Letter | Published:

Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards

Abstract

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples1,2. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97–98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.

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Acknowledgements

This work was supported by funds from New Energy and Industrial Technology Development Organization, Japan (NEDO).

Author information

Competing interests

The authors declare no competing financial interests.

Correspondence to Yoshiya Oda.

Supplementary information

  1. Supplementary Table 1

    Reproducibility of quantitative procedures using CDIT (including from affinity purification to MS analysis) (PDF 62 kb)

  2. Supplementary Table 2

    Linearity and its reproducibility of quantitative procedures using CDIT (PDF 65 kb)

  3. Supplementary Table 3

    Dependence of MS signal ratios (heavy/light) on the mixing ratios (brain and Neuro2A) (PDF 16 kb)

  4. Supplementary Table 4

    Protein list quantified by CDIT and ICAT (PDF 112 kb)

  5. Supplementary Table 5

    Same sequence pair versus different sequence pair as internal standard (PDF 18 kb)

  6. Supplementary Table 6

    Number of quantified proteins in mouse hippocampus (PDF 92 kb)

  7. Supplementary Table 7

    Hippocampal proteins showing more than 2-fold change between kainate-treated and untreated mice (PDF 21 kb)

  8. Supplementary Table 8

    Absolute amount of proteins in Neuro2A cells and mouse brains (PDF 16 kb)

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Further reading

Figure 1: Strategy of quantitative mouse brain proteomics using CDITs.
Figure 2: Validation data of CDIT strategy.
Figure 3: Total scheme for absolute quantification using CDITs.