P66-T Efficient Identification of Phosphorylation Sites in AMPK: Autoradiographical Visualization of Microfractionated Phosphopeptides using 384-PAC MALDI Plates

摘要

Reversible phosphorylation of proteins plays an important role in many cellular processes. To investigate the functions of kinases and their substrates in vivo and in vitro—for example, production of phospho-specific antibodies or generation of phospho-site mutants—the precise identification of phosphorylation sites is mandatory.A rapid and efficient workflow has been elaborated using prespotted 384 alpha-cyano-4-hydroxycinnamic acid MALDI targets as fractionation units; these are ideally suited to visualize ³²P-labeled phosphopeptides. Ingel-tryptic digests of phosphorylated AMP-activated protein kinase (either by protein kinase B or autophosphorylation in the presence of γ³²P-ATP) were separated on an Agilent 1100 capillary LC system coupled to a microfractionation unit. The tryptic peptides were deposited in 1-μL portions onto a PAC-MALDI target with 384 prespotted matrix preparations as well as calibrant spots. In order to visualize ³²P-labeled peptides, the PAC plate was then exposed to a Kodak MR autoradiography film. Accordingly, the ³²P-positive spots were screened for phosphorylation sites by performing MS and MSMS with the Ultraflex II TOF/ TOF.This new workflow for phosphorylation site identification eliminates tedious manual handling of digested samples, thus reducing the initial sample material dramatically.Furthermore, we observe that the separated peptides can be stored on the PAC plate for several weeks without any significant loss of resolution and signal intensity.