Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines.
Neuroendocrine tumors (NET) often develop asymptomatically and are detected at a late stage. Currently, there exist certain markers of NET that occur only in the advanced stages of the disease. Still, there is need to develop markers specific of the early stage of cancer development. Nevertheless, biomarkers are mostly low‑abundant proteins and require separation from complex protein mixtures, which remains a major challenge. The goal of the present study was to optimize one‑dimensional‑polyacrylamide gel electrophoresis (1D‑PAGE) for separation and comparison of protein composition from neuroendocrine tumor samples. 1D‑PAGE was optimized by modification of the gel concentration and by comparison of different gel staining protocols. In addition, several steps prior to electrophoresis were carried out to purify and preliminarily reduce the complexity of the sample. The results of these optimization steps indicated that use of an albumin removal kit can considerably decrease the amount of albumin in the samples, thereby allowing to detect proteins of low abundance. Optimal separation of the sample was obtained using a 12% polyacrylamide gel. Furthermore, the use of silver staining allowed detection of proteins at nanogram levels, whereas for Coomassie Brilliant Blue staining, the detection limit was 10 times higher. Optimization of the sample preparation workflow and parameters of the electrophoretic separation allowed to reduce the complexity of the studied material and facilitated further identification of proteins of low abundance in the sample. This study demonstrated that analysis of the secreted proteome of NET cells by 1D‑PAGE is a simple and suitable tool for the identification of potential NET protein biomarkers.
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gdańsk 80‑416, Poland.
Natalia
N
Miękus
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gdańsk 80‑416, Poland.
Ilona
I
Olędzka
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gdańsk 80‑416, Poland.
Alina
A
Plenis
Department of Endocrinology and Internal Medicine, Medical University of Gdańsk, Gdańsk 80‑210, Poland.
Zofia
Z
Woźniak
Department of Endocrinology and Internal Medicine, Medical University of Gdańsk, Gdańsk 80‑210, Poland.
Anna
A
Lewczuk
Department of Medical Biotechnology, Laboratory of Cell Biology, Medical University of Gdańsk, Gdańsk 80‑210, Poland.
Patrycja
P
Koszałka
Bank of Frozen Tissues and Genetic Specimens, Medical University of Gdańsk, Gdańsk 80‑210, Poland.
Barbara
B
Seroczyńska
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gdańsk 80‑416, Poland.
Tomasz
T
Bączek
D000096:Acetone / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000432:Methanol / Q000737:chemistry; D018358:Neuroendocrine Tumors / Q000378:metabolism* / Q000473:pathology; D020543:Proteome / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D014407:Tumor Cells, Cultured
D015415:Biomarkers; D020543:Proteome; D000096:Acetone; D000432:Methanol