High spatial and temporal resolution cell manipulation techniques in microchannels

The advent of microfluidics has enabled thorough control of cell manipulation experiments in so called lab on chips. Lab on chips foster the integration of actuation and detection systems, and require minute sample and reagent amounts. Typically employed microfluidic structures have similar dimensions as cells, enabling precise spatial and temporal control of individual cells and their local environments. Several strategies for high spatio-temporal control of cells in microfluidics have been reported in recent years, namely methods relying on careful design of the microfluidic structures (e.g. pinched flow), by integration of actuators (e.g. electrodes or magnets for dielectro-, acousto- and magneto-phoresis), or integrations thereof. This review presents the recent developments of cell experiments in microfluidics divided into two parts: an introduction to spatial control of cells in microchannels followed by special emphasis in the high temporal control of cell-stimulus reaction and quenching. In the end, the present state of the art is discussed in line with future perspectives and challenges for translating these devices into routine applications

Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin

Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, nonunique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as Glu-C, Asp-N, Lys-N, Arg-C, LysargiNase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1–5 min. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8363 vs 6807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14 628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either Asp-N, Arg-C, Glu-C, Lys-C, or Lys-N. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsindespite broad specificity and fast digestion that may increase technical variance