Absolute Quantification to Study Neurodegenerative Diseases by QconCAT Strategy

Junjun Chen, Illarion V. Turko

 

Absolute quantification plays an important role in proteomics because it determines protein abundances. However, absolute quantification was hampered by the availability of standard labeled signature peptides in accurately known amounts.  In this project, we designed peptide standards containing concatenated sequences of specific proteotypic peptides that are derived from various neuron disease related proteins. The concatenated peptides were generated in in vitro translation system. Isotopically labeled Lys and Arg were introduced to produce stable isotope-labeled peptides. The concatenated peptides were purified by Ni-NTA column followed by a buffer exchange introducing 2 % SDS. Isotope incorporation was measured by MALDI-TOF. A known amount of isotope-labeled peptides standards were co-digested with tissue samples containing target proteins.  Absolute quantification of these neuron disease related proteins will be performed by multiple reaction monitoring (MRM) with a triple quadrupole mass spectrometry.