A2_Quantitative Proteomics

 TOPLAB GmbH (Martinsried, München)
Dr. Thomas Halder (PL), Dr. Franka Pluder (PhD), Kristina Teichmann (TA)

Project summary:

Hepatocyte regeneration requires cell proliferation. Two cell signalling cascades, JAK1-STAT3, and NFĸB, are the key players for the transition from the priming towards the proliferative state. The goal of this project is to quantify key proteins from these two signalling pathways in primary murine hepatocyte cells by mass spectrometry (MS) based methods. For this purpose, we used proteotypic peptides, specific and unique for a given protein. They can be quantified by different quantitative, MS-based advanced techniques. A non-labelling quantification method based on LC-MALDI-MS has been established and successfully tested for two candidate proteins, SMAD2/3. For normalisation of the signals an internal standard peptide is added to the MALDI-MS matrix in a known amount. An alternative method was successfully introduced based upon multiple reaction monitoring using proteotypic peptides labelled with stable isotopes as internal standards, and LC-ESI-MS. This method allows highly specific and sensitive detection of proteotypic peptides by the use of quadrupoles as mass filters. First, a quadrupole restricts the entrance of peptides with a defined mass into the MS, then these peptides are fragmented in a collision cell, and only defined peptide fragments derived from the original proteotypic peptides are detected. By the use of proteotypic peptides with stable isotopes in a known concentration an absolute quantification of the non-modified peptides from the cell samples is possible.

Former co-workers: Michael Kersten