| Autor: |
Pinho JPC; Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E Fowler Ave, ISA 2015, Tampa, FL, 33620, USA., Bell-Temin H; Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA., Liu B; Department of Pharmacodynamics, University of Florida, 1345 Center Drive, Box 100487, Gainesville, FL, 32610, USA. liu@cop.ufl.edu., Stevens SM Jr; Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E Fowler Ave, ISA 2015, Tampa, FL, 33620, USA. smstevens@usf.edu. |
| Abstrakt: |
Stable isotope labeling by amino acids in cell culture (SILAC) is a versatile mass spectrometry-based proteomic approach that can achieve accurate relative protein quantitation on a global scale. In this approach, proteins are labeled while being synthesized by the cell due to the presence of certain amino acids exclusively as heavier mass analogs than their regular (light) counterparts. This differential labeling allows for the identification of heavy and light forms of each peptide corresponding to two or more different experimental groups upon mass spectrometric analysis, the intensities of which reflect their abundance in the sample analyzed. Relative quantitation is straightforward when SILAC labeling efficiency is high (>99%) and the same cell proteome is used as the quantitation reference, which is typically the case for immortalized cell lines. However, the SILAC methodology for the proteomic analysis of primary cells isolated after in vivo experimentation is more challenging given the low labeling efficiency that would be achieved post-isolation. Alternatively, a stable-isotope-labeled cell line representing the cell type can be used as an internal standard (spike-in SILAC); however, adequate representation of the primary cell proteome with the stable-isotope-labeled internal standard may limit overall protein quantitation, especially for cell types that exhibit a broad range of phenotypes such as microglia, the resident immune cells in the brain. Here, we present a way to circumvent this limitation by combining multiple phenotypes of a single-cell type (the immortalized mouse BV2 microglial cell line) into a single spike-in standard using primary mouse microglia as our model system. We describe the preparation of media, incorporation of labels, induction of four different activation states (plus resting), isolation of primary microglia from adult mice brains, preparation of lysates for analysis, and general guidelines for data processing. |
