Virtual Liver Retreat 2012: Quantitative Proteomics of liver

Virtual Liver Retreat 2012 (Poster)
Quantitative Proteomics of liver

S. Babak Azimifar, Kirti Sharma, Mara Monetti, Nagarjuna Nagaraj and Matthias Mann*.
*Corresponding author; contact: S. Babak Azimifar (Max Planck Institute of Biochemistry, Martinsried, Germany; Azimifar@biochem.mpg.de).

Despite enormous progress in MS based proteomics, accurate quantification of in vivo proteomic and phosphoproteomic changes such as those in liver remains a challenge. We developed a new strategy which we termed as a “spike-in” SILAC methodology that employs SILAC based quantitation but uses phosphopeptides derived from a mouse liver cell line as an internal standard for in vivo measurements.
We focused on optimizing all the steps in the method, ranging from sample preparation, to SILAC quantitation. Mouse liver samples were processed by filter aided sample preparation (FASP) wherein the proteins are extracted under denaturing conditions and then placed on a filter for the digestion. This simple method enables digestion of protein in an unbiased way and membrane proteins are digested under conditions previously applied only to soluble proteins. We achieved technical progress through Q Exactive, a novel Orbitrap mass spectrometer, and nano-ultra-high pressure liquid chromatography system using optimized home-made 50 cm long columns.
Our spike in SILAC approach has been successfully adapted to primary hepatocytes as well as mouse liver. This approach enabled identification of about 15,000 class I phosphosites and quantitative comparison of 10,000 phosphosites in mouse liver as a function of insulin treatment by high resolution MS. The spike-in SILAC technique can be used to study different signaling pathways in primary hepatocytes and mouse liver under normal or altered physiological conditions.

Filename: 2012_Poster_Azimifar.pdf

Format: PDF document

Size: 1.04 MB

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[S. Babak Azimifar] [Matthias Mann]

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Cell and Liver
Views: 2042    Downloads: 3
  • Created: 28th Nov 2012 at 08:35
  • Last updated: 28th Nov 2012 at 09:28
  • Last used: 29th Nov 2020 at 17:38

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