Characterization of proteome profile data of chemicals based on data-independent acquisition MS with SWATH method.

Transcriptomic data of cultured cells treated with a chemical are widely recognized as useful numeric information that describes the effects of the chemical. This property is due to the high coverage and low arbitrariness of the transcriptomic data as profiles of chemicals. Considering the importance of posttranslational regulation, proteomic profiles could provide insights into the unrecognized aspects of the effects of chemicals. Therefore, this study aimed to address the question of how well the proteomic profiles obtained using data-independent acquisition (DIA) with the sequential window acquisition of all theoretical mass spectra, which can achieve comprehensive and arbitrariness-free protein quantification, can describe chemical effects. We demonstrated that the proteomic data obtained using DIA-MS exhibited favorable properties as profile data, such as being able to discriminate chemicals like the transcriptomic profiles. Furthermore, we revealed a new mode of action of a natural compound, harmine, through profile data analysis using the proteomic profile data. To our knowledge, this is the first study to investigate the properties of proteomic data obtained using DIA-MS as the profiles of chemicals. Our 54 (samples) × 2831 (proteins) data matrix would be an important source for further analyses to understand the effects of chemicals in a data-driven manner.

Proteomics-based investigation of cerebrovascular molecular mechanisms in cerebral amyloid angiopathy by the FFPE-LMD-PCT-SWATH method.

BACKGROUND: Cerebral amyloid angiopathy (CAA) occurs in 80% of patients with Alzheimer's disease (AD) and is mainly caused by the abnormal deposition of Aß in the walls of cerebral blood vessels. Cerebrovascular molecular mechanisms in CAA were investigated by using comprehensive and accurate quantitative proteomics. METHODS: Concerning the molecular mechanisms specific to CAA, formalin-fixed paraffin-embedded (FFPE) sections were prepared from patients having AD neuropathologic change (ADNC) with severe cortical Aß vascular deposition (ADNC +/CAA +), and from patients having ADNC without vascular deposition of Aß (ADNC +/CAA -; so called, AD). Cerebral cortical vessels were isolated from FFPE sections using laser microdissection (LMD), processed by pressure cycling technology (PCT), and applied to SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. RESULTS: The protein expression levels of 17 proteins in ADNC +/CAA +/H donors (ADNC +/CAA + donors with highly abundant Aß in capillaries) were significantly different from those in ADNC +/CAA - and ADNC -/CAA - donors. Furthermore, we identified 56 proteins showing more than a 1.5-fold difference in average expression levels between ADNC +/CAA + and ADNC -/CAA - donors, and were significantly correlated with the levels of Aß or Collagen alpha-2(VI) chain (COL6A2) (CAA markers) in 11 donors (6 ADNC +/CAA + and 5 ADNC -/CAA -). Over 70% of the 56 proteins showed ADNC +/CAA + specific changes in protein expression. The comparative analysis with brain parenchyma showed that more than 90% of the 56 proteins were vascular-specific pathological changes. A literature-based pathway analysis showed that 42 proteins are associated with fibrosis, oxidative stress and apoptosis. This included the increased expression of Heat shock protein HSP 90-alpha, CD44 antigen and Carbonic anhydrase 1 which are inhibited by potential drugs against CAA. CONCLUSIONS: The combination of LMD-based isolation of vessels from FFPE sections, PCT-assisted sample processing and SWATH analysis (FFPE-LMD-PCT-SWATH method) revealed for the first time the changes in the expression of many proteins that are involved in fibrosis, ROS production and cell death in ADNC +/CAA + (CAA patients) vessels. The findings reported herein would be useful for developing a better understanding of the pathology of CAA and for promoting the discovery and development of drugs and biomarkers for CAA.

Establishment and validation of highly accurate formalin-fixed paraffin-embedded quantitative proteomics by heat-compatible pressure cycling technology using phase-transfer surfactant and SWATH-MS.

The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of not only non-membrane proteins, but also membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. Protein extraction from FFPE sections of mouse liver was increased 3.33-fold by pressure cycling technology (PCT) and reached the same level as protein extraction from frozen sections. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. Compared to the conventional method using urea buffer, the present method using phase-transfer surfactant (PTS) buffer at 95 °C showed better agreement of peptide peak areas between FFPE and fresh samples. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2-fold range between FFPE and fresh samples. This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples.