Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision.
The effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and prevent proteome degradation has been evaluated. Mouse brains were bisected immediately following excision, with one hemisphere being heat treated followed by snap freezing in liquid nitrogen while the other hemisphere was snap frozen immediately. Sections were cut by cryostatic microtome and analyzed by MALDI-MS imaging and minimal label 2-D DIGE, to monitor time-dependent relative changes in intensities of protein and peptide signals. Analysis by MALDI-MS imaging demonstrated that the relative intensities of markers varied across a time course (0-5 min) when the tissues were not stabilized by heat treatment. However, the same markers were seen to be stabilized when the tissues were heat treated before snap freezing. Intensity profiles for proteins indicative of both degradation and stabilization were generated when samples of treated and nontreated tissues were analyzed by 2-D DIGE, with protein extracted before and after a 10-min warming of samples. Thus, heat treatment of tissues at the time of excision is shown to prevent subsequent uncontrolled degradation of tissues at the proteomic level before any quantitative analysis, and to be compatible with downstream proteomic analysis.
Goodwin
Richard J A
RJ
Division of Integrative and Systems Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Lang
Alastair M
AM
Allingham
Heather
H
Borén
Mats
M
Pitt
Andrew R
AR
D000818:Animals; D001921:Brain / Q000166:cytology; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006358:Hot Temperature; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome