RESUMO
We present a framework for the analysis of multiplexed mass spectrometry proteomics data that reduces estimation error when combining multiple isobaric batches. Variations in the number and quality of observations have long complicated the analysis of isobaric proteomics data. Here we show that the power to detect statistical associations is substantially improved by utilizing models that directly account for known sources of variation in the number and quality of observations that occur across batches.In a multibatch benchmarking experiment, our open-source software (msTrawler) increases the power to detect changes, especially in the range of less than twofold changes, while simultaneously increasing quantitative proteome coverage by utilizing more low-signal observations. Further analyses of previously published multiplexed datasets of 4 and 23 batches highlight both increased power and the ability to navigate complex missing data patterns without relying on unverifiable imputations or discarding reliable measurements.
Assuntos
Proteômica , Software , Proteômica/métodos , Espectrometria de Massas/métodos , Proteoma/análiseRESUMO
Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2-4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.
Assuntos
Doença de Alzheimer , Encefalite , Doença de Alzheimer/metabolismo , Animais , Clusterina/genética , Clusterina/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , ProteômicaRESUMO
The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.
Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Gênica , Transferrina/metabolismoRESUMO
Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM sensitivity, peptide abundance, binding affinity and motif, peptide length, and choice of binding register along the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.
Assuntos
Apresentação de Antígeno , Antígenos HLA-D , Antígenos de Histocompatibilidade Classe II , Proteômica , Células Apresentadoras de Antígenos , Linhagem Celular , Antígenos HLA-DR , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Peptídeos/metabolismoRESUMO
Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Região Variável de Imunoglobulina/imunologia , Linfoma de Célula do Manto/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/imunologia , Citotoxicidade Imunológica , Análise Mutacional de DNA , Epitopos de Linfócito T/imunologia , Exoma/genética , Genômica , Antígenos HLA-D/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Imunoterapia/tendências , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/patologia , Linfoma de Célula do Manto/terapia , Mutação , ProteômicaRESUMO
Systemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes. Methodologies used included immunohistochemistry, immunoglobulin isotyping, multiplexed proteome profiling, immunoglobulin immunoprecipitation with glycoproteome profiling, and size exclusion chromatography (SEC) profiling to enable a comprehensive proteome profiling of proteins and protein complexes. We also used a novel native multiplexed plasma proteome profiling (NativeMP3) method that relies on native enrichment of plasma proteins enabling ultra-deep single shot profiling where we identified 922 plasma proteins at 1% false discovery rate (FDR) in a single shot mass spectrometry run. We observed many large plasma protein differences between the MRL-lpr and control strain including differences in the immunoglobulins, immunoglobulins against specific antigens, chemokines, and proteases as well as changes in protein complexes such as the immunoproteasome.
Assuntos
Doenças Autoimunes , Doenças do Complexo Imune , Camundongos , Animais , Camundongos Endogâmicos MRL lpr , Complexo Antígeno-Anticorpo , Proteômica , Proteoma , Autoanticorpos , Modelos Animais de Doenças , Peptídeo HidrolasesRESUMO
Performing large-scale plasma proteome profiling is challenging due to limitations imposed by lengthy preparation and instrument time. We present a fully automated multiplexed proteome profiling platform (AutoMP3) using the Hamilton Vantage liquid handling robot capable of preparing hundreds to thousands of samples. To maximize protein depth in single-shot runs, we combined 16-plex Tandem Mass Tags (TMTpro) with high-field asymmetric waveform ion mobility spectrometry (FAIMS Pro) and real-time search (RTS). We quantified over 40 proteins/min/sample, doubling the previously published rates. We applied AutoMP3 to investigate the naked mole-rat plasma proteome both as a function of the circadian cycle and in response to ultraviolet (UV) treatment. In keeping with the lack of synchronized circadian rhythms in naked mole-rats, we find few circadian patterns in plasma proteins over the course of 48 h. Furthermore, we quantify many disparate changes between mice and naked mole-rats at both 48 h and one week after UV exposure. These species differences in plasma protein temporal responses could contribute to the pronounced cancer resistance observed in naked mole-rats. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD022891.
Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Animais , Proteínas Reguladoras de Apoptose , Espectrometria de Massas , Camundongos , Ratos-Toupeira , ProteomaRESUMO
BACKGROUND: Cytokine-induced killer (CIK) cells are an ex vivo-expanded cellular therapy product with potent anti-tumor activity in a subset of patients with solid and hematologic malignancies. We hypothesize that directing CIK cells to a specific tumor antigen will enhance CIK cell anti-tumor cytotoxicity. METHODS: We present a newly developed method for affixing antibodies directly to cell surface proteins. First, we evaluated the anti-tumor potential of CIK cells after affixing tumor-antigen targeting monoclonal antibodies. Second, we evaluated whether this antibody-conjugation method can profile the surface proteome of CIK cells. RESULTS: We demonstrated that affixing rituximab or daratumumab to CIK cells enhances cytotoxic killing of multiple lymphoma cell lines in vitro. These 'armed' CIK cells exhibited enhanced intracellular signaling after engaging tumor targets. Cell surface proteome profiling suggested mechanisms by which antibody-armed CIK cells concurrently activated multiple surface proteins, leading to enhanced cytolytic activity. Our surface proteome analysis indicated that CIK cells display enhanced protein signatures indicative of immune responses, cellular activation and leukocyte migration. CONCLUSIONS: Here, we characterize the cell surface proteome of CIK cells using a novel methodology that can be rapidly applied to other cell types. Our study also demonstrates that without genetic modification CIK cells can be rapidly armed with monoclonal antibodies, which endows them with high specificity to kill tumor targets.
Assuntos
Anticorpos/metabolismo , Células Matadoras Induzidas por Citocinas/imunologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Proteoma/metabolismo , Proteômica , Linfócitos T/imunologiaRESUMO
Immunopeptidomes promise novel surface markers as ideal immunotherapy targets, but their characterization by mass spectrometry (MS) remains challenging. Until recently, cell numbers exceeding 109 were needed to survey thousands of HLA ligands. Such limited analytical sensitivity has historically constrained the types of clinical specimens that can be evaluated to cell cultures or bulk tissues. Measuring immunopeptidomes from purified cell subpopulations would be preferable for many applications, particularly those evaluating rare, primary hematopoietic cell lineages. Here, we test the feasibility of immunopeptidome profiling from limited numbers of primary purified human regulatory T cells (TReg ), conventional T cells (Tconv ), and activated T cells. The combined T cell immunopeptide dataset reported here contains 13 804 unique HLA ligands derived from 5049 proteins. Of these, more than 700 HLA ligands were derived from 82 proteins that we exclusively identified from TReg -enriched cells. This study 1) demonstrates that primary, lineage-enriched T cell subpopulations recovered from single donors are compatible with immunopeptidome analysis; 2) presents new TReg -biased ligand candidates; and 3) supports immunopeptidome surveys' value for revealing T cell biology that may not be apparent from expression data alone. Taken together, these findings open up new avenues for targeting TReg and abrogating their suppressive functions to treat cancer.
Assuntos
Biomarcadores/análise , Epitopos/metabolismo , Antígenos HLA/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T/classificação , Linfócitos T/metabolismo , Apresentação de Antígeno/imunologia , Epitopos/imunologia , Antígenos HLA/análise , Antígenos HLA/imunologia , Humanos , Imunoterapia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Doadores de TecidosRESUMO
Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.
Assuntos
Metionina tRNA Ligase/genética , Proteoma/genética , Proteômica/métodos , Tirosina-tRNA Ligase/genética , Alcinos/química , Aminoácidos/química , Aminoácidos/genética , Animais , Azidas/química , Sequência de Bases , Células CHO , Química Click , Cricetulus , Reação de Cicloadição , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/enzimologiaRESUMO
The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock'n'flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by ß-cyclodextrin (ß-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized ß-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.
RESUMO
Tumor progression and prognosis in breast cancer patients are difficult to assess using current clinical and laboratory parameters, where a pathological grading is indicative of tumor aggressiveness. This grading is based on assessments of nuclear grade, tubule formation, and mitotic rate. We report here the first protein signatures associated with histological grades of breast cancer, determined using a novel affinity proteomics approach. We profiled 52 breast cancer tissue samples by combining nine antibodies and label-free LC-MS/MS, which generated detailed quantified proteomic maps representing 1,388 proteins. The results showed that we could define in-depth molecular portraits of histologically graded breast cancer tumors. Consequently, a 49-plex candidate tissue protein signature was defined that discriminated between histological grades 1, 2, and 3 of breast cancer tumors with high accuracy. Highly biologically relevant proteins were identified, and the differentially expressed proteins indicated further support for the current hypothesis regarding remodeling of the tumor microenvironment during tumor progression. The protein signature was corroborated using meta-analysis of transcriptional profiling data from an independent patient cohort. In addition, the potential for using the markers to estimate the likelihood of long-term metastasis-free survival was also indicated. Taken together, these molecular portraits could pave the way for improved classification and prognostication of breast cancer.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/genética , Carcinoma/patologia , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Carcinoma/diagnóstico , Carcinoma/mortalidade , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Proteômica/métodos , Proteômica/estatística & dados numéricos , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5-1000 ng/mL. Linear responses were demonstrated down to low ng/mL concentrations with high reproducibility. The platform allows for high throughput measurements in 96-well format, and all steps are amendable to automation and scale-up. We believe the use of recombinant antibody technology in combination with SRM MS analysis provides a powerful way to reach sensitivity, specificity, and reproducibility as well as the opportunity to build resources for fast on-demand implementation of novel assays.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteômica/métodos , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Afinidade de Anticorpos/imunologia , Proteína BRCA1/sangue , Proteína BRCA1/imunologia , Proteína BRCA1/metabolismo , Humanos , Queratina-19/sangue , Queratina-19/imunologia , Queratina-19/metabolismo , Mucina-1/sangue , Mucina-1/imunologia , Mucina-1/metabolismo , Peptídeos/sangue , Peptídeos/imunologia , Peptídeos/metabolismo , Proteoma/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey, where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the global proteome survey technology by targeting stable isotope labeling with amino acids in cell culture-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Humanos , Marcação por Isótopo/métodos , Peptídeos/química , Peptídeos/metabolismo , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
One of the main hallmarks of aging is aging-associated inflammation, also known as inflammaging. In this study, by comparing plasma and kidney proteome profiling of young and old mice using LC-MS profiling, we discovered that immunoglobulins are the proteins that exhibit the highest increase with age. This observation seems to have been disregarded because conventional proteome profiling experiments typically overlook the expression of high-abundance proteins or employ depletion methods to remove them before LC-MS analysis. We show that proteome profiling of immunoglobulins will likely be a useful biomarker of aging. Spatial profiling using immunofluorescence staining of kidney sections indicates that the main increases in immunoglobulins with age are localized in the glomeruli of the kidney. Using laser capture microdissection coupled with LC-MS, we show an increase in multiple immune-related proteins in glomeruli from aged mice. Increased deposition of immunoglobulins, immune complexes, and complement proteins in the kidney glomeruli may be a factor leading to reduced filtering capacity of the kidney with age. Therapeutic strategies to reduce the deposition of immunoglobulins in the kidney may be an attractive strategy for healthy aging.
RESUMO
Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv antibody microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.
Assuntos
Neoplasias da Mama/genética , Região Variável de Imunoglobulina , Linfoma Folicular/genética , Linfoma de Célula do Manto/genética , Proteínas de Neoplasias/análise , Análise Serial de Proteínas/instrumentação , Anticorpos de Cadeia Única , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Fixadores , Formaldeído , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/imunologia , Limite de Detecção , Linfoma Folicular/diagnóstico , Linfoma Folicular/imunologia , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Inclusão em Parafina , Análise Serial de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/imunologia , Fixação de TecidosRESUMO
Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.
Assuntos
Motivos de Aminoácidos/imunologia , Afinidade de Anticorpos/imunologia , Análise Serial de Proteínas/métodos , Proteoma/análise , Proteômica/métodos , Animais , Colo/imunologia , Colo/metabolismo , Proteínas Fúngicas/análise , Humanos , Fígado/imunologia , Fígado/metabolismo , Espectrometria de Massas/métodos , Camundongos , Anticorpos de Cadeia Única/imunologia , Especificidade da EspécieRESUMO
We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.
Assuntos
Proteoma , Espectrometria de Massas em Tandem , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Cromatografia em Gel , Fracionamento QuímicoRESUMO
All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires - particularly novel MHC presentation of mutation-bearing peptides (neoantigens) - can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells' antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.