RESUMO
Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach, called LRP-IS-PRM, is a new modality for generating protein-level evidence of alternative isoforms - a critical first step in designing functional studies and eventually clinical assays.
RESUMO
Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoformsâa critical first step in designing functional studies and eventually clinical assays.
RESUMO
Monitoring the renal arterial Doppler flow velocity indices, the resistive index and pulsatility index, with ultrasound may help predict renal dysfunction. However, such monitoring has been done intermittently by transcutaneous ultrasound in the postoperative intensive care setting. In the operating room, transesophageal echocardiography (TEE) is an alternative to transcutaneous ultrasound for obtaining indices of renal perfusion. However, it is difficult to locate the right kidney using TEE. We propose a new technique to locate the left kidney that, in our experience, is simple and easy to perform. We believe, starting from a transgastric left ventricular short-axis view, turning left to locate the abdominal aorta, and following it to the origin of the left renal artery may help locate the left kidney faster than previously described techniques. We also propose a new technique to monitor these Doppler indices using TEE during the intraoperative period.
Assuntos
Ecocardiografia Transesofagiana/métodos , Rim/irrigação sanguínea , Rim/diagnóstico por imagem , Monitorização Intraoperatória/métodos , Velocidade do Fluxo Sanguíneo/fisiologia , Ecocardiografia Transesofagiana/instrumentação , Humanos , Rim/fisiologia , Monitorização Intraoperatória/instrumentaçãoAssuntos
Átrios do Coração/diagnóstico por imagem , Neoplasias Cardíacas/diagnóstico por imagem , Veias Hepáticas/diagnóstico por imagem , Mixoma/diagnóstico por imagem , Veia Cava Inferior/diagnóstico por imagem , Adulto , Ecocardiografia/métodos , Feminino , Neoplasias Cardíacas/complicações , Neoplasias Cardíacas/cirurgia , Veias Hepáticas/fisiopatologia , Humanos , Monitorização Intraoperatória/métodos , Mixoma/complicações , Mixoma/cirurgia , Veia Cava Inferior/fisiopatologiaAssuntos
Septo Interatrial/diagnóstico por imagem , Ecocardiografia Transesofagiana , Defeitos dos Septos Cardíacos/diagnóstico por imagem , Achados Incidentais , Tromboembolia/etiologia , Septo Interatrial/cirurgia , Ecocardiografia Doppler em Cores , Feminino , Defeitos dos Septos Cardíacos/complicações , Defeitos dos Septos Cardíacos/cirurgia , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fatores de RiscoAssuntos
Comunicação Interatrial/complicações , Comunicação Interatrial/diagnóstico por imagem , Estenose da Valva Mitral/complicações , Estenose da Valva Mitral/diagnóstico por imagem , Pré-Escolar , Feminino , Comunicação Interatrial/cirurgia , Humanos , Estenose da Valva Mitral/cirurgia , UltrassonografiaAssuntos
Ponte de Artéria Coronária , Doença da Artéria Coronariana/cirurgia , Ecocardiografia Doppler em Cores , Ecocardiografia Transesofagiana , Comunicação Interatrial/diagnóstico por imagem , Idoso , Doença da Artéria Coronariana/diagnóstico , Feminino , Humanos , Achados Incidentais , Cuidados Intraoperatórios , Valor Preditivo dos TestesAssuntos
Ecocardiografia Tridimensional , Endocardite Bacteriana , Endocardite , Humanos , Valva Tricúspide/diagnóstico por imagem , Valva Tricúspide/cirurgia , Endocardite/diagnóstico por imagem , Valva Aórtica/diagnóstico por imagem , Ecocardiografia Transesofagiana , Endocardite Bacteriana/diagnóstico por imagemRESUMO
A 68-year-old gentleman presented with a recent history of exertional dyspnea associated with anginal chest pain. Transthoracic echocardiography revealed a well-circumscribed mass in the left atrium attached to the inter-atrial septum. A provisional diagnosis of left atrial (LA) myxoma was made. Coronary angiography revealed significant single-vessel disease. The patient underwent coronary artery bypass grafting and resection of the LA tumor. The histopathological diagnosis of the tumor came out to be a cardiac hemangioma.
Assuntos
Diagnóstico Diferencial , Átrios do Coração/patologia , Neoplasias Cardíacas/diagnóstico , Hemangioma/diagnóstico , Mixoma/diagnóstico , Idoso , Ecocardiografia Transesofagiana , Neoplasias Cardíacas/cirurgia , Hemangioma/cirurgia , Humanos , MasculinoRESUMO
A 35-year-old woman presented with 4 months history of progressively increasing intermittent dyspnea and hemoptysis. Transthoracic echocardiography revealed a loculated mass in the left atrium (LA). A provisional diagnosis of LA myxoma was made. Intraoperatively the tumor was found extending into and closely adherent to the left pulmonary vein and could not be completely cleared off from the pulmonary venous wall. The histopathological examination of the tumor revealed it to be a myxoid malignant fibrous histiocytoma.