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Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins.
Electrophoresis. 2000 Jul; 21(13):2622-36.E

Abstract

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.

Authors+Show Affiliations

Proteinstrukturlabor, Institut für Physiologische Chemie, Ruhr-Universität Bochum, Germany.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article

Language

eng

PubMed ID

10949139

Citation

Marcus, K, et al. "Identification of Platelet Proteins Separated By Two-dimensional Gel Electrophoresis and Analyzed By Matrix Assisted Laser Desorption/ionization-time of Flight-mass Spectrometry and Detection of Tyrosine-phosphorylated Proteins." Electrophoresis, vol. 21, no. 13, 2000, pp. 2622-36.
Marcus K, Immler D, Sternberger J, et al. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis. 2000;21(13):2622-36.
Marcus, K., Immler, D., Sternberger, J., & Meyer, H. E. (2000). Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. Electrophoresis, 21(13), 2622-36.
Marcus K, et al. Identification of Platelet Proteins Separated By Two-dimensional Gel Electrophoresis and Analyzed By Matrix Assisted Laser Desorption/ionization-time of Flight-mass Spectrometry and Detection of Tyrosine-phosphorylated Proteins. Electrophoresis. 2000;21(13):2622-36. PubMed PMID: 10949139.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins. AU - Marcus,K, AU - Immler,D, AU - Sternberger,J, AU - Meyer,H E, PY - 2000/8/19/pubmed PY - 2001/2/28/medline PY - 2000/8/19/entrez SP - 2622 EP - 36 JF - Electrophoresis JO - Electrophoresis VL - 21 IS - 13 N2 - Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/10949139/Identification_of_platelet_proteins_separated_by_two_dimensional_gel_electrophoresis_and_analyzed_by_matrix_assisted_laser_desorption/ionization_time_of_flight_mass_spectrometry_and_detection_of_tyrosine_phosphorylated_proteins_ L2 - https://doi.org/10.1002/1522-2683(20000701)21:13<2622::AID-ELPS2622>3.0.CO;2-3 DB - PRIME DP - Unbound Medicine ER -