Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins.
An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.
González
Luis Javier
LJ
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Castellanos-Serra
Lila
L
Badock
Volker
V
Díaz
Maylín
M
Moro
Alejandro
A
Perea
Silvio
S
Santos
Alicia
A
Paz-Lago
Dalila
D
Otto
Albrecht
A
Müller
Eva-Christina
EC
Kostka
Susanne
S
Wittmann-Liebold
Brigitte
B
Padrón
Gabriel
G
D000595:Amino Acid Sequence; D018288:Carcinoma, Small Cell / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012834:Silver; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012834:Silver