Characterization of heat shock protein 27 phosphorylation sites in renal cell carcinoma.
The phosphorylation of heat shock protein 27 (HSP27) occurs differently in human renal cell carcinoma (RCC) compared to homologous normal kidney tissue. Two-dimensional electrophoresis was used to separate and visualize HSP27, via immunostaining with anti-HSP27 antibody, in tumor and normal renal samples, obtained after surgery resection from patients with RCC. The mean number of protein species was 21 in RCC and 15 in normal tissues. Selected spots were in-gel digested with trypsin, extracted and analyzed by microcapillary liquid chromatography (LC) electrospray ionization tandem mass spectrometry to confirm HSP27 protein identity and reveal phosphorylation sites. Loss of phosphopeptides due to extensive plumbing and/or metal components in automated LC-systems was limited by manual loading of samples directly onto the LC system using a homemade pressure vessel. Mass spectrometry (MS) analysis revealed that in three of the HSP27 protein species phosphorylation occurred at Serine 15 and in five at Serine 82 in a different pattern. The phosphorylation of Serine 15 and 82 was also investigated by immunohistochemistry on tissue sections. The data obtained using anti-HSP27Serine82phos-antibody are consistent with MS results, while the variance between results achieved by anti-HSP27Serine15phos-antibody and by MS is probably due to the low specificity of the antibody. Knowledge of the diversity and modulation of HSP27 phosphorylation protein species might represent useful markers involved in the differentiation of RCC.
Tremolada
Lucia
L
Department of Experimental, Environmental Medicine and Medical Biotechnologies, University of Milano-Bicocca, Monza, Italy.
Magni
Fulvio
F
Valsecchi
Cristina
C
Sarto
Cecilia
C
Mocarelli
Paolo
P
Perego
Roberto
R
Cordani
Nicoletta
N
Favini
Paolo
P
Galli Kienle
Marzia
M
Sanchez
Jean-Charles
JC
Hochstrasser
Denis F
DF
Corthals
Garry L
GL
D002292:Carcinoma, Renal Cell; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D010748:Phosphopeptides; D012694:Serine