Background: When dealing with rare samples of which only minute amounts are available, e.g. human heart tissue, simultaneous extraction of DNA, RNA, and proteins from the same sample is crucial for a comprehensive analysis on the physiological or pathological state of such precious tissue. In this study we provethe efficacy of a modified TRIzol protocol to extract proteins from samples of small size, such as endomyocardial biopsies (EMBs).
Method: Initially, we compared TRIzol protein extraction efficacy to urea/thiourea extraction from total murine left ventricles and then small amounts of left and right murine ventricles. Finally, we applied the modified TRIzol protocol to the proteomic profiling of EMBs from human left and right ventricles.
Results: Analysis of the proteins extracted from mouse and human samples revealed sufficient protein amount for downstream applications. Thus, LC-tandem mass spectrometry permitted highly sensitive protein identifications and comparable protein patterns and coverage of cellular components as a standard extraction protocol. 2D gel-based analysis confirmed the high quality and reproducibility of the TRIzol derived protein extracts.
Conclusion: Our results prove the utility of the modified TRIzol protocol for proteomics analyses involving minute amounts of precious samples.
Keywords: 2D PAGE; Heart biopsies; LC–MS/MS; Protein extraction; Proteomics; TRIzol.
Copyright © 2014 Elsevier B.V. All rights reserved.