RESUMO
Large RNA including mRNA (mRNA) has emerged as an important new class of therapeutics. Recently, this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study, we have developed an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilized on magnetic particles were performed in conjunction with high-resolution liquid chromatography-mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach, a >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS-CoV-2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation, can be completed within 90 min. The ability to rapidly identify, characterize, and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation, and impurity analysis.
Assuntos
COVID-19 , Espectrometria de Massas em Tandem , COVID-19/terapia , Humanos , RNA/química , RNA Mensageiro/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Espectrometria de Massas em Tandem/métodosRESUMO
The accurate diagnosis of Trisomy 21 requires invasive procedures that carry a risk of miscarriage. The current state-of-the-art maternal serum screening tests measure levels of PAPP-A, free bhCG, AFP, and uE3 in various combinations with a maximum sensitivity of 60-75% and a false positive rate of 5%. There is currently an unmet need for noninvasive screening tests with high selectivity that can detect pregnancies at risk, preferably within the first trimester. The aim of this study was to apply proteomics and mass spectrometry techniques for the discovery of new putative biomarkers for Trisomy 21 in first trimester maternal serum coupled with the immediate development of quantitative selective reaction monitoring (SRM) assays. The results of the novel workflow were 2-fold: (1) we identified a list of differentially expressed proteins in Trisomy 21 vs Normal samples, including PAPP-A, and (2) we developed a multiplexed, high-throughput SRM assay for verification of 12 new putative markers identified in the discovery experiments. To narrow down the initial large list of differentially expressed candidates resulting from the discovery experiments, we incorporated receiver operating characteristic (ROC) curve algorithms early in the data analysis process. We believe this approach provides a substantial advantage in sifting through the large and complex data typically obtained from discovery experiments. The workflow efficiently mined information derived from high-resolution LC-MS/MS discovery data for the seamless construction of rapid, targeted assays that were performed on unfractionated serum digests. The SRM assay lower limit of detection (LLOD) for the target peptides in a background of digested serum matrix was approximately 250-500 attomoles on column and the limit of accurate quantitation (LOQ) was approximately 1-5 femtomoles on column. The assay error as determined by coefficient of variation at LOQ and above ranged from 0 to 16%. The workflow developed in this study bridges the gap between proteomic biomarker discovery and translation into a clinical research environment. Specifically, for Trisomy 21, the described multiplexed SRM assay provides a vehicle for high-throughput verification of these, and potentially other, peptide candidates on larger sample cohorts.
Assuntos
Biomarcadores/sangue , Síndrome de Down/diagnóstico , Espectrometria de Massas/métodos , Primeiro Trimestre da Gravidez , Diagnóstico Pré-Natal/métodos , Proteômica/métodos , Área Sob a Curva , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Feminino , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Gravidez , Curva ROCRESUMO
We report on a proteomic analysis of ex vivo human immunodeficiency virus (HIV) type 1 infection in human primary CD4 cells by shotgun liquid chromatography-tandem mass spectrometry analysis, revealing two distinct proteomic profiles at two phases of virus replication. Relative to mock-infected cells, 168 signature proteins exhibited abundance changes at the first sign of Gag p24 production (8 h postinfection [p.i.]) or the peak of virus replication (24 h p.i.); interestingly, most of the changes were exclusive to only one phase of virus replication. Based on characterization by functional ontology and known human-HIV protein interactions, we observed the enrichment for protein abundance increases pertaining to protein synthesis and nucleasomal reorganization amid an otherwise placid cellular proteome at the first sign of HIV replication. In contrast, we observed indications of decreased protein turnover, concomitant with heightened DNA repair activities and preludes to apoptosis, in the presence of robust virus replication. We also observed hints of disruptions in protein and small molecule trafficking. Our label-free proteomic strategy allowed us to perform multiplexed comparisons-we buttressed our detection specificity with the use of a reverse transcriptase inhibitor as a counterscreen, enabling highlighting of cellular protein abundance changes unique to robust virus replication as opposed to viral entry. In conjunction with complementary high-throughput screens for cellular partners of HIV, we put forth a model pinpointing specific rerouting of cellular biosynthetic, energetic, and trafficking pathways as HIV replication accelerates in human primary CD4 cells.
Assuntos
Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/virologia , Proteínas do Citoesqueleto/análise , Metabolismo Energético , Infecções por HIV/patologia , Espectrometria de Massas em Tandem/métodos , Células Cultivadas , Cromatografia Líquida , HIV-1 , Humanos , Redes e Vias Metabólicas , Transporte Proteico , Proteoma , Proteômica/métodos , Fatores de Tempo , Replicação ViralRESUMO
Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/administração & dosagem , Grupo dos Citocromos c/administração & dosagem , Proteínas Mitocondriais/administração & dosagem , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cavalos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Espectrometria de Massas , Microinjeções , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Morfolinas/farmacologia , Testes de Precipitina , Neoplasias da Próstata/tratamento farmacológico , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Ensaios de Triagem em Larga Escala , Imunoensaio/métodos , Espectrometria de Massas/métodos , Doença de Alzheimer/sangue , Doenças Cardiovasculares/sangue , Transtornos do Crescimento/sangue , Humanos , Neoplasias/sangue , Insuficiência Renal/sangueRESUMO
The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (nâ=â10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.
Assuntos
Esmalte Dentário/química , Durapatita/química , Fluoretos/química , Histatinas/química , Saliva/química , Cárie Dentária/metabolismo , Cárie Dentária/patologia , Esmalte Dentário/metabolismo , Feminino , Fluoretos/farmacologia , Histatinas/metabolismo , Humanos , Masculino , Saliva/metabolismoRESUMO
PURPOSE: Typically, apolipoproteins are individually measured in blood by immunoassay. In this report, we describe the development of a multiplexed selected reaction monitoring (SRM) based assay for a panel of apolipoproteins and its application to a clinical cohort of samples derived from acute stroke patients. EXPERIMENTAL DESIGN: An SRM assay for a panel of nine apolipoproteins was developed on a triple quadrupole mass spectrometer. Quantitative data for each apolipoprotein were analyzed to determine expression ratio and receiver operating characteristic (ROC) values for ischemic versus hemorrhagic stroke. RESULTS: The optimized SRM assay was used to interrogate a small cohort of well-characterized plasma samples obtained from patients with acute ischemic and hemorrhagic strokes. The ROC analyses demonstrated good classification power for several single apolipoproteins, most notably apoC-III and apoC-I. When a novel multi-marker ROC algorithm was applied, the ischemic versus hemorrhagic groups were best differentiated by a combination of apoC-III and apoA-I with an area under the curve (AUC) value of 0.92. CONCLUSIONS AND CLINICAL RELEVANCE: This proof-of-concept study provides interesting and provocative data for distinguishing ischemic versus hemorrhage within first week of symptom onset. However, the observations are based on one cohort of patient samples and further confirmation will be required.
Assuntos
Algoritmos , Apolipoproteínas/sangue , Proteínas Sanguíneas/análise , Transtornos Hemorrágicos/diagnóstico , Espectrometria de Massas/métodos , Curva ROC , Acidente Vascular Cerebral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Apolipoproteínas/classificação , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Transtornos Hemorrágicos/patologia , Humanos , Isquemia/diagnóstico , Isquemia/patologia , Limite de Detecção , Masculino , Espectrometria de Massas/normas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Acidente Vascular Cerebral/patologia , Adulto JovemRESUMO
Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.
Assuntos
Forame Oval Patente/sangue , Regulação da Expressão Gênica , Proteômica/métodos , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/sangue , Espectrometria de Massas em Tandem , Adulto , Encéfalo/fisiologia , Cromatografia Líquida/métodos , Estudos de Coortes , Feminino , Forame Oval Patente/epidemiologia , Forame Oval Patente/cirurgia , Coração/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/epidemiologia , Espectrometria de Massas em Tandem/métodos , Adulto JovemRESUMO
The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)-encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family-associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.
Assuntos
Epitopos/imunologia , Antígeno HLA-A3/imunologia , Melanoma/imunologia , Fragmentos de Peptídeos/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Epitopos/isolamento & purificação , Epitopos/metabolismo , Análise de Fourier , Antígeno HLA-A3/metabolismo , Humanos , Espectrometria de Massas , Melanoma/metabolismo , Melanoma/patologia , Fragmentos de Peptídeos/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.
Assuntos
Focalização Isoelétrica/métodos , Proteoma , Soro/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Quimotripsina/metabolismo , Eletroforese , Humanos , Tripsina/metabolismoRESUMO
A novel reversed-phase (RP-) HPLC gradient profile applicable to multidimensional separations of complex protein mixtures is reported. This gradient profile elutes small numbers of proteins from the RP-HPLC column in discrete intervals while minimizing the amount of band broadening between elution intervals and maintaining constant flow through the HPLC column. Eluting the proteins in discrete intervals eases the instrumental requirements necessary for performing multidimensional separations and can be used to aid in the collection of well-defined fractions. The saw-tooth gradient was applied to the successful isolation of albumin from less abundant proteins in whole human serum and provides adequate separation of proteins in a low-molecular weight (LMW) fraction of human serum with resolution comparable to that achieved using a typical linear gradient profile.