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1.
bioRxiv ; 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38979258

RESUMO

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.

2.
J Proteome Res ; 23(6): 1926-1936, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38691771

RESUMO

Data-independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimized to minimize the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings, we analyzed Mus musculus macrophages and compared their proteome to macrophages spiked withCandida albicans. This experimental system enabled the identification of "false positives" as Candida albicans peptides and proteins should not be present in the Mus musculus-only samples. We show that adjusting the search parameters reduced "false positive" identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimized parameters incurred a moderate cost, only reducing the overall number of "true positive" identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analyzing heterogeneous populations of cell types or tissues.


Assuntos
Candida albicans , Macrófagos , Proteoma , Proteômica , Animais , Camundongos , Proteoma/análise , Proteômica/métodos , Macrófagos/metabolismo , Macrófagos/imunologia , Confiabilidade dos Dados , Peptídeos/análise
3.
Cell Mol Life Sci ; 81(1): 90, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38353833

RESUMO

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC-DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.


Assuntos
Vesículas Extracelulares , Melanoma , Humanos , Proteoma , Proteômica , Cromatografia em Gel
4.
Methods Mol Biol ; 2554: 69-89, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36178621

RESUMO

Metabolite-protein interactions regulate diverse cellular processes, prompting the development of methods to investigate the metabolite-protein interactome at a global scale. One such method is our previously developed structural proteomics approach, limited proteolysis-mass spectrometry (LiP-MS), which detects proteome-wide metabolite-protein and drug-protein interactions in native bacterial, yeast, and mammalian systems, and allows identification of binding sites without chemical modification. Here we describe a detailed experimental and analytical workflow for conducting a LiP-MS experiment to detect small molecule-protein interactions, either in a single-dose (LiP-SMap) or a multiple-dose (LiP-Quant) format. LiP-Quant analysis combines the peptide-level resolution of LiP-MS with a machine learning-based framework to prioritize true protein targets of a small molecule of interest. We provide an updated R script for LiP-Quant analysis via a GitHub repository accessible at https://github.com/RolandBruderer/MiMB-LiP-Quant .


Assuntos
Proteoma , Proteômica , Animais , Mamíferos/metabolismo , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteólise , Proteoma/metabolismo , Proteômica/métodos
5.
Nat Struct Mol Biol ; 29(10): 978-989, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36224378

RESUMO

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis-mass spectrometry (LiP-MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein α-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes.


Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Biomarcadores , Humanos , Proteoma/metabolismo , alfa-Sinucleína/metabolismo
6.
STAR Protoc ; 3(4): 101725, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36166358

RESUMO

Here, we describe an optimized protocol to analyze murine bone-marrow-derived macrophages using label-free data-independent acquisition (DIA) proteomics. We provide a complete step-by-step protocol describing sample preparation utilizing the S-Trap approach for on-column digestion and peptide purification. We then detail mass spectrometry data acquisition and approaches for data analysis. Single-shot DIA protocols achieve comparable proteomic depth with data-dependent MS approaches without the need for fractionation. This allows for better scaling for large sample numbers with high inter-experimental reproducibility. For complete details on the use and execution of this protocol, please refer to Ryan et al. (2022).


Assuntos
Medula Óssea , Proteômica , Animais , Camundongos , Proteômica/métodos , Reprodutibilidade dos Testes , Peptídeos , Espectrometria de Massas/métodos
7.
Nat Commun ; 13(1): 4146, 2022 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842429

RESUMO

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.


Assuntos
Adenosina Trifosfatases , Doenças Neurodegenerativas , Proteína com Valosina , Adenosina Trifosfatases/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Fosfatos de Fosfatidilinositol , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
8.
J Proteome Res ; 21(7): 1718-1735, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35605973

RESUMO

The plasma proteome has the potential to enable a holistic analysis of the health state of an individual. However, plasma biomarker discovery is difficult due to its high dynamic range and variability. Here, we present a novel automated analytical approach for deep plasma profiling and applied it to a 180-sample cohort of human plasma from lung, breast, colorectal, pancreatic, and prostate cancers. Using a controlled quantitative experiment, we demonstrate a 257% increase in protein identification and a 263% increase in significantly differentially abundant proteins over neat plasma. In the cohort, we identified 2732 proteins. Using machine learning, we discovered biomarker candidates such as STAT3 in colorectal cancer and developed models that classify the diseased state. For pancreatic cancer, a separation by stage was achieved. Importantly, biomarker candidates came predominantly from the low abundance region, demonstrating the necessity to deeply profile because they would have been missed by shallow profiling.


