TY - JOUR
T1 - A multidimensional approach to an in-depth proteomics analysis of transcriptional regulators in neuroblastoma cells
AU - Li, Qing
AU - Jain, Mohit Raja
AU - Chen, Wei
AU - Li, Hong
N1 - Funding Information:
The project described was supported in part by a grant P30NS046593 from the National Institute of Neurological Disorders and Stroke . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. The authors report no conflict of interest.
PY - 2013/6/5
Y1 - 2013/6/5
N2 - The dynamic regulation of transcriptional events is fundamental to many aspects of neuronal cell functions. However, proteomics methods have not been routinely used in global neuroproteomics analyses of transcriptional regulators because they are much less abundant than the "house-keeping" proteins in cells and tissues. Recent improvements in both biochemical preparations of nuclear proteins and detection sensitivities of proteomics technologies have made the global analysis of nuclear transcriptional regulators possible. We report here an optimised neuroproteomic method for the analysis of transcriptional regulators in the nuclear extracts of SHSY-5Y neuroblastoma cells by combining an improved nuclear protein extraction procedure with multidimensional peptide separation approaches. We found that rigorous removal of cytoplasmic proteins and solubilisation of DNA-associated proteins improved the number of nuclear proteins identified. Furthermore, we discovered that multidimensional peptide separations by either strong cation exchange (SCX) chromatography or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) analysis detected more than 1800 nuclear proteins, which constitutes one of the largest datasets of nuclear proteins reported for a neuronal cell. Thus, in-depth analysis of transcriptional regulators for studying neurological diseases are increasingly feasible.
AB - The dynamic regulation of transcriptional events is fundamental to many aspects of neuronal cell functions. However, proteomics methods have not been routinely used in global neuroproteomics analyses of transcriptional regulators because they are much less abundant than the "house-keeping" proteins in cells and tissues. Recent improvements in both biochemical preparations of nuclear proteins and detection sensitivities of proteomics technologies have made the global analysis of nuclear transcriptional regulators possible. We report here an optimised neuroproteomic method for the analysis of transcriptional regulators in the nuclear extracts of SHSY-5Y neuroblastoma cells by combining an improved nuclear protein extraction procedure with multidimensional peptide separation approaches. We found that rigorous removal of cytoplasmic proteins and solubilisation of DNA-associated proteins improved the number of nuclear proteins identified. Furthermore, we discovered that multidimensional peptide separations by either strong cation exchange (SCX) chromatography or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) analysis detected more than 1800 nuclear proteins, which constitutes one of the largest datasets of nuclear proteins reported for a neuronal cell. Thus, in-depth analysis of transcriptional regulators for studying neurological diseases are increasingly feasible.
KW - Electrostatic repulsion-hydrophilic interaction chromatographic (ERLIC)
KW - Neuroblastoma
KW - Neuroproteomics
KW - SHSY-5Y cell
KW - Strong cation exchange (SCX)
KW - Transcription factor
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U2 - 10.1016/j.jneumeth.2013.03.016
DO - 10.1016/j.jneumeth.2013.03.016
M3 - Article
C2 - 23558336
AN - SCOPUS:84878238118
SN - 0165-0270
VL - 216
SP - 118
EP - 127
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -