Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated proteins detected by immunostaining.
We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.
Nakanishi
Tsuyoshi
T
Life Science Laboratory, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
Ando
Eiji
E
Furuta
Masaru
M
Tsunasawa
Susumu
S
Nishimura
Osamu
O
D045163:Antibodies, Phospho-Specific / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D002294:Carcinoma, Squamous Cell; D002457:Cell Extracts / Q000737:chemistry*; D045744:Cell Line, Tumor / Q000737:chemistry / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology; D019000:Phosphotyrosine / Q000032:analysis* / Q000276:immunology; D011506:Proteins / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D045163:Antibodies, Phospho-Specific; D002457:Cell Extracts; D010750:Phosphoproteins; D011506:Proteins; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor