RESUMO
Proteomics analysis of circulating exosomes derived from cancer cells represents a promising approach to the elucidation of cell-cell communication and the discovery of putative biomarker candidates for cancer diagnosis and treatment. Nonetheless, the proteome of exosomes derived from cell lines with different metastatic capabilities still warrants further investigation. Here, we present a comprehensive quantitative proteomics investigation of exosomes isolated from immortalized mammary epithelial cells and matched tumor lines with different metastatic potentials in an attempt to discover exosome markers specific to breast cancer (BC) metastasis. A total of 2135 unique proteins were quantified with a high confidence level from 20 isolated exosome samples, including 94 of the TOP 100 exosome markers archived by ExoCarta. Moreover, 348 altered proteins were observed, among which several metastasis-specific markers, including cathepsin W (CATW), magnesium transporter MRS2 (MRS2), syntenin-2 (SDCB2), reticulon-4 (RTN), and UV excision repair protein RAD23 homolog (RAD23B), were also identified. Notably, the abundance of these metastasis-specific markers corresponds well with the overall survival of BC patients in clinical settings. Together, these data provide a valuable dataset for BC exosome proteomics investigation and prominently facilitate the elucidation of the molecular mechanisms underlying primary tumor development and progression.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Exossomos , Feminino , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Exossomos/metabolismo , Proteômica , Metástase Neoplásica , Biomarcadores Tumorais/metabolismoRESUMO
Reproducible quantification of large biological cohorts is critical for clinical/pharmaceutical proteomics yet remains challenging because most prevalent methods suffer from drastically declined commonly quantified proteins and substantially deteriorated quantitative quality as cohort size expands. MS2-based data-independent acquisition approaches represent tremendous advancements in reproducible protein measurement, but often with limited depth. We developed IonStar, an MS1-based quantitative approach enabling in-depth, high-quality quantification of large cohorts by combining efficient/reproducible experimental procedures with unique data-processing components, such as efficient 3D chromatographic alignment, sensitive and selective direct ion current extraction, and stringent postfeature generation quality control. Compared with several popular label-free methods, IonStar exhibited far lower missing data (0.1%), superior quantitative accuracy/precision [â¼5% intragroup coefficient of variation (CV)], the widest protein abundance range, and the highest sensitivity/specificity for identifying protein changes (<5% false altered-protein discovery) in a benchmark sample set (n = 20). We demonstrated the usage of IonStar by a large-scale investigation of traumatic injuries and pharmacological treatments in rat brains (n = 100), quantifying >7,000 unique protein groups (>99.8% without missing data across the 100 samples) with a low false discovery rate (FDR), two or more unique peptides per protein, and high quantitative precision. IonStar represents a reliable and robust solution for precise and reproducible protein measurement in large cohorts.
Assuntos
Biomarcadores/análise , Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Metanfetamina/farmacologia , Proteoma/análise , Proteômica/métodos , Animais , Encéfalo/efeitos dos fármacos , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Estimulantes do Sistema Nervoso Central/farmacologia , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1-Adx complex. We report that differential cross-linking internal to CYP24A1 points toward an Adx-induced conformational change that perturbs the F and G helices, which are required for substrate binding. Moreover, the modeled complex suggests that a semiconserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. Taken together, these findings contribute to our understanding of Adx recognition in a critical vitamin D-inactivating enzyme and provide broader insight regarding the variability inherent in CYP-Adx interactions.
Assuntos
Adrenodoxina/análise , Vitamina D3 24-Hidroxilase/química , Adrenodoxina/metabolismo , Regulação Alostérica , Sítios de Ligação , Humanos , Modelos Moleculares , Especificidade por Substrato , Vitamina D3 24-Hidroxilase/metabolismoRESUMO
Monocarboxylate transporter 6 [(MCT6), SLC16A5] is an orphan transporter with no known endogenous substrates or physiological role. Previous in vitro and in vivo experiments investigated MCT6 substrate/inhibitor specificity in Xenopus laevis oocytes; however, these data remain limited. Transcriptomic changes in the livers of mice undergoing different dieting schemes have suggested that Mct6 plays a role in glucose and lipid metabolism. The objectives of this study were 1) to develop a novel knockout (KO) mouse model (Mct6-/-) using CRISPR/Cas9 technology, 2) to characterize the KO animal model by examining physiological and biochemical parameters, and 3) to understand the physiological role of MCT6 in vivo through global proteomic and liver transcriptomic profiling. mRNA tissue analysis demonstrated knockout of Mct6, which showed greater than 90% knockdown of Mct6 (Slc16a5) gene expression in all major tissues analyzed when normalized to Mct6+/+ mice. Proteomic analyses identified greater than 4000 unique proteins in kidney, liver, and colon tissues, among which 51, 38, and 241 proteins were significantly altered, respectively (for each tissue), between Mct6+/+ and Mct6-/- mice. Additionally, Mct6-/- mice demonstrated significant changes in 199 genes in the liver compared with Mct6+/+ mice. In silico biological pathway analyses revealed significant changes in proteins and genes involved in glucose and lipid metabolism-associated pathways. This study is the first to provide evidence for an association of Mct6 in the regulation of glucose and lipid metabolism. SIGNIFICANCE STATEMENT: This paper focuses on elucidating the innate biological role of an orphan transporter in vivo, which has not been investigated thus far. Using efficient and high-throughput technologies, such as CRISPR/Cas9 gene editing, liquid chromatography-tandem mass spectrometry-based proteomic and RNA-sequencing transcriptomic analyses, our laboratory provides the first existence and characterization of a Mct6 knockout mouse model. The evidence gathered in this paper, as well as other laboratories, support the importance of MCT6 in regulating a variety of glucose and lipid metabolic pathways, which may indicate its significance in metabolic diseases.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Fígado/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteômica/métodos , Animais , Cromatografia Líquida , Técnicas de Inativação de Genes , Glucose/metabolismo , Metabolismo dos Lipídeos , Camundongos , Mapas de Interação de Proteínas , Análise de Sequência de RNA , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
Failure to properly repair damaged due to myocardial infarction is a major cause of heart failure. In contrast with adult mammals, zebrafish hearts show remarkable regenerative capabilities after substantial damage. To characterize protein dynamics during heart regeneration, we employed an HPLC-ESI-MS/MS (mass spectrometry) approach. Myocardium tissues were taken from sham-operated fish and ventricle-resected sample at three different time points (2, 7, and 14 days); dynamics of protein expression were analyzed by an ion-current-based quantitative platform. More than 2000 protein groups were quantified in all 16 experiments. Two hundred and nine heart-regeneration-related protein groups were quantified and clustered into six time-course patterns. Functional analysis indicated that multiple molecular function and metabolic pathways were involved in heart regeneration. Interestingly, Ingenuity Pathway Analysis revealed that P53 signaling was inhibited during the heart regeneration, which was further verified by real-time quantitative polymerase chain reaction (Q-PCR). In summary, we applied systematic proteomics analysis on regenerating zebrafish heart, uncovered the dynamics of regenerative genes expression and regulatory pathways, and provided invaluable insight into design regenerative-based strategies in human hearts.
Assuntos
Proteínas de Peixes/genética , Traumatismos Cardíacos/genética , Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Proteômica/métodos , Regeneração/genética , Animais , Cromatografia Líquida de Alta Pressão , Proteínas de Peixes/metabolismo , Ontologia Genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/reabilitação , Ventrículos do Coração/lesões , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Proteômica/instrumentação , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização por Electrospray , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-ZebraRESUMO
Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Células Epiteliais/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proteômica/métodos , Testosterona/farmacologia , Adsorção , Animais , Proteínas Sanguíneas/química , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Carvão Vegetal/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feto , Expressão Gênica , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/isolamento & purificação , Fator de Crescimento Insulin-Like II/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptor IGF Tipo 1/isolamento & purificação , Receptor de Insulina/isolamento & purificação , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Testosterona/isolamento & purificaçãoRESUMO
For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail (SC) containing multiple nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible protein extraction, including membrane-bound proteins; (ii) effective removal of detrimental nonprotein matrix components (e.g., >94% of phospholipids); (iii) rapid/efficient proteolytic digestion owing to dual (surfactants + precipitation) denaturation. The optimal SC composition and concentrations were determined by Orthogonal-Array-Design investigation of their collective/individuals effects on protein extraction/denaturation. Key parameters for cleanup and digestion were experimentally identified as well. The optimized SEPOD procedures allowed a rapid 6 h digestion providing a clean digest with high peptide yields and excellent quantitative reproducibility (especially low-abundance proteins). Compared with filter-assisted sample preparation (FASP) and in-solution digestion, SEPOD showed superior performance by recovering substantially more peptide/proteins (including integral membrane proteins), yielding significantly higher peptide intensities and improving quantification for peptides with extreme physicochemical properties. SEPOD was further applied in a large-cohort temporal investigation of 44 IAV-infected mouse lungs, providing efficient and reproducible peptide yields (77.9 ± 4.6%) across all samples. With the IonStar pipeline, >6â¯400 unique protein groups were quantified (≥2 peptide/protein, peptide-FDR < 0.05%), â¼99% without missing data in any sample with <7% technical median-intragroup CV. Altered proteome patterns revealed interesting novel insights into pathophysiological changes by IAV infection. In summary, SEPOD offers a feasible solution for rapid, efficient, and reproducible preparation of biological samples, facilitating high-quality proteomic quantification of large sample cohorts.
Assuntos
Proteômica/métodos , Tensoativos/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Camundongos , Peptídeos/química , Reprodutibilidade dos Testes , Solventes/química , Espectrometria de Massas em TandemRESUMO
Arsenic is a known potent risk factor for bladder cancer. Increasing evidence suggests that epigenetic alterations, e.g., DNA methylation and histones posttranslational modifications (PTMs), contribute to arsenic carcinogenesis. Our previous studies have demonstrated that exposure of human urothelial cells (UROtsa cells) to monomethylarsonous acid (MMAIII), one of arsenic active metabolites, changes the histone acetylation marks across the genome that are correlated with MMAIII-induced UROtsa cell malignant transformation. In the current study, we employed a high-resolution and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitatively measure various PTM patterns during the MMAIII-induced malignant transformation. Our data showed that MMAIII exposure caused a time-dependent increase in histone H3 acetylation on lysine K4, K9, K14, K18, K23, and K27, but a decrease in acetylation on lysine K5, K8, K12, and K16 of histone H4. Consistent with this observation, H3K18ac was increased while H4K8ac was decreased in the leukocytes collected from people exposed to high concentrations of arsenic compared to those exposed to low concentrations. MMAIII was also able to alter histone methylation patterns: MMAIII transformed cells experienced a loss of H3K4me1, and an increase in H3K9me1 and H3K27me1. Collectively, our data shows that arsenic exposure causes dynamic changes in histone acetylation and methylation patterns during arsenic-induced cancer development. Exploring the genomic location of the altered histone marks and the resulting aberrant expression of genes will be of importance in deciphering the mechanism of arsenic-induced carcinogenesis.
Assuntos
Arsênio/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Código das Histonas/efeitos dos fármacos , Histonas/metabolismo , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/patologia , Bexiga Urinária/patologia , Acetilação , Animais , Células Cultivadas , Humanos , Leucócitos/efeitos dos fármacos , Lisina/metabolismo , Camundongos Nus , Compostos Organometálicos/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key features include: (i) an optimized, surfactant-aided sample preparation approach provides highly efficient (>75% recovery) and reproducible (<15% CV) peptide recovery across large cell/tissue cohorts; (ii) a long column with modest gradient length (2.5 h) yields the optimal balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID trap not only enables highly reproducible gradient delivery as for the first time observed via real-time conductivity monitoring, but also increases quantitative loading capacity by >8-fold and quantified >25% more proteins; (iv) an optimized HCD-OT markedly outperforms HCD-IT when analyzing large cohorts with high loading amounts; (v) selective removal of hydrophobic/hydrophilic matrix components using a novel selective trapping/delivery approach enables reproducible, robust LC-MS analysis of >100 biological samples in a single set, eliminating batch effect; (vi) MS1 acquired at higher resolution (fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision for low-abundance species. We examined this pipeline by analyzing a 5 group, 20 samples biological benchmark sample set, and quantified 6273 unique proteins (≥2 peptides/protein) under stringent cutoffs without fractionation, 6234 (>99.4%) without missing data in any of the 20 samples. The strategy achieved high quantitative accuracy (3-6% media error), low intragroup variation (6-9% media intragroup CV) and low false-positive biomarker discovery rates (3-8%) across the five groups, with quantified protein abundances spanning >6.5 orders of magnitude. Finally, this strategy is straightforward, robust, and broadly applicable in pharmaceutical/biomedical investigations.
Assuntos
Fracionamento Químico/métodos , Peptídeos/análise , Proteoma/isolamento & purificação , Proteômica/métodos , Linhagem Celular Tumoral , Fracionamento Químico/instrumentação , Cromatografia Líquida , Misturas Complexas/química , Humanos , Ductos Pancreáticos/química , Ductos Pancreáticos/patologia , Proteômica/instrumentação , Reprodutibilidade dos Testes , Tamanho da Amostra , Tensoativos/química , Espectrometria de Massas em TandemRESUMO
Intrinsically disordered proteins (IDPs) play a variety of important physiological roles in all living organisms. However, there is no comprehensive analysis of the abundance of IDPs associated with environmental stress in plants. Here, we show that a set of heat-stable proteins (i.e., proteins that do not denature after boiling at 100 °C for 10 min) was present in R0mm and R15mm radicles (i.e., before radicle emergence and 15 mm long radicles) of soybean (Glycine max) seeds. This set of 795 iTRAQ-quantified heat-stable proteins contained a high proportion of wholly or highly disordered proteins (15%), which was significantly higher than that estimated for the whole soybean proteome containing 55,787 proteins (9%). The heat-stable proteome of soybean radicles that contain many IDPs could protect lactate dehydrogenase (LDH) during freeze-thaw cycles. Comparison of the 795 heat-stable proteins in the R0mm and R15mm soybean radicles revealed that many of these proteins changed abundance during seedling growth with 170 and 89 proteins being more abundant in R0mm and R15mm, respectively. KEGG analysis identified 18 proteins from the cysteine and methionine metabolism pathways and nine proteins from the phenylpropanoid biosynthesis pathway. As an important type of IDP related to stress, 30 late embryogenesis abundant proteins were also found. Ten selected proteins with high levels of predicted intrinsic disorder were able to efficiently protect LDH from the freeze-thaw-induced inactivation, but the protective ability was not correlated with the disorder content of these proteins. These observations suggest that protection of the enzymes and other proteins in a stressed cell can be one of the biological functions of plant IDPs.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max/genética , Proteínas Intrinsicamente Desordenadas/genética , Chaperonas Moleculares/genética , Proteínas de Plantas/genética , Proteoma/genética , Sementes/genética , Cisteína/metabolismo , Dessecação , Ontologia Genética , Temperatura Alta , Proteínas Intrinsicamente Desordenadas/metabolismo , L-Lactato Desidrogenase/metabolismo , Metionina/metabolismo , Chaperonas Moleculares/metabolismo , Anotação de Sequência Molecular , Proteínas de Plantas/metabolismo , Propanóis/metabolismo , Estabilidade Proteica , Proteoma/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Estresse FisiológicoRESUMO
Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteômica/métodos , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Humanos , Metabolismo/efeitos dos fármacos , Mioblastos , Proteínas/análise , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Given the tremendous detriments of cocaine dependence, effective diagnosis and patient stratification are critical for successful intervention yet difficult to achieve due to the largely unknown molecular mechanisms involved. To obtain new insights into cocaine dependence and withdrawal, we employed a reproducible, reliable, and large-scale proteomics approach to investigate the striatal proteomes of rats (n = 40, 10 per group) subjected to chronic cocaine exposure, followed by either short- (WD1) or long- (WD22) term withdrawal. By implementing a surfactant-aided precipitation/on-pellet digestion procedure, a reproducible and sensitive nanoLC-Orbitrap MS analysis, and an optimized ion-current-based MS1 quantification pipeline, >2000 nonredundant proteins were quantified confidently without missing data in any replicate. Although cocaine was cleared from the body, 129/37 altered proteins were observed in WD1/WD22 that are implicated in several biological processes related closely to drug-induced neuroplasticity. Although many of these changes recapitulate the findings from independent studies reported over the last two decades, some novel insights were obtained and further validated by immunoassays. For example, significantly elevated striatal protein kinase C activity persisted over the 22 day cocaine withdrawal. Cofilin-1 activity was up-regulated in WD1 and down-regulated in WD22. These discoveries suggest potentially distinct structural plasticity after short- and long-term cocaine withdrawal. In addition, this study provides compelling evidence that blood vessel narrowing, a long-known effect of cocaine use, occurred after long-term but not short-term withdrawal. In summary, this work developed a well-optimized paradigm for ion-current-based quantitative proteomics in brain tissues and obtained novel insights into molecular alterations in the striatum following cocaine exposure and withdrawal.
Assuntos
Cocaína/farmacologia , Corpo Estriado/química , Proteoma/efeitos dos fármacos , Proteômica/métodos , Síndrome de Abstinência a Substâncias , Animais , Transtornos Relacionados ao Uso de Cocaína , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Fatores de TempoRESUMO
Investigation of influenza-A-virus (IAV)-infected lung proteomes will greatly promote our understanding on the virus-host crosstalk. Using a detergent-cocktail extraction and digestion procedure and a reproducible ion-current-based method, we performed the first comprehensive temporal analysis of mouse IAV infection. Mouse lung tissues at three time points post-inoculation were compared with controls (n = 4/group), and >1600 proteins were quantified without missing value in any animal. Significantly changed proteins were identified at 4 days (n = 144), 7 days (n = 695), and 10 days (n = 396) after infection, with low false altered protein rates (1.73-8.39%). Functional annotation revealed several key biological processes involved in the systemic host responses. Intriguingly, decreased levels of several cell junction proteins as well as increased levels of tissue metalloproteinase MMP9 were observed, reflecting the IAV-induced structural breakdown of lung epithelial barrier. Supporting evidence of MMP9 activation came from immunoassays examining the abundance and phosphorylation states of all MAPKs and several relevant molecules. Importantly, IAV-induced MMP gelatinase expression was suggested to be specific to MMP9, and p38 MAPK may contribute predominantly to MMP9 elevation. These findings help to resolve the long-lasting debate regarding the signaling pathways of IAV-induced MMP9 expression and shed light on the molecular mechanisms underlying pulmonary capillary-alveolar leak syndrome that can occur during influenza infection.
Assuntos
Barreira Alveolocapilar/metabolismo , Pulmão/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Barreira Alveolocapilar/virologia , Western Blotting , Cromatografia de Fase Reversa , Vírus da Influenza A Subtipo H3N2/fisiologia , Modelos Lineares , Pulmão/irrigação sanguínea , Pulmão/virologia , Masculino , Espectrometria de Massas , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologiaRESUMO
Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.
Assuntos
Proteínas do Olho/isolamento & purificação , Espectrometria de Massas/métodos , Proteoma/isolamento & purificação , Retina/química , Degeneração Retiniana/genética , Animais , Animais Recém-Nascidos , Anexina A3/genética , Anexina A3/isolamento & purificação , Anexina A3/metabolismo , Clusterina/genética , Clusterina/isolamento & purificação , Clusterina/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/isolamento & purificação , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Neovascularização Patológica/genética , Oxigênio , Proteoma/genética , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/isolamento & purificação , Fator de Transcrição STAT1/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/isolamento & purificação , Proteínas rab de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/isolamento & purificação , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/isolamento & purificação , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator-associated combinations; therefore, Percolator enhanced the reliability for both identification and quantification. The analyses were performed using the specific programs embedded in Proteome Discoverer, Scaffold, and an in-house algorithm (BuildSummary). These results provide valuable guidelines for the optimal interpretation of proteomic results and the development of fit-for-purpose protocols under different situations.
Assuntos
Algoritmos , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Ferramenta de Busca/métodos , Software , Linhagem Celular Tumoral , Bases de Dados de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteoma/genética , Proteoma/metabolismo , Proteômica/instrumentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em TandemRESUMO
For decades, epidemiological studies have found significant differences in the susceptibility to disease progression among HIV-carrying patients. One unique group of HIV-1-positive patients, the long-term-nonprogressors (LTNP), exhibits far superior ability in virus control compared with normal-progressors (NP), which proceed to Acquired Immune Deficiency Syndrome (AIDS) much more rapidly. Nonetheless, elucidation of the underlying mechanisms of virus control in LTNP is highly valuable in disease management and treatment but remains poorly understood. Peripheral blood mononuclear cells (PBMC) have been known to play important roles in innate immune responses and thereby would be of great interest for the investigation of the mechanisms of virus defense in LTNP. Here, we described the first comparative proteome analysis of PBMC from LTNP (n = 10) and NP (n = 10) patients using a reproducible ion-current-based MS1 approach, which includes efficient and reproducible sample preparation and chromatographic separation followed by an optimized pipeline for protein identification and quantification. This strategy enables analysis of many biological samples in one set with high quantitative precision and extremely low missing data. In total, 925 unique proteins were quantified under stringent criteria without missing value in any of the 20 subjects, and 87 proteins showed altered expressions between the two patient groups. These proteins are implicated in key processes such as cytoskeleton organization, defense response, apoptosis regulation, intracellular transport, etc., which provided novel insights into the control of disease progressions in LTNP versus NP, and the expression and phosphorylation states of key regulators were further validated by immunoassay. For instance, (1) SAMH1, a potent and "hot" molecule facilitating HIV-1 defense, was for the first time found elevated in LTNP compared with NP or healthy controls; elevated proteins from IFN-α response pathway may also contribute to viral control in LTNP; (2) decreased proapoptotic protein ASC along with the elevation of antiapoptotic proteins may contribute to the less apoptotic profile in PBMC of LTNP; and (3) elevated actin polymerization and less microtubule assembly that impede viral protein transport were first observed in LTNP. These results not only enhanced the understanding of the mechanisms for nonprogression of LTNP, but also may afford highly valuable clues to direct therapeutic efforts. Moreover, this work also demonstrated the ion-current-based MS1 approach as a reliable tool for large-scale clinical research.
Assuntos
Infecções por HIV/sangue , Infecções por HIV/etiologia , Sobreviventes de Longo Prazo ao HIV , HIV-1 , Proteômica/métodos , Adulto , Idoso , Proteínas Reguladoras de Apoptose/sangue , Proteínas Reguladoras de Apoptose/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/metabolismo , Proteínas do Citoesqueleto/sangue , Proteínas do Citoesqueleto/isolamento & purificação , Progressão da Doença , Feminino , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteoma/isolamento & purificação , Proteínas Virais/metabolismo , Adulto JovemRESUMO
Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ~40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.
Assuntos
Proteínas do Olho/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteoma/isolamento & purificação , Retina/metabolismo , Degeneração Retiniana/genética , Síndrome de Smith-Lemli-Opitz/genética , Animais , Morte Celular , Cromatografia de Fase Reversa , Modelos Animais de Doenças , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Anotação de Sequência Molecular , Proteoma/genética , Proteoma/metabolismo , Ratos , Reprodutibilidade dos Testes , Retina/química , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Síndrome de Smith-Lemli-Opitz/metabolismo , Síndrome de Smith-Lemli-Opitz/patologia , Visão Ocular/fisiologiaRESUMO
Survey-scan-based label-free method have shown no compelling benefit over fragment ion (MS2)-based approaches when low-resolution mass spectrometry (MS) was used, the growing prevalence of high-resolution analyzers may have changed the game. This necessitates an updated, comparative investigation of these approaches for data acquired by high-resolution MS. Here, we compared survey scan-based (ion current, IC) and MS2-based abundance features including spectral-count (SpC) and MS2 total-ion-current (MS2-TIC), for quantitative analysis using various high-resolution LC/MS data sets. Key discoveries include: (i) study with seven different biological data sets revealed only IC achieved high reproducibility for lower-abundance proteins; (ii) evaluation with 5-replicate analyses of a yeast sample showed IC provided much higher quantitative precision and lower missing data; (iii) IC, SpC, and MS2-TIC all showed good quantitative linearity (R(2) > 0.99) over a >1000-fold concentration range; (iv) both MS2-TIC and IC showed good linear response to various protein loading amounts but not SpC; (v) quantification using a well-characterized CPTAC data set showed that IC exhibited markedly higher quantitative accuracy, higher sensitivity, and lower false-positives/false-negatives than both SpC and MS2-TIC. Therefore, IC achieved an overall superior performance than the MS2-based strategies in terms of reproducibility, missing data, quantitative dynamic range, quantitative accuracy, and biomarker discovery.
Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bases de Dados de Proteínas , Humanos , Íons/análise , Íons/química , Proteínas/análise , Proteínas/química , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The rapidly expanding availability of high-resolution mass spectrometry has substantially enhanced the ion-current-based relative quantification techniques. Despite the increasing interest in ion-current-based methods, quantitative sensitivity, accuracy, and false discovery rate remain the major concerns; consequently, comprehensive evaluation and development in these regards are urgently needed. Here we describe an integrated, new procedure for data normalization and protein ratio estimation, termed ICan, for improved ion-current-based analysis of data generated by high-resolution mass spectrometry (MS). ICan achieved significantly better accuracy and precision, and lower false-positive rate for discovering altered proteins, over current popular pipelines. A spiked-in experiment was used to evaluate the performance of ICan to detect small changes. In this study E. coli extracts were spiked with moderate-abundance proteins from human plasma (MAP, enriched by IgY14-SuperMix procedure) at two different levels to set a small change of 1.5-fold. Forty-five (92%, with an average ratio of 1.71 ± 0.13) of 49 identified MAP protein (i.e., the true positives) and none of the reference proteins (1.0-fold) were determined as significantly altered proteins, with cutoff thresholds of ≥ 1.3-fold change and p ≤ 0.05. This is the first study to evaluate and prove competitive performance of the ion-current-based approach for assigning significance to proteins with small changes. By comparison, other methods showed remarkably inferior performance. ICan can be broadly applicable to reliable and sensitive proteomic survey of multiple biological samples with the use of high-resolution MS. Moreover, many key features evaluated and optimized here such as normalization, protein ratio determination, and statistical analyses are also valuable for data analysis by isotope-labeling methods.
Assuntos
Proteoma/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Proteínas de Escherichia coli/química , Humanos , Espectrometria de Massas/normas , Proteoma/química , Proteoma/normas , Padrões de Referência , Sensibilidade e Especificidade , Soroalbumina Bovina/químicaRESUMO
Proteomic analysis of bronchoalveolar lavage fluid (BALF) in chronic obstructive pulmonary disease (COPD) patients may provide new biomarkers and deeper understanding of the disease mechanisms but remains challenging. Here we describe an ion-current-based strategy for comparative analysis of BALF proteomes from patients with moderate and stable COPD versus healthy controls. The strategy includes an efficient preparation procedure providing quantitative recovery and a nano-LC/MS analysis with a long, heated column. Under optimized conditions, high efficiency and reproducibility were achieved for each step, enabling a "20-plex" comparison of clinical subjects (n = 10/group). Without depletion/fractionation, a total of 423 unique protein groups were quantified under stringent criteria with at least two quantifiable peptides. Seventy-six proteins were determined as significantly altered in COPD, which represent a diversity of biological processes such as alcohol metabolic process, gluconeogenesis/glycolysis, inflammatory response, proteolysis, and oxidation reduction. Interestingly, altered alcohol metabolism responding to oxidant stress is a novel observation in COPD. The prominently elevated key enzymes involved in alcohol metabolism (e.g., ADH1B, ALDH2, and ALDH3A1) may provide a reasonable explanation for a bewildering observation in COPD patients known for decades: the underestimation of the blood alcohol concentrations through breath tests. These discoveries could provide new insights for identifying novel biomarkers and pathological mediators in clinical studies.