RESUMO
Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified â¼8000 protein groups and â¼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.
Assuntos
Proteômica , Fluxo de Trabalho , Humanos , Proteômica/métodos , Células HeLa , Cromatografia Líquida , Automação , Proteoma/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Reprodutibilidade dos Testes , Melanoma/metabolismo , Fosfopeptídeos/metabolismoRESUMO
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.
Assuntos
Proteoma , Zigoto , Humanos , Animais , Camundongos , Proteoma/análise , Células HeLa , Zigoto/química , Reprodutibilidade dos Testes , Espectrometria de Massas/métodosRESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.
Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidrazinas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triazóis , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.
Assuntos
Proteínas Quinases Ativadas por Mitógeno , Músculo Esquelético , Animais , MAP Quinase Quinase Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
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 .
Assuntos
Proteoma/metabolismo , Proteômica , Transdução de Sinais , Animais , Fenômenos Biológicos , Fracionamento Celular , Células HeLa , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Pressão Osmótica , Fosforilação , Frações Subcelulares/metabolismo , Fluxo de TrabalhoRESUMO
p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14-3-3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.
Assuntos
MAP Quinase Quinase 4/metabolismo , Estresse Fisiológico/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Complexo de Golgi/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 4/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
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.
Assuntos
Proteômica , Cromatografia Líquida , Proteoma , Reprodutibilidade dos Testes , Software , Espectrometria de Massas em TandemRESUMO
Neuroblastoma is a highly heterogeneous embryonal solid tumor of the sympathetic nervous system. As some tumors can be treated to undergo differentiation, investigating this process can guide differentiation-based therapies of neuroblastoma. Here, we studied the role of E3 ubiquitin ligases Cbl and Cbl-b in regulation of long-term signaling responses associated with extracellular signal-regulated kinase phosphorylation and neurite outgrowth, a morphological marker of neuroblastoma cell differentiation. Using quantitative mass spectrometry (MS)-based proteomics, we analyzed how the neuroblastoma cell line proteome, phosphoproteome, and ubiquitylome were affected by Cbl and Cbl-b depletion. To quantitatively assess neurite outgrowth, we developed a high-throughput microscopy assay that was applied in combination with inhibitor studies to pinpoint signaling underlying neurite outgrowth and to functionally validate proteins identified in the MS data sets. Using this combined approach, we identified a role for SHP-2 and CDK16 in Cbl/Cbl-b-dependent regulation of extracellular signal-regulated kinase phosphorylation and neurite outgrowth, highlighting their involvement in neuroblastoma cell differentiation.
RESUMO
Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.
Assuntos
Carcinoma de Células Renais/genética , Biologia Computacional/métodos , Redes Reguladoras de Genes , Neoplasias Renais/genética , Carcinoma de Células Renais/metabolismo , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Metabolômica , FosfoproteínasRESUMO
Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , Peptidil Transferases/metabolismo , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ativação Enzimática , Células HeLa , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Conformação Proteica , Domínios Proteicos , RNA Ribossômico 18S/genética , Homologia de Sequência , Transdução de SinaisRESUMO
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.
Assuntos
Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Proteoma/metabolismo , Animais , Cromatografia Líquida , Células HeLa , Humanos , Masculino , Fases de Leitura Aberta/genética , Especificidade de Órgãos , Peptídeos/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Algoritmos , Biologia Computacional/métodos , Fosfopeptídeos/análise , Proteínas Quinases/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Fator de Crescimento Epidérmico/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Fosfopeptídeos/metabolismo , Fosfoproteínas/análise , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Reprodutibilidade dos TestesRESUMO
Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
Assuntos
Carcinogênese/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animais , Humanos , Espectrometria de Massas/métodos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Proteômica , Ratos , Ratos Sprague-Dawley , Peixe-ZebraRESUMO
Alternative translation is an important mechanism of post-transcriptional gene regulation leading to the expression of different protein isoforms originating from the same mRNA. Here, we describe an abundant long isoform of the stress/p38MAPK-activated protein kinase MK2. This isoform is constitutively translated from an alternative CUG translation initiation start site located in the 5' UTR of its mRNA. The RNA helicase eIF4A1 is needed to ensure translation of the long and the known short isoforms of MK2, of which the molecular properties were determined. Only the short isoform phosphorylated Hsp27 in vivo, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling revealed short-isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains at least one additional phosphorylatable serine in its unique N terminus. In sum, our data reveal a longer isoform of MK2 with distinct physiological properties.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Células RAW 264.7 , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente PequenoRESUMO
Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.
Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Neuroblastoma/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Biologia Computacional , Proteína Forkhead Box O3/metabolismo , Humanos , Espectrometria de Massas , Peptídeos/química , Fosforilação , Proteoma , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Proteômica/métodos , Proteínas Sanguíneas/metabolismo , Células HeLa , Humanos , Proteoma/metabolismo , Raios UltravioletaRESUMO
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.
Assuntos
Lisina/química , Ubiquitina/imunologia , Ubiquitina/metabolismo , Ubiquitinação , Especificidade de Anticorpos , Sítios de Ligação , Linhagem Celular , Humanos , Células Jurkat , Espectrometria de Massas , Proteoma/química , Proteoma/metabolismo , Ubiquitina/químicaRESUMO
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 MS2- and MS3-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. MS2-based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS3-based TMT can partly rescue. However, MS2-based TMT outperforms MS3-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 MS3-based TMT.
Assuntos
Fosfopeptídeos/análise , Proteômica/métodos , Algoritmos , Marcação por Isótopo , Espectrometria de Massas/métodos , Fosforilação , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em TandemRESUMO
Progress in proteomics is mainly driven by advances in mass spectrometric (MS) technologies. Here we benchmarked the performance of the latest MS instrument in the benchtop Orbitrap series, the Q Exactive HF-X, against its predecessor for proteomics applications. A new peak-picking algorithm, a brighter ion source, and optimized ion transfers enable productive MS/MS acquisition above 40 Hz at 7500 resolution. The hardware and software improvements collectively resulted in improved peptide and protein identifications across all comparable conditions, with an increase of up to 50 percent at short LC-MS gradients, yielding identification rates of more than 1000 unique peptides per minute. Alternatively, the Q Exactive HF-X is capable of achieving the same proteome coverage as its predecessor in approximately half the gradient time or at 10-fold lower sample loads. The Q Exactive HF-X also enables rapid phosphoproteomics with routine analysis of more than 5000 phosphopeptides with short single-shot 15 min LC-MS/MS measurements, or 16â¯700 phosphopeptides quantified across ten conditions in six gradient hours using TMT10-plex and offline peptide fractionation. Finally, exciting perspectives for data-independent acquisition are highlighted with reproducible identification of 55â¯000 unique peptides covering 5900 proteins in half an hour of MS analysis.