Selective enrichment in phosphopeptides for the identification of phosphorylated mitochondrial proteins.
In vivo protein phosphorylation is a reversible and dynamic process controlled by protein kinases and phosphatases for the addition and removal of the phosphate group to serine, threonine, and tyrosine residues. In addition to other regulation events, increasing evidence indicates that reversible protein phosphorylation plays an important role in regulating mitochondrial function. Detecting changes in the state of protein phosphorylation is a difficult task since phosphorylation on a specific protein is typically transient and usually presents in substoichiometric concentration. Moreover, what makes mass spectrometry (MS)-based phosphopeptide analysis difficult is that ionization efficiency of phosphopeptides is lower than their nonphosphorylated analogues. Different strategies have been proposed for the selective enrichment of low abundance phosphoproteins and phosphopeptides present in biological samples. As a result of recent advances, phosphoproteomics has become one of the most rapidly developing areas of proteomics. In the last few years, a number of studies have focused on the analysis of phosphoproteome purified from yeast, liver and heart mitochondria, as well as on the identification of endogenously phosphorylated subunits of mitochondrial oxidative phosphorylation system complexes. This chapter describes methods for the selective enrichment of phosphoserine, phosphothreonine, and phosphotyrosine containing peptides and proteins and phosphate-specific MS strategy generally applicable to phosphoprotein analysis but focusing specifically on mitochondrial samples.
Istituto di Biochimica delle Proteine-Consiglio Nazionale delle Ricerche, Naples, Italy.
Gabriella
G
Pocsfalvi
D000818:Animals; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000302:isolation & purification; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000302:isolation & purification; D010769:Phosphothreonine / Q000032:analysis / Q000302:isolation & purification; D019000:Phosphotyrosine / Q000032:analysis / Q000302:isolation & purification
D024101:Mitochondrial Proteins; D010748:Phosphopeptides; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine