Preparação da amostra para análise de Endopeptidomic no líquido cefalorraquidiano humano

The goal of this protocol is to prepare and analyze human cerebrospinal fluid samples for LC-MS analysis of endogenous peptides.A peptide pre-fractionation step prior to LC-MS increases the number of identified peptides and enables detection of relatively low abundance CSF peptides.Endogenous peptides in CSF are calcium molecules that have not been extensively explored and may potentially include important new biomarkers of neurodegenerative disorders.This method enables extensive characterization of the CSF peptidome by mass spectrometry.The main advantages of this method are that it enables injection of low abundance CSF peptides and is compatible with multiplex isobaric labeling techniques, making it applicable for clinical studies.So far we have mainly applied this method to the analysis of cerebrospinal fluid.But we have preliminary data indicating that with minor modifications, it can also be used for analysis of plasma and tissue samples.Begin by thawing 1.5 milliliter aliquots of CSF at room temperature on roller mixer.Once thawed, transfer the contents to 10 milliliter polypropylene tubes.And add 80 microliters of one molar TAB as a buffering agent.Then add 0.65 milliliters of a eight molar guanidinium hydrochloride to dissolve protein aggregates and vortex gently at room temperature for 10 minutes.Next, at 60 microliters of 200 millimolar aqueous TCEP, and incubate at 55