High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics
In this project we report 133 biologically distinct mouse liver cell (Hepa 1-6 and FL83B) and mouse tissue (brain, kidney, and liver) phosphoproteomes prepared using a newly developed scalable, single-run phosphoproteomics platform, using titanium dioxide (TiO2) based phosphopeptide enrichment and performed in 96-well format. We combine this technology with in situ hepatic stimulation, to generate time-resolved maps of insulin signaling in the mouse liver.
Sample Processing Protocol
Samples were processed according to the protocol described in detail in the Supplementary Methods provided with the associated manuscript. Briefly, protein was digested in a TFE-based buffer directly compatible with downstream phosphopeptide enrichment. Phosphopeptides were enriched in a single-step, 96-well plate format, followed by single-run LC-MS/MS analysis as described in the manuscript.
Data Processing Protocol
Raw mass spectrometry data were processed using MaxQuant version 18.104.22.168, with FDR < 0.01 at the level of proteins, peptides and modification sites. Proteins and peptides (minimum six amino acids) were identified using a target-decoy approach with a reversed database, using the Andromeda search engine integrated into the MaxQuant environment. Searches were performed against the Mouse UniProt FASTA database (September 2014) containing 51,573 entries. Quantification of peptides and proteins was performed by MaxQuant, match between runs was enabled with a matching time window of 1 min.
Humphrey SJ, Azimifar SB, Mann M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat Biotechnol. 2015 Aug 17 PubMed: 26280412