Assuntos
Neoplasias Pancreáticas , Proteômica , Biomarcadores , Proteínas Sanguíneas/análise , Humanos , Masculino , Proteoma/metabolismo
9.
Sci Rep ; 12(1): 3278, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35228603

RESUMO

Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8+-T-cells, CD4+-T-cells, Treg, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8+ cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4+ Teff TILs played a key role. TIL-Treg also played a critical role in supporting tumor growth in all four syngeneic models as well as M2-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.


Assuntos
Antineoplásicos , Imunoterapia , Animais , Antineoplásicos/metabolismo , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Humanos , Linfócitos do Interstício Tumoral , Camundongos , Linfócitos T Reguladores , Microambiente Tumoral
10.
ACS Chem Biol ; 17(1): 54-67, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-34955012

RESUMO

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteômica , Linhagem Celular Tumoral , Fracionamento Químico , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de Massas
11.
Mol Cell Proteomics ; 21(1): 100178, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34798331

RESUMO

MS-based immunopeptidomics is maturing into an automatized and high-throughput technology, producing small- to large-scale datasets of clinically relevant major histocompatibility complex (MHC) class I-associated and class II-associated peptides. Consequently, the development of quality control (QC) and quality assurance systems capable of detecting sample and/or measurement issues is important for instrument operators and scientists in charge of downstream data interpretation. Here, we created MhcVizPipe (MVP), a semiautomated QC software tool that enables rapid and simultaneous assessment of multiple MHC class I and II immunopeptidomic datasets generated by MS, including datasets generated from large sample cohorts. In essence, MVP provides a rapid and consolidated view of sample quality, composition, and MHC specificity to greatly accelerate the "pass-fail" QC decision-making process toward data interpretation. MVP parallelizes the use of well-established immunopeptidomic algorithms (NetMHCpan, NetMHCIIpan, and GibbsCluster) and rapidly generates organized and easy-to-understand reports in HTML format. The reports are fully portable and can be viewed on any computer with a modern web browser. MVP is intuitive to use and will find utility in any specialized immunopeptidomic laboratory and proteomics core facility that provides immunopeptidomic services to the community.


Assuntos
Antígenos de Histocompatibilidade Classe I , Software , Peptídeos , Proteômica , Controle de Qualidade
12.
Nat Commun ; 11(1): 4200, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826910

RESUMO

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Aprendizado de Máquina , Proteoma , Proteômica/métodos , Sítios de Ligação , Botrytis , Sobrevivência Celular , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Células HeLa , Humanos , Ligantes , Espectrometria de Massas , Fosfotransferases/metabolismo , Ligação Proteica , Proteólise , Saccharomyces cerevisiae
13.
Mol Cell Proteomics ; 19(2): 421-430, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31888964

RESUMO

In bottom-up, label-free discovery proteomics, biological samples are acquired in a data-dependent (DDA) or data-independent (DIA) manner, with peptide signals recorded in an intact (MS1) and fragmented (MS2) form. While DDA has only the MS1 space for quantification, DIA contains both MS1 and MS2 at high quantitative quality. DIA profiles of complex biological matrices such as tissues or cells can contain quantitative interferences, and the interferences at the MS1 and the MS2 signals are often independent. When comparing biological conditions, the interferences can compromise the detection of differential peptide or protein abundance and lead to false positive or false negative conclusions.We hypothesized that the combined use of MS1 and MS2 quantitative signals could improve our ability to detect differentially abundant proteins. Therefore, we developed a statistical procedure incorporating both MS1 and MS2 quantitative information of DIA. We benchmarked the performance of the MS1-MS2-combined method to the individual use of MS1 or MS2 in DIA using four previously published controlled mixtures, as well as in two previously unpublished controlled mixtures. In the majority of the comparisons, the combined method outperformed the individual use of MS1 or MS2. This was particularly true for comparisons with low fold changes, few replicates, and situations where MS1 and MS2 were of similar quality. When applied to a previously unpublished investigation of lung cancer, the MS1-MS2-combined method increased the coverage of known activated pathways.Since recent technological developments continue to increase the quality of MS1 signals (e.g. using the BoxCar scan mode for Orbitrap instruments), the combination of the MS1 and MS2 information has a high potential for future statistical analysis of DIA data.


