Phosphoproteomic analysis of liver homogenates.
Regulation of protein function via reversible phosphorylation is an essential component of cell signaling. Our ability to understand complex phosphorylation networks in the physiological context of a whole organism or tissue remains limited. This is largely due to the technical challenge of isolating serine/threonine phosphorylated peptides from a tissue sample. In the present study, we developed a phosphoproteomic strategy to purify and identify phosphopeptides from a tissue sample by employing protein gel filtration, protein strong anion exchange and strong cation exchange (SCX) chromatography, peptide SCX chromatography, and TiO(2) affinity purification. By applying this strategy to the mass spectrometry-based analysis of rat liver homogenates, we were able to identify with high confidence and quantify over 4,000 unique phosphopeptides. Finally, the reproducibility of our methodology was demonstrated by its application to analysis of the mammalian Target of Rapamycin (mTOR) signaling pathways in liver samples obtained from rats in which hepatic mTOR was activated by refeeding following a period of fasting.
Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA.
Gokhan
G
Demirkan
Arthur R
AR
Salomon
Philip A
PA
Gruppuso
D000478:Alkylation; D000818:Animals; D002852:Chromatography, Ion Exchange; D007475:Ion Exchange Resins / Q000737:chemistry; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D007475:Ion Exchange Resins; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; C009495:titanium dioxide; D014025:Titanium; D014357:Trypsin