Localization of Proteins within a Cell or a Tissue
A major influence on the function of a protein is its distribution and localization within the cell. The number of methods for the analysis of protein subcellular localization in cells and tissues is large and diverse.
The most frequently used subcellular fractionation methods include differential centrifugation followed by MS analysis. These techniques are used for either the isolation of specific organelles or for protein correlation profiling (PCP). The major issue associated with any biochemical subcellular fractionation technique is the occurrence of cross-fraction contaminants, i.e. proteins redistribute to subcellular fractions they do not belong to. This issue can be caused for example by using a too harsh lysis buffer (non-physiological salt concentrations or inappropriate detergents, etc.).
An alternative method to visualize proteins directly within a cell or a tissue is MALDI-Imaging Mass Spectrometry (MALDI-IMS). With this method a thin tissue section is moved in two dimensions, while continuously mass spectra are recorded using MALDI-MS. The most critical and most difficult step in the MALDI-IMS workflow is the optimal preparation of the tissue sample itself, which must happen fast and at low temperatures, in order to prevent protein degradation and other artefacts. Once an optimally sliced tissue sample is on the surface of a conductive slide, the material must be coated with the correct amount of MALDI matrix in a highly homogenous way. This can be achieved using a specialized “MALDI Matrix Deposition Device”, which BayBioMS can provide to its customers (ImagePrepTM Bruker). MALDI-IMS has provided important contributions to the understanding of diseases and to obtain histological information in cancer research. However, MALDI-IMS in the context of proteomics remains challenging because of its limited sensitivity, poor ionization and matrix fragmentation effects, which make this technology biased towards the most abundant and rather small protein species in a tissue. A further challenge in using MALDI-IMS in the context of proteomics can be the final identification of the underlying protein and/or peptide species from the detected mass values.
The outcome of a subcellular fractionation analysis is a list of confidently identified proteins per subcellular fraction.
The outcome of a MALDI-IMS analysis is a visual tissue image with spatial indication of protein or peptide or mass-to-charge-value localization.