Assuntos
Proteômica/métodos , Animais , Caenorhabditis elegans , Cerebelo/metabolismo , Interpretação Estatística de Dados , Células HeLa , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas , Camundongos , Saccharomyces cerevisiae
14.
J Proteome Res ; 19(1): 371-381, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31738065

RESUMO

In mammalian cells, the lysosome is the main organelle for the degradation of macromolecules and the recycling of their building blocks. Correct lysosomal function is essential, and mutations in every known lysosomal hydrolase result in so-called lysosomal storage disorders, a group of rare and often fatal inherited diseases. Furthermore, it is becoming more and more apparent that lysosomes play also decisive roles in other diseases, such as cancer and common neurodegenerative disorders. This leads to an increasing interest in the proteomic analysis of lysosomes for which enrichment is a prerequisite. In this study, we compared the four most common strategies for the enrichment of lysosomes using data-independent acquisition. We performed centrifugation at 20,000 × g to generate an organelle-enriched pellet, two-step sucrose density gradient centrifugation, enrichment by superparamagnetic iron oxide nanoparticles (SPIONs), and immunoprecipitation using a 3xHA tagged version of the lysosomal membrane protein TMEM192. Our results show that SPIONs and TMEM192 immunoprecipitation outperform the other approaches with enrichment factors of up to 118-fold for certain proteins relative to whole cell lysates. Furthermore, we achieved an increase in identified lysosomal proteins and a higher reproducibility in protein intensities for label-free quantification in comparison to the other strategies.


Assuntos
Lisossomos/química , Proteínas/isolamento & purificação , Proteômica/métodos , Centrifugação com Gradiente de Concentração , Células HEK293 , Humanos , Proteínas de Membrana Lisossomal/análise , Nanopartículas Magnéticas de Óxido de Ferro/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas/análise , Fluxo de Trabalho
15.
Nat Commun ; 10(1): 5734, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844046

RESUMO

System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when samples are limited. Here, we miniaturize and automate the previously described Cell Surface Capture (CSC) technology, increasing sensitivity, reproducibility and throughput. We use this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and use targeted flow cytometry to uncover developmental cell subpopulations.


Assuntos
Subpopulações de Linfócitos B/classificação , Diferenciação Celular , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana/análise , Proteômica/métodos , Animais , Subpopulações de Linfócitos B/metabolismo , Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Células HT29 , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Cultura Primária de Células , Reprodutibilidade dos Testes
16.
Mol Omics ; 15(5): 348-360, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31465043

RESUMO

Comprehensive proteome quantification is crucial for a better understanding of underlying mechanisms of diseases. Liquid chromatography mass spectrometry (LC-MS) has become the method of choice for comprehensive proteome quantification due to its power and versatility. Even though great advances have been made in recent years, full proteome coverage for complex samples remains challenging due to the high dynamic range of protein expression. Additionally, when studying disease regulatory proteins, biomarkers or potential drug targets are often low abundant, such as for instance kinases and transcription factors. Here, we show that with improvements in chromatography and data analysis the single shot proteome coverage can go beyond 10 000 proteins in human tissue. In a testis cancer study, we quantified 11 200 proteins using data independent acquisition (DIA). This depth was achieved with a false discovery rate of 1% which was experimentally validated using a two species test. We introduce the concept of hybrid libraries which combines the strength of direct searching of DIA data as well as the use of large project-specific or published DDA data sets. Remarkably deep proteome coverage is possible using hybrid libraries without the additional burden of creating a project-specific library. Within the testis cancer set, we found a large proportion of proteins in an altered expression (in total: 3351; 1453 increased in cancer). Many of these proteins could be linked to the hallmarks of cancer. For example, the complement system was downregulated which helps to evade the immune response and chromosomal replication was upregulated indicating a dysregulated cell cycle.


Assuntos
Cromatografia Líquida/instrumentação , Espectrometria de Massas/instrumentação , Células-Tronco Neoplásicas/química , Proteômica/métodos , Cromatografia Líquida/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Espectrometria de Massas/métodos , Células-Tronco Neoplásicas/metabolismo , Proteoma , Neoplasias Testiculares/metabolismo
17.
Mol Cell Proteomics ; 18(6): 1242-1254, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948622

