores, carnivores, small mammals and birds. The high proportion of anthropogenic modifications on the bone surfaces suggests that Denisovans were the primary agent of faunal accumulation. The chaîne opératoire of carcass processing indicates that animal taxa were exploited for their meat, marrow and hides, while bone was also used as raw material for the production of tools. Our results shed light on the behaviour of Denisovans and their adaptations to the diverse and fluctuating environments of the late Middle and Late Pleistocene of eastern Eurasia. Zooarchaeological and proteomic analyses of bones from Baishiya Karst Cave on the Tibetan Plateau identify a hominin rib specimen, and provide insight into the ways Denisovans interacted with their surrounding environment and made use of animal resources.

Deregulated cellular signalling is a common hallmark of disease, and delineating tissue phosphoproteomes is key to unravelling the underlying mechanisms. Here we present the broadest tissue catalogue of phosphoproteins to date, covering 31,480 phosphorylation sites on 7,280 proteins quantified across 14 rat organs and tissues. We provide the data set as an easily accessible resource via a web-based database, the CPR PTM Resource. A major fraction of the presented phosphorylation sites are tissue-specific and modulate protein interaction networks that are essential for the function of individual organs. For skeletal muscle, we find that phosphotyrosines are over-represented, which is mainly due to proteins involved in glycogenolysis and muscle contraction, a finding we validate in human skeletal muscle biopsies. Tyrosine phosphorylation is involved in both skeletal and cardiac muscle contraction, whereas glycogenolytic enzymes are tyrosine phosphorylated in skeletal muscle but not in the liver. The presented phosphoproteomic method is simple and rapid, making it applicable for screening of diseased tissue samples. The function of proteins is often regulated by their phosphorylation at specific amino-acid residues. The authors of this article have catalogued phosphoproteins and their phosphorylation sites in 14 rat organs and tissues, and provide these data as a resource for researchers.

Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employ a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which causes global proteome remodeling. Pulsed-SILAC experiments reveals that NatA-deficient strains consistently increase degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovers decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding. N-terminal acetylation is a common modification with unclear function. Here, using multidimensional proteomics, the authors found that NatA-deficient yeast show increased ribosomal protein degradation and decreased ribosome thermostability, suggesting that N-terminal acetylation enhances proteome stability.