抄録
The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy of hierarchical use of peptide-concatenated standards (PCSs) to quantify more proteins over a wider dynamic range. Multiple primary PCSs were used for quantification of many target proteins. Unique "ID-tag peptides" were introduced into individual primary PCSs, allowing us to monitor the exact amounts of individual PCSs using a "secondary PCS" in which all "ID-tag peptides" were concatenated. Furthermore, we varied the copy number of the "ID-tag peptide" in each PCS according to a range of expression levels of target proteins. This strategy accomplished absolute quantification over a wider range than that of the measured ratios. The quantified abundance of budding yeast proteins showed a high reproducibility for replicate analyses and similar copy numbers per cell for ribosomal proteins, demonstrating the accuracy and precision of this strategy. A comparison with the absolute abundance of transcripts clearly indicated different post-transcriptional regulation of expression for specific functional groups. Thus, the approach presented here is a faithful method for the absolute quantification of proteomes and provides insights into biological mechanisms, including the regulation of expressed protein abundance.
本文言語 | 英語 |
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ページ(範囲) | 1457-1473 |
ページ数 | 17 |
ジャーナル | Proteomics |
巻 | 16 |
号 | 10 |
DOI | |
出版ステータス | 出版済み - 5月 1 2016 |
!!!All Science Journal Classification (ASJC) codes
- 生化学
- 分子生物学