RESUMO

Comprehensive, high throughput analysis of the plasma proteome has the potential to enable holistic analysis of the health state of an individual. Based on our own experience and the evaluation of recent large-scale plasma mass spectrometry (MS) based proteomic studies, we identified two outstanding challenges: slow and delicate nano-flow liquid chromatography (LC) and irreproducibility of identification of data-dependent acquisition (DDA). We determined an optimal solution reducing these limitations with robust capillary-flow data-independent acquisition (DIA) MS. This platform can measure 31 plasma proteomes per day. Using this setup, we acquired a large-scale plasma study of the diet, obesity and genes dietary (DiOGenes) comprising 1508 samples. Proving the robustness, the complete acquisition was achieved on a single analytical column. Totally, 565 proteins (459 identified with two or more peptide sequences) were profiled with 74% data set completeness. On average 408 proteins (5246 peptides) were identified per acquisition (319 proteins in 90% of all acquisitions). The workflow reproducibility was assessed using 34 quality control pools acquired at regular intervals, resulting in 92% data set completeness with CVs for protein measurements of 10.9%.The profiles of 20 apolipoproteins could be profiled revealing distinct changes. The weight loss and weight maintenance resulted in sustained effects on low-grade inflammation, as well as steroid hormone and lipid metabolism, indicating beneficial effects. Comparison to other large-scale plasma weight loss studies demonstrated high robustness and quality of biomarker candidates identified. Tracking of nonenzymatic glycation indicated a delayed, slight reduction of glycation in the weight maintenance phase. Using stable-isotope-references, we could directly and absolutely quantify 60 proteins in the DIA.In conclusion, we present herein the first large-scale plasma DIA study and one of the largest clinical research proteomic studies to date. Application of this fast and robust workflow has great potential to advance biomarker discovery in plasma.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteômica , Reologia , Redução de Peso , Adulto , Bases de Dados de Proteínas , Glicosilação , Humanos , Marcação por Isótopo , Proteoma/metabolismo , Padrões de Referência
18.
J Proteome Res ; 18(3): 1340-1351, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30726097

RESUMO

Label-free quantification (LFQ) and isobaric labeling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT SPS/MS3 10-plex workflow to a label free single shot data-independent acquisition (DIA) workflow on a controlled sample set. The sample set consisted of ten samples derived from 10 biological replicates of mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. For a fair comparison, we matched the instrument time for the two workflows. The LC-MS data were acquired at two facilities to assess interlaboratory reproducibility. Both methods resulted in a high proteome coverage (>5000 proteins) with low missing values on protein level (<2%). The TMT workflow led to 15-20% more identified proteins and a slightly better quantitative precision, whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA showed a similar performance. The quantitative performance of the DIA data stayed in a similar range when searching the spectra against a fasta database directly, instead of using a project-specific library. Our experiments also demonstrated that both workflows are readily transferrable between facilities.


Assuntos
Cerebelo/metabolismo , Proteoma/genética , Proteômica/métodos , Animais , Cromatografia Líquida , Camundongos , Proteômica/normas , Coloração e Rotulagem , Espectrometria de Massas em Tandem , Fluxo de Trabalho
19.
Sci Rep ; 8(1): 4346, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531254

RESUMO

Quantitative proteomics is key for basic research, but needs improvements to satisfy an increasing demand for large sample series in diagnostics, academia and industry. A switch from nanoflowrate to microflowrate chromatography can improve throughput and reduce costs. However, concerns about undersampling and coverage have so far hampered its broad application. We used a QTOF mass spectrometer of the penultimate generation (TripleTOF5600), converted a nanoLC system into a microflow platform, and adapted a SWATH regime for large sample series by implementing retention time- and batch correction strategies. From 3 µg to 5 µg of unfractionated tryptic digests that are obtained from proteomics-typical amounts of starting material, microLC-SWATH-MS quantifies up to 4000 human or 1750 yeast proteins in an hour or less. In the acquisition of 750 yeast proteomes, retention times varied between 2% and 5%, and quantified the typical peptide with 5-8% signal variation in replicates, and below 20% in samples acquired over a five-months period. Providing precise quantities without being dependent on the latest hardware, our study demonstrates that the combination of microflow chromatography and data-independent acquisition strategies has the potential to overcome current bottlenecks in academia and industry, enabling the cost-effective generation of precise quantitative proteomes in large scale.


Assuntos
Cromatografia Líquida/métodos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Análise Custo-Benefício , Humanos , Células K562 , Software , Espectrometria de Massas em Tandem/métodos
20.
Mol Cell Proteomics ; 16(12): 2296-2309, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29070702

RESUMO

Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.


Assuntos
Peptídeos/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular , Cromatografia Líquida , Células HEK293 , Células HeLa , Humanos , Camundongos , Peptídeos/genética , Reprodutibilidade dos Testes , Análise de Sequência de Proteína
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