Combining Data Independent Acquisition with Spike-in SILAC (DIA-SiS) Improves Proteome Coverage and Quantification.

Data Independent Acquisition (DIA) is increasingly preferred over Data Dependent Acquisition (DDA) due to its higher throughput and fewer missing values. Whereas DDA often utilizes stable isotope labeling to improve quantification, DIA mostly relies on label-free approaches. Efforts to integrate DIA with isotope labeling include chemical methods like mTRAQ and dimethyl labeling, which, while effective, complicate sample preparation. Stable isotope labeling by amino acids in cell culture (SILAC) achieves high labeling efficiency through the metabolic incorporation of heavy labels into proteins in vivo. However, the need for metabolic incorporation limits the direct use in clinical scenarios and certain high-throughput experiments. Spike-in SILAC methods utilize an externally generated heavy sample as an internal reference, enabling SILAC-based quantification even for samples that cannot be directly labeled. Here, we combine DIA with spike-in SILAC (DIA-SiS), leveraging the robust quantification of SILAC without the complexities associated with chemical labeling. We developed DIA-SiS and rigorously assessed its performance with mixed-species benchmark samples on bulk and single cell-like amount level. We demonstrate that DIA-SiS substantially improves proteome coverage and quantification compared to label-free approaches and reduces incorrectly quantified proteins. Additionally, DIA-SiS proves effective in analyzing proteins in low-input formalin-fixed paraffin-embedded (FFPE) tissue sections. DIA-SiS combines the precision of stable isotope-based quantification with the simplicity of label-free sample preparation, facilitating simple, accurate and comprehensive proteome profiling.

High-Throughput GLP-Capable Target Cell Visualization Assay for Measuring Cell-Mediated Cytotoxicity.

One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.