期刊
JOURNAL OF PROTEOME RESEARCH
卷 7, 期 6, 页码 2215-2221出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr700575m
关键词
comparative phosphoproteomics; immobilized metal-ion affinity chromatography (IMAC); mass spectrometry; 50 mu m ID reversed phase column; 1D SDS-PAGE; non-metal nano-HPLC
资金
- NCRR NIH HHS [P41 RR018522-05, P41 RR018522, RR018522] Funding Source: Medline
- NINDS NIH HHS [NS031221, R01 NS031221] Funding Source: Medline
Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 Uniquely identified peptides per data set) affords the optimal collection of peptide information.
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