Targeted Mass Spectrometry of Brain and Pancreatic tissues
- Organism: Homo sapiens
- Instrument: Q Exactive HF
Targeted Mass Spectrometry, QPrEST, PRM, Brain, Pancreas, CZI
- Lab head:
QPrESTs are heavy labelled recombinant protein fragments covering regions of proteins with low homology and span over 50-150 amino acids of the sequence. They were produced as a part of Human Protein Atlas project in auxotrophic Escherichia coli and individually purified, serving as an excellent resource for MS-based protein quantification.
In this project, a workflow for sample preparation of brain and pancreatic tissues with use of QPrESTs was developed together with a panel of parallel reaction monitoring (PRM) methods for quantification of more than 50 proteins using targeted mass spectrometry. In total, methods for quantification of 125 and 126 peptides in cortex and pancreas respectively were established. The technical robustness of the workflow was investigated on three technical replicates of pancreatic and three biological replicates of brain tissues. The results are reported in fmol/µg of protein content in a tissue lysate and span with dynamic range over 5-6 orders of magnitude. The combination of QPrEST standards being added before digestion and state of the art mass spectrometry performing in PRM mode showed high degree of reproducibility where 78.8 % and 81.1 % of quantified proteins had coefficient of variation (CV) below 20 % in Cortex and Pancreas respectively.
Brain and Pancreatic tissues were lysed and spiked in with heavy labelled protein fragments QPrESTs, enzymaticaly digested and analysed on the LC-MS/MS in order to quantify 50 clinically relevant targets.
Fresh frozen Brain and Pancreatic tissues
Created on 3/1/19, 2:06 PM