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.
| 元の言語 | 英語 |
|---|---|
| ページ（範囲） | 1457-1473 |
| ページ数 | 17 |
| ジャーナル | Proteomics |
| 巻 | 16 |
| 発行部数 | 10 |
| DOI | |
| 出版物ステータス | 出版済み - 5 1 2016 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
これを引用
A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards. / Kito, Keiji; Okada, Mitsuhiro; Ishibashi, Yuko; Okada, Satoshi; Ito, Takashi.
：: Proteomics, 巻 16, 番号 10, 01.05.2016, p. 1457-1473.研究成果: ジャーナルへの寄稿 › 記事
}
TY - JOUR
T1 - A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards
AU - Kito, Keiji
AU - Okada, Mitsuhiro
AU - Ishibashi, Yuko
AU - Okada, Satoshi
AU - Ito, Takashi
PY - 2016/5/1
Y1 - 2016/5/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=84964931417&partnerID=8YFLogxK
U2 - 10.1002/pmic.201500414
DO - 10.1002/pmic.201500414
M3 - Article
C2 - 27030420
AN - SCOPUS:84964931417
VL - 16
SP - 1457
EP - 1473
JO - Proteomics
JF - Proteomics
SN - 1615-9853
IS - 10
ER -