Mass Spectrometry

Proteomics reveals the global drafts of proteins in biological systems, with the recent discovery and identification of 92% proteins encoded by human genome. The deeper insights of proteins in living systems call for the compliment with dynamic changes in localization, abundance, and interactions of proteins. Trials have been made to perform in situ and dynamic profiling of proteome/peptidome at the subcellular range by the combination of pre-fraction of subcellular components in vitro with isotope labeling at different time points and quantitative proteomics. Other than utilizing in vitro protein collection and labeling strategies, proteins obtained from physiological ambient without the chemical derivatizations such as in situ extraction with live cells are preferred for proteomic investigation containing high temporal and spatial resolution. 

To achieve the in situ and timing harvest of intracellular proteome and peptidome from a living system, the extraction probes should meet following essential requirements: 1) biocompatible with live cells; 2) suitable for both general or selective extraction of intracellular proteome/peptidome; and capatible to harvest compounds at 3) designated intracellular loci, and at 4) appointed time period. So far, the conventional proteome/peptidome sampling methods including micro extraction, electroporation could hardly satisfy all the above requirements. Nanoprobes, owning high biocompatibility and abilities for subcellular targeting and controllable intracellular transportation, hint promising potentials to extract cellular components from live cells in situ. In earlier attempts, heparin sulfate proteoglycans (HSPGs) antibody functionalized magnetic particles were employed to collect targeted endocytotic vesicles for proteomic analysis of HSPGs induced endocytosis,[5] methotrexate (MTX) linked dendrimers were applied in proteomic discovery of drug substrates from live cells,[6] and magnetic carbon nanotubes were used for harvest nucleic acid associated proteins in situ coupled with proteomic exploration[7]. However, nanocarriers for dynamic extraction of proteins are not satisfiable yet, since the proteins would be adsorbed by nanocarriers as soon as the cellular internalization. In this paper, a photo-switchable mesoporous silica nanocarrier (MSN with coumarin gates, MSNcg) was constructed as a general nano platform to collect cytosol polypeptides from live HeLa cells for the in situ and timing proteomic investigation.