A strategy for absolute proteome quantification with mass spectrometry by hierarchical use of peptide-concatenated standards

作者：Kito Keiji^*; Okada Mitsuhiro; Ishibashi Yuko; Okada Satoshi; Ito Takashi

来源：Proteomics, 2016, 16(10): 1457-1473.

DOI：10.1002/pmic.201500414

• Absolute quantification
• Budding yeast
• Mass spectrometry
• Peptide-concatenated standard (PCS)
• Technology

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.

• 出版日期2016-5