Proteomic comparison of human brain tissue preservation methods

This study investigated the impact of tissue preservation methods on protein profiles analyzed by reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using data-independent acquisition (DIA). Proteomic profiles from formalin-fixed, formalin-fixed and paraffin-embedded (FFPE), and fresh-frozen human brain tissues (cortex and hippocampus, n = 6) were compared, including an FFPE-specific protein extraction kit (n = 4). Formalin-fixed samples more closely resembled fresh-frozen profiles than FFPE or FFPE-Kit samples, while still showing high correlation and overlap with FFPE tissues in principal component analyses. A core set of 1753 proteins was consistently detected across all sample preparation methods. A total of 35 proteins were identified exclusively in fresh-frozen samples, but without functional enrichment. Quantitative comparisons to the proteome of fresh-frozen tissue revealed an underrepresentation of cellular processes, energy metabolism, signaling, and transport related to protein properties such as length, location, and hydrophobicity. In contrast, neuronal development and phagosome-related pathways were overrepresented in fixed tissues. In a pilot study comparing low (Braak 0-II, n = 4) and high (Braak IV-VI, n = 4) Alzheimer's disease (AD) stages using formalin-fixed samples, we identified 12 potential protein biomarkers, primarily nucleosomal proteins and carboxypeptidase M (CPM). These findings suggest that formalin-fixed brain tissue provides reliable proteomic information, making it a valuable resource for neurodegenerative disease research. SIGNIFICANCE: Proteomics offers enormous potential for investigating the molecular regulation of the human brain. Valuable tissue samples are often preserved in formalin or additionally with paraffin for later analysis. The potential value of these preserved samples for proteomic analysis has already been recognized. However, tissue preservation poses a challenge for proteome analysis. Consequently, several studies have compared different protein extraction protocols for fixed samples. In addition, studies have been published comparing protein extraction from FFPE samples with fresh-frozen samples. To our knowledge, this is the first study to compare protein extraction across all three tissue preservation methods with subsequent functional analysis using samples obtained from the same donors, thereby eliminating inter-donor variability and enabling a direct comparison of preservation effects. This study validates a protein extraction protocol from formalin-fixed samples, laying the groundwork for future research into potential biomarkers in formalin-fixed samples.