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High-throughput and sensitive non-targeted proteomics: a method development and its application for characterizing non-small cell lung cancer
Mass spectrometry-based proteomics is an indispensable tool for molecular, cellular biology and for the emerging field of clinical medicine. It is recently required to analyze samples in a high throughput fashion as well as in a sensitive fashion.
StageTips have advantages on its high sample recovery rate, throughput and cost as a device for sample purification, enrichment and fractionation1). However, StageTip-based fractionation for large-scale phosphoproteome analysis has not been succeeded yet. We found that acid-based step-wise elution from C18-SCX StageTips works well for peptides/phosphopeptide fractionation compared with traditional salt-based elution method.
Furthermore, we succeeded to enhance throughput by employing Tandem Mass Tag (TMT) labeling technology and C18-SCX StageTip fractionation method (Figure 1).
Recent advances in genome sequencing technology have revealed that non-small-cell lung cancer (NSCLC) has subsets with specific genetic alterations that are critical to the growth and survival of these cancers. In this study, we tried to characterize NSCLC cell lines by proteome and phosphoproteome analysis using our developed TMT-StageTip fractionation system to access how proteome analysis can discriminate the differences of drug (erlotinib)-sensitive and drug-resistant NSCLC cell lines.