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Human tissues are an important source of biological material for the discovery of novel biomarkers. Fresh-frozen tissue could represent an ideal supply of archival material for molecular investigations. However, immediate flash freezing is usually not possible, especially for rare or valuable tissue samples such as biopsies. Here, we investigated the compatibility of RCL2/CS100, a non-cross-linking, nontoxic, and nonvolatile organic fixative, with shotgun proteomic analyses. Several protein extraction protocols compatible with mass spectrometry were investigated from RCL2/CS100-fixed and fresh-frozen colonic mucosa, breast, and prostate tissues. The peptides and proteins identified from RCL2/CS100 tissue were then comprehensively compared with those identified from matched fresh-frozen tissues using a bottom-up strategy based on nano-reversed phase liquid chromatography coupled with tandem mass spectrometry (nanoRPLC-MS/MS). Results showed that similar peptides could be identified in both archival conditions and the proteome coverage was not obviously compromised by the RCL2/CS100 fixation process. NanoRPLC-MS/MS of laser capture microdissected RCL2/CS100-fixed tissues gave the same amount of biological information as that recovered from whole RCL2/CS100-fixed or frozen tissues. We next performed MALDI tissue profiling and imaging mass spectrometry and observed a high level of agreement in protein expression as well as excellent agreement between the images obtained from RCL2/CS100-fixed and fresh-frozen tissue samples. These results suggest that RCL2/CS100-fixed tissues are suitable for shotgun proteomic analyses and tissue imaging. More importantly, this alternate fixative opens the door to the analysis of small, valuable, and rare target lesions that are usually inaccessible to complementary biomarker-driven genomic and proteomic research.

journal of proteome research

Liquid Chromatography-Tandem and MALDI Imaging Mass Spectrometry Analyses of RCL2/CS100-Fixed, Paraffin-Embedded Tissues: Proteomics Evaluation of an Alternate Fixative for Biomarker Discovery