The Secretome Analysis by High-Throughput Proteomics and Multiple Reaction Monitoring (MRM)

Abstract

The secretome is a sub-proteome of great interest in several fields of biomedical sciences, especially as a source of diagnostics and therapeutic targets. Proteomics has been contributing significantly to elucidate the secretome of a great diversity of cells, tissues, and organisms, turning profiles of thousands of proteins a usual practice. After elucidation of long protein lists, targeted proteomics also plays important roles in accurate quantification and validation of such secreted proteins. Here we present detailed protocols to explore and quantify the secretome of cancer cells, even though this protocol can be employed to any kind of biological material.

Hypothesis-free secretome analysis of thoracic aortic aneurysm reinforces the central role of TGF-β cascade in patients with bicuspid aortic valve

“…Previous studies have focused on the association of certain genes and proteins with aneurysm by using genomics and proteomics approaches [6][7][8]. The secretome analysis by highthroughput proteomics has been demonstrated to have a great advantage because, mimicking the in vivo secretion mechanism, it shows traces of tissue-specific biological events although implying a reduced complexity compared to the whole tissue proteomics and thus allowing the detection of under-represented potential biomarkers [9]. Accordingly, the purpose of the present study was to evaluate secreted/released proteins from ATAA by liquid chromatography coupled with mass spectrometry in order to gain further insights into molecular mechanisms leading to ascending aortic aneurysm formation in patients with BAV or TAV.…”

Metabolic alterations derived from absence of Two-Pore Channel 1 at cardiac level

“…In the last years, the mainstream proteomics approaches that involve 'shotgun' proteomics, in which complex protein mixtures are digested into peptides, which are subsequently separated through reverse-phase liquid chromatography (LC) and analysed using mass spectrometry (MS), have been widely used and improved (Roca-Rivada et al 2015;Tuli and Ressom 2009). This technique resolves the peptides prior to mass spectrometry analysis, thus dramatically increasing the number of protein identifications and sequence coverage in complex samples (Faça et al 2014). LC/MS has good selectivity and sensitivity and is a fast and accurate bioanalytical tool for proteomics (Zhang et al 2010).…”

Purificação e caracterização de inibidores de proteases dos soros de Crotalus durissus terrificus e Didelphis marsupialis

“…Blast. www.matrixscience.com), para identificação de proteínas homólogas aos peptídeos trípticos sequenciados.No laboratório do Professor Faça a tripsinização do inibidor e análise dos peptídeos obtidos foram realizadas segundo procedimento estabelecido no laboratório(Faça et al 2014). Utilizando-se espectrometria de massas em modo positivo, em um espectrômetro XEVO TQ-S (Waters), e voltagem constante de ionização de 3,5 kv.…”