Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule.
We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3.
Nakanishi
Tsuyoshi
T
Life Science Laboratory, Analytical & Measuring Instruments Division, Shimadzu Corp., 1, Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604-8511, Japan.
Ando
Eiji
E
Furuta
Masaru
M
Kinoshita
Eiji
E
Kinoshita-Kikuta
Emiko
E
Koike
Tohru
T
Tsunasawa
Susumu
S
Nishimura
Osamu
O
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007281:Ink; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D015032:Zinc / Q000378:metabolism
D008565:Membrane Proteins; D010710:Phosphates; D010748:Phosphopeptides; D010750:Phosphoproteins; D015032:Zinc