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bioRxiv · 10.1101/2023.06.05.543682

One thousand samples per day capillary-flow LC/MS/MS for high-speed, high-sensitivity and in-depth proteomics

Tomioka, A.·Tomioka, R.·Mori, I.·Isobe, Y.·Arita, M.·Imami, K.·Kanao, E.·Ogata, K.·Ishihama, Y.
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Abstract

We developed a capillary-flow LC/MS/MS system with ultrahigh speed, enabling a throughput of 1,000 samples per day while maintaining high sensitivity and depth of analysis. In targeted LC/MS mode, 36 endogenous phosphopeptides in HeLa cells, including EphA2- derived phosphopeptide isomers, were successfully quantified with high selectivity and linearity by combining ion mobility separation. When 500 ng of HeLa cell digest was measured 100 times repeatedly in data-dependent acquisition mode, the coefficient of variation of retention time, peak intensity and number of identified peptides were on average 3.4%, 19.8%, and 6.0%, respectively. In data-independent acquisition mode, this system achieved the identification and quantification of 3,139 protein groups from a 100 ng HeLa cell digest and 2,145 protein groups from a sample of only 10 ng. The coefficient of variation of protein commonly quantified in the triplicate analysis ranged from 12 to 24% for HeLa digest samples ranging from 10 to 1000 ng. Finally, we applied this high-speed system to the spatial proteomics of the mouse brain, and succeeded in capturing the proteome distribution along a 96-sectioned brain structure in 135 minutes. This is the first LC/MS/MS system to achieve both more than 500 samples per day and more than 3000 identified protein groups ID with less than 100 ng human cultured cells simultaneously.

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Tomioka, A., Tomioka, R., Mori, I., Isobe, Y., Arita, M., Imami, K., Kanao, E., Ogata, K., Ishihama, Y.. 2023-06-07. One thousand samples per day capillary-flow LC/MS/MS for high-speed, high-sensitivity and in-depth proteomics. https://doi.org/10.1101/2023.06.05.543682

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