3.1. Sample Preparation
Tissue samples that are suitable for RPPA can be fresh, frozen, or fixed. So far it is a world-wide standard to use formalin-fixation and paraffin-embedded (FFPE) tissue samples for histopathological diagnosis. Huge archives of patient samples are available in the Institutes of Pathology. The advantage of FFPE-tissues compared to frozen tissue samples are multiple: (i) routine method of all Institutes of Pathology worldwide, (ii) broadly available, (iii) linkage of molecular data with clinical information, (iv) tissues of rare diseases are available, (v) cheap and (vi) easy storage of tissue blocks for many years or even decades at room temperature [34]. The problem of formalin-fixed tissues is that extensive cross-linking of macromolecules hinders the easy extraction of proteins. With the introduction of antigen retrieval techniques in the early 1990s in the pathology laboratories [35], IHC has become manifest as the only routinely used technique to analyze proteins in FFPE-tissue. This may change in the future due to the development of protocols allowing the extraction of full-length proteins from FFPE-tissues. Thus, extraction-based protein analysis is likely to complement IHC data. Histologic evaluation of the samples by a pathologist before protein extraction is needed to estimate the cellular composition. Often it is desired to analyze a specific subpopulation of cells in a tissue section, e.g., tumor cells. Tissue microdissection for cell enrichment can be performed either laser‑assisted or manually. The purification of pure tumor cell populations by laser-capture microdissection (LCM) has been efficiently coupled to RPPA for studying tumor biopsies and detection of clinically relevant information on the molecular characteristics of tumor cells [36]. The so‑called “guided protein extraction” using manual tissue microdissection is an alternative technology [37]. So far, several groups have reported different protocols for extractions of proteins from FFPE-tissues [34,38,39,40,41,42,43]. We recommend deparaffinization (e.g., with xylol) and rehydration with a graded series of alcohol (see also [38]). For protein extraction a heating step is crucial in all mentioned protocols. Additionally, Chu et al. propose an optional implementation of microwave or ultrasound to improve the reversal of protein-crosslinking and the penetration of buffer into the tissue [39]. Chung et al. use a combination of heat and pressure to obtain optimal protein extraction [40]. Most buffers used for protein extraction usually include detergents (e.g., 1%–2% SDS) for optimal solubilization of hydrophobic proteins. In our lab, we use Qproteome FFPE-Tissue Kit-Buffer (Qiagen, Hilden, Germany), because it is compatible with our antibodies and allows high quality protein extraction [34]. Furthermore, it does not interfere with Sypro Ruby staining which is necessary for measuring the amount of total protein printed on nitrocellulose-coated glass slides. It should be emphasized that different applications may require different extraction conditions and that samples or buffers with high viscosity are not suitable for certain printing platforms. With our protein extraction method, it could be shown that the HER2 score of breast cancer samples obtained by RPPA is of good concordance with the HER2 score obtained with IHC [44]. Beyond that, it could be demonstrated that data obtained from RPPA of cryo‑frozen samples were in agreement with data of RPPA analyzed FFPE samples, indicating that FFPE-tissues are a valuable source for protein level analysis by RPPA [45]. Although most investigators accept that proteins extracted from FFPE tissue are suitable for downstream proteomic analysis, including RPPA [46], the workflows for collecting specimens are not standardized between hospitals, or even within single institutions. Thus, the pre-analytical phase must be improved in order to develop more reliable biomarkers and more effective treatments. There are major efforts to meet the needs as the European Committee for Standardization (CEN) will publish Technical Specifications in 2015, aiming to reduce errors in the pre-analytical phase [47]. There are indications that these European Standards will proceed to global Standards on the ISO (International Organization for Standardization) level. In addition to fresh, frozen or FFPE-tissue samples, types of samples that have already been used for RPPA analysis include cellular lysates obtained by laser capture microdissection [36], serum [48,49], body fluids [50], cell culture lysates [17,51], low molecular weight serum protein fractions [52], peptides [53] and fine needle aspirates [54,55], which reflects the sample diversity for which RPPA can be used.