Presentation by Ákos Végvári at the single-cell proteomics conference [ Ссылка ] [ Ссылка ]
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Today’s proteomics affords identification and quantification of over 10,000 proteins in bulk biological samples. However, the large dynamic range of protein concentrations together with the limited sensitivity of current mass spectrometers do not permit the analysis of full cellular proteomes in mammalian samples. Additionally, capturing even the abundant part of the proteome of a single mammalian cell (ca 0.2 ng of protein) turned out to be quite challenging.
The single cell proteomic strategy recently introduced by the Boston group is based on the use of isobaric tandem mass tag (TMT) together with the “carrier proteome” (CP). This strategy seems to have for the first time allowed one to identify and quantify hundreds of proteins obtained from isolated cells and probe the heterogeneity of their proteomes. However, the magic of this approach remained largely unexplained; in particular, the CP’s role in boosting the sensitivity. Questions also remain about the quantitative nature of single cell proteomics.
Hence, we have designed experiments to investigate the correlations of the measured fold changes (FCs) of proteins in single cells with the corresponding FCs in bulk samples. Two TMT-based methods, with and without CP, were compared. The samples were obtained in four replicates from RKO cancer cells treated with methotrexate (MTX) at IC50 level for 48 h. Experiments were performed on a brand-new Orbitrap Lumos using a range of protein concentrations obtained by serial dilution.
In the CP-free method, 8, 40, 200 and 1000 ng samples were injected on column (corresponding to 5, 25, 125 and 625 cells in each TMT channel, respectively), resulting in linearly decreased numbers of identified proteins and peptides from 200 ng to 8 ng loaded, in proportion with the number of acquired MS2 spectra. The quantitative similarity with the bulk sample (1 µg loaded), measured as Pearson’s correlation between the FCs, was also decreased, as expected, but maintained significance to the lowest loaded level for both protein and peptide FCs. In addition, the significantly upregulated MTX target protein (DHFR) used as an indicator of the analytical performance, showed a gradually decreasing rank with lowering of the amount injected (ranked 8 out of 3728 in 1000 ng and 20 out of 3045 in 200 ng loaded), not being identified for 40 ng and 8 ng sample loads (1347 and 199 protein IDs, respectively).
For the CP-based method with a 200-fold enhanced load in the TMT-Zero channel, the numbers of identified proteins were always higher for the same sample amounts in the TMT-10 channels (“single-cell channels”) in comparison with the CP-free method. At a single cell level, the CP-based method quantified 774 proteins, while the CP-free method produced no identifications. On average, it was found that the 200-fold CP offers an order of magnitude improvement in detection threshold for “single-cell channels”. The quantitative aspect, i.e. statistically significant correlation with bulk proteome FCs, was preserved in the CP-based method down to the level of single cells.
Our results indicate that the FC-based approach has a great analytical potential. However, further improvements in methodology are desirable to obtain reliable quantification for over 1000 proteins at a single cell level.
