Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

iSanXoT: A standalone application for the integrative analysis of mass spectrometry-based quantitative proteomics data.

Many bioinformatics tools are available for the quantitative analysis of proteomics experiments. Most of these tools use a dedicated statistical model to derive absolute quantitative protein values from mass spectrometry (MS) data. Here, we present iSanXoT, a standalone application that processes relative abundances between MS signals and then integrates them sequentially to upper levels using the previously published Generic Integration Algorithm (GIA). iSanXoT offers unique capabilities that complement conventional quantitative software applications, including statistical weighting and independent modeling of error distributions in each integration, aggregation of technical or biological replicates, quantification of posttranslational modifications, and analysis of coordinated protein behavior. iSanXoT is a standalone, user-friendly application that accepts output from popular proteomics pipelines and enables unrestricted creation of quantification workflows and fully customizable reports that can be reused across projects or shared among users. Numerous publications attest the successful application of diverse integrative workflows constructed using the GIA for the analysis of high-throughput quantitative proteomics experiments. iSanXoT has been tested with the main operating systems. Download links for the corresponding distributions are available at https://github.com/CNIC-Proteomics/iSanXoT/releases.

ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.

Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling.

Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al.1 and Martínez-Val et al.2.

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

PRMT5-mediated regulatory arginine methylation of RIPK3.

The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context-dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found to be a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling.

Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.

In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based fractionation strategies against single-shot label-free quantification with spectral library-free data independent acquisition (LFQ-DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reversed-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.5 to 1 µg per sample. Our results indicate that, for input amounts higher than 5 µg per sample, TMT labeling, followed by microflow fractionation (MF) and phospho-enrichment, achieves the deepest phosphoproteome coverage, even compared to single shot direct-DIA analysis. Conversely, STF of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 µg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of sample amount.

Obtaining Complete Human Proteomes.

Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io .

Data Processing and Analysis for DIA-Based Phosphoproteomics Using Spectronaut.

Data-independent acquisition (DIA) for liquid chromatography tandem mass spectrometry (LC-MS/MS) can improve the depth and reproducibility of the acquired proteomics datasets. DIA solves some limitations of the conventional data-dependent acquisition (DDA) strategy, for example, bias in intensity-dependent precursor selection and limited dynamic range. These advantages, together with the recent developments in speed, sensitivity, and resolution in MS technology, position DIA as a great alternative to DDA. Recently, we demonstrated that the benefits of DIA are extendable to phosphoproteomics workflows, enabling increased depth, sensitivity, and reproducibility of our analysis of phosphopeptide-enriched samples. However, computational data analysis of phospho-DIA samples have some specific challenges and requirements to the software and downstream processing workflows. A step-by-step guide to analyze phospho-DIA raw data using either spectral libraries or directDIA in Spectronaut is presented here. Furthermore, a straightforward protocol to perform differential phosphorylation site analysis using the output results from Spectronaut is described.

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures.

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.

Dissection of two routes to naïve pluripotency using different kinase inhibitors.

Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.

Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases.

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF-MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF-MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.

Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries.

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.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients.

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.

Proteotyping pluripotency with mass spectrometry.

INTRODUCTION: Pluripotency emerges transiently during embryogenesis in two main forms with different developmental potential, termed naïve and primed states. Importantly, these pluripotent states can be recapitulated in vitro under specific culture conditions, representing a unique model to study the regulatory principles of development and cellular plasticity. Areas covered: A complex network of signaling pathways that senses intrinsic and extrinsic cues controls the fine balance between self-renewal and differentiation. Much of our knowledge on this tight regulation originates from epigenetic and transcriptomic approaches. However, the presence of post-transcriptional and post-translational mechanisms demands a direct assessment of the proteome in its multiple facets. Mass spectrometry-based proteomics is now a mature technique and has started to deliver new insights in the stem cell field. Expert opinion: Here, we review our current understanding on the mechanisms that dictate the spectrum of pluripotency levels. We put special emphasis on the emerging proteomic studies that focused on the molecular properties behind the naïve and primed states. In addition, we hypothesize on the impact that future developments in proteomic technologies can have to improve our view of pluripotency.

The RNA Polymerase II Factor RPAP1 Is Critical for Mediator-Driven Transcription and Cell Identity.

The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation.

Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.