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Characterization of heat shock protein 27 phosphorylation sites in renal cell carcinoma
Author(s) -
Tremolada Lucia,
Magni Fulvio,
Valsecchi Cristina,
Sarto Cecilia,
Mocarelli Paolo,
Perego Roberto,
Cordani Nicoletta,
Favini Paolo,
Galli Kienle Marzia,
Sanchez JeanCharles,
Hochstrasser Denis F.,
Corthals Garry L.
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401134
Subject(s) - hsp27 , phosphorylation , renal cell carcinoma , heat shock protein , serine , microbiology and biotechnology , tandem mass spectrometry , chemistry , protein phosphorylation , phosphoprotein , peptide mass fingerprinting , proteomics , gel electrophoresis , immunostaining , biology , biochemistry , mass spectrometry , immunohistochemistry , chromatography , hsp70 , protein kinase a , pathology , immunology , medicine , gene
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.