{"id":7666,"date":"2016-02-15T14:07:04","date_gmt":"2016-02-15T19:07:04","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=7666"},"modified":"2016-04-29T16:52:56","modified_gmt":"2016-04-29T16:52:56","slug":"indigestion-relief-with-digestif","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/proteomics\/indigestion-relief-with-digestif\/","title":{"rendered":"Indigestion Relief with DIGESTIF"},"content":{"rendered":"

\"TrypsinEspecially for quantitative label-free proteomics studies, consistency of workflow, methodology <\/span><\/span>and <\/span><\/span>sample preparation <\/span><\/span>in a multi-stage<\/span><\/span> experimental process is <\/span><\/span>key<\/span><\/span> for obtaining meaningful, reproducible data. With bottom-up proteomics, this also includes consistent sample digestion prior to mass spectrometry analysis. <\/span><\/span>Lebert<\/span><\/span> et al. (2014) propose a universal protein standard that monitors this step, providing essential feedback <\/span><\/span>about workflow optimization<\/span><\/span> <\/span><\/span>to researchers.1<\/sup><\/span><\/span> <\/span><\/p>\n

Although other methods for monitoring experimental conditions exist, <\/span><\/span>Lebert<\/span><\/span> et <\/span><\/span>al. argue that these are <\/span><\/span>stage- <\/span><\/span>and sample-specific<\/span><\/span>. In creating DIGESTIF, a soluble recombinant protein scaffold holding 11 artificial peptides, the researchers provide a way for scientists to monitor overall digestion during sample preparation.<\/span><\/span> <\/span><\/p>\n

The researchers used <\/span><\/span>PyMOL<\/span><\/span> to design the protein standard, creating a synthetic gene that they expressed in a PET33b vector, which they amplified using <\/em><\/span><\/span>Escherichia coli<\/span><\/span><\/em>.<\/span><\/span><\/span> Following this, <\/span><\/span>Lebert<\/span><\/span> et al. transformed the product into <\/span><\/span>BL21(<\/span><\/span>DE3) <\/span><\/span>E. coli<\/span><\/span>, a strain that is auxotrophic for lysine and arginine, thus allowing isotopic labeling. Following synthesis, the research team <\/span><\/span>isolat<\/span><\/span>ed the DIGESTIF protein before evaluating it<\/span><\/span>s purity<\/span><\/span> using sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE).<\/span><\/span> <\/span><\/p>\n

The research team examined the synthetic protein standard initially by digesting it using trypsin, then analyzing it using liquid-chromatography tandem <\/span><\/span>mass<\/span><\/span> spectrometry (LC-MS\/MS). <\/span><\/span>They also analyzed it after spiking the digest<\/span><\/span> into commercially available human serum<\/span><\/span>. Instrumentation used included an <\/span><\/span><\/span><\/span>UltiMate<\/a> 3000 liquid chromatography system<\/a> and a TSQ Quantum Ultra EMR triple quadrupole mass spectrometer<\/a> (both T<\/span><\/span><\/span>hermo<\/span><\/span> Scientific).<\/span><\/span> <\/span><\/p>\n

From the synthetic protein <\/span><\/span>digest <\/span><\/span>alone<\/span><\/span>, the team determined proteolytic characteristics and experimental kinetics by <\/span><\/span>mass<\/span><\/span> spectrometry evaluation, generating mass spectral profiles for future reference. <\/span><\/span>Using LC-selected reaction monitoring (LC-SRM), they gathered MS data on DIGESTIF performance in <\/span><\/span>various <\/span><\/span>matrices such as human serum, urine and mouse liver lysate. <\/span><\/span>Lebert<\/span><\/span> et al. then used the protein standard in samples of human plasma from a patient with drug-induced liver injury to examine a real-life scenario where clinicians analyze biomarker levels.<\/span><\/span> <\/span><\/p>\n

<\/span><\/span><\/span><\/span>The research team <\/span><\/span>designed <\/span><\/span>DIGESTIF to reflect varying degrees of proteolytic treatment by <\/span><\/span>progressively <\/span><\/span>releasing indicator peptides from its scaffold. In doing so, t<\/span><\/span>hey <\/span><\/span>found that as an internal standard <\/span><\/span>the recombinant<\/span><\/span> protein reliably indicated the completeness of the digestion process<\/span><\/span><\/span>. <\/span><\/span>Lebert<\/span><\/span> et al. conclude <\/span><\/span>that DIGESTIF is a useful tool for proteomics researchers that will assist with experimental design and in monitoring workflows. They suggest that it is useful in the following areas:<\/span><\/span> <\/span><\/p>\n