Application Note 17

The Use of Adenosine 5’-Triphosphate (γ-P¹⁸O₄, 97%) for the Unambiguous Identification of Phosphopeptides

Ming Zhou, Zhaojing Meng, and Timothy D. Veenstra

SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702-1201 USA

Phosphorylation is arguably the key signaling event that occurs within cells controlling processes such as metabolism, growth, proliferation, motility, differentiation, and division. Current data predicts that approximately 2% of the human genome encodes for kinases represented just over 500 individual protein species.² The importance of kinases (and phosphorylation) is underscored by the fact that 30% of all drug-discovery efforts target this class of proteins. Determining kinase specificity has long been a key research area in molecular biology and is important to a variety of fields, including cancer research, cell and developmental biology, and drug discovery. 

Identifying phosphorylated residues within proteins has historically been accomplished using in vitro studies in which a kinase is mixed with a potential substrate in the presence of ³²P-labeled adenosine triphosphate ATP (γ-³²P). The reaction mixture is then digested into peptides that are analyzed using scintillation counting. Radioactive peptides are then sequenced to reveal the identity of the phosphopeptide. While radioactive isotopes have been used quite successfully for a number of years, they have a number of drawbacks that are not simply limited to safety and regulatory issues. To overcome the need for radioactivity, investigators have turned to mass spectrometry (MS) for the identification of phosphopeptides. While MS data is extremely useful, sequence database search engines and statistical models for data validation are not optimized for the specific MS fragmentation properties exhibited by phosphopeptides. The result is a large, indeterminable rate of false-positive and false-negative values. 

In this note, we describe a stable isotope-labeling approach that provides unambiguous identification of phosphorylated peptides produced through in vitro kinase reactions. The method utilizes adenosine triphosphate in which four oxygen-¹⁶ atoms of the terminal phosphate group are substituted with oxygen-¹⁸ ATP (γ-P¹⁸O₄). In vitro kinase reactions were conducted using a 1:1 mixture of ATP (γ-P¹⁸O₄, 97%) and normal isotopic abundance ATP. Phosphopeptides produced during the reaction present themselves in the mass spectrum as peaks separated by 6.01 Da due to the presence of both normal and ¹⁸O-labeled phosphate groups.