Cellular function can be controlled through the gene expression program, but often protein post-translational modifications (PTMs) provide a more precise and elegant mechanism. Key functional roles ...of specific modification events—for instance, during the cell cycle—have been known for decades, but only in the past 10 years has mass-spectrometry-(MS)-based proteomics begun to reveal the true extent of the PTM universe. In this overview for the special PTM issue of Molecular and Cellular Proteomics, we take stock of where MS-based proteomics stands in the large-scale analysis of protein modifications. For many PTMs, including phosphorylation, ubiquitination, glycosylation, and acetylation, tens of thousands of sites can now be confidently identified and localized in the sequence of the protein. The quantification of PTM levels between different cellular states is likewise established, with label-free methods showing particular promise. It is also becoming possible to determine the absolute occupancy or stoichiometry of PTM sites on a large scale. Powerful software for the bioinformatic analysis of thousands of PTM sites has been developed. However, a complete inventory of sites has not been established for any PTM, and this situation will persist into the foreseeable future. Furthermore, although PTM coverage by MS-based methods is impressive, it still needs to be improved, especially in tissues and in clinically relevant systems. The central challenge for the field is to develop streamlined methods for determining biological functions for the myriad of modifications now known to exist.
The phenomenon of protein aggregation capture (PAC) on a wide range of different microparticles is described. Exploiting this mechanism enables generation of clean peptide mixtures from cell lines, ...tissues, and protein pulldowns for proteomics, phosphoproteomics, and secretomics analysis. The findings vastly increase the accessibility of the method that may ultimately lead to low cost and automated proteomics workflows.
Display omitted
Highlights
•Insoluble protein aggregates preferentially precipitate on microparticles.•The protein aggregation capture (PAC) occurs irrespective of microparticle surface chemistry.•This process can be exploited for multi-purpose proteomics sample preparation.•Facilitates potential for low cost, efficient and high-sensitivity proteomics workflows.
Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.
Orbitrap Exploris 480 MS with FAIMS Pro provides fast, sensitive and robust profiling of proteomes when combined with Evosep One. The combination of Data Independent Acquisition (DIA) and FAIMS with ...single compensation voltages enables analysis of up to 2000 peptides per LC gradient minute and more than 5000 protein groups in twenty minutes. DIA-FAIMS based label-free quantitation achieves similar depth and quantitative accuracy as the well-established isobaric labelling approaches, but with less LC-MS measurement time, making it a good choice for large cohort of samples.
Display omitted
Highlights
•Increased proteome coverage with Orbitrap Exploris 480 MS and FAIMS using single compensation voltages and short LC gradients.•Towards single-cell proteomics with high-sensitivity analysis of 5 ng HeLa with more than 1,000 proteins identified in 5 minutes using FAIMS and DIA.•Deep proteome profiling across twelve rat organs tissues by label-free quantitation using DIA compared to TMT-multiplexing and turboTMT acquisition using phi-SDM.•Rapid and sensitive phosphoproteomics with automated enrichment using Ti-IMAC magnetic beads and direct DIA analysis.
State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.
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.
Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made ...in liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC–MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC–MS/MS gradients. From 14 HpH fractions, we routinely identified over 30 000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404).
The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide ...response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair.
► Global quantification of DDR-regulated phosphoproteome, acetylome, and proteome ► Phosphorylation is regulated more robustly than acetylation after DNA damage ► Much DDR-induced phosphorylation depends on non-ATM/ATR/DNA-PK kinases ► THRAP3 is phosphorylated and excluded from sites of DNA damage
Protein phosphorylation is a key posttranslational modification, which reversibly regulates almost all processes in the living cell. Deregulated signaling is a hallmark of cancer and other diseases, ...and protein kinases are prominent drug targets. Phosphorylation events are commonly probed in a targeted manner by phosphorylation-specific antibodies. In contrast, advances in proteomics technology, including phosphopeptide enrichment, high-accuracy mass spectrometry, and associated bioinformatics now make it possible to analyze entire phosphoproteomes. Quantitative methods can assess the relative change in phosphorylation for several thousand sites in a single experiment. Here we review enrichment strategies and methods for mass spectrometric fragmentation and analysis of phosphopeptides. We also describe different quantitative methods and their application to problems in cell signaling and drug target discovery. Emerging phosphoproteomics technologies are becoming more comprehensive, robust, and generically applicable to a wide range of questions, including areas outside traditional eukaryotic cell signaling such as Ser/Thr/Tyr signaling in bacteria.
Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation 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 dephosphorylation. 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.
Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues ...analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.
Display omitted
► Identification of 15,474 lysine acetylation sites on 4,541 proteins in tissues ► Lysine acetylation sequence motifs differ between subcellular compartments ► Acetylation of lysine residues can switch off enzymatic activity ► The subcellular distribution of acetylation differs from phosphorylation
Lysine acetylation is a major posttranslational modification best known for its role in gene regulation. By means of high-resolution tandem mass spectrometry, Olsen and colleagues have generated an organ-wide compendium of lysine acetylation sites in rat tissues. They quantify 15,474 modification sites on 4,541 proteins and thereby demonstrate that lysine acetylation is much more prevalent than previously appreciated. Lysine acetylation targets proteins involved in diverse physiological functions, and the regulatory implications of the modification extend far beyond histone modifications.
Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but ...the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes.
Display omitted
•The PP2A-B56 holoenzyme binds to short linear LxxIxE motifs in eukaryotes•LxxIxE motifs are recognized by a conserved binding pocket on B56 regulatory subunits•LxxIxE motifs can provide PP2A-B56 substrate specificity
The molecular details of substrate recognition by the protein phosphatase 2A (PP2A) are unknown. Hertz, Kruse et al. identify a short linear motif, which they term the LxxIxE motif, that mediates binding to a complementary pocket conserved in the B56 family of regulatory subunits of PP2A.
PDF
