Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues.
There are vast archives of formalin-fixed tissues spanning many conceivable conditions such as different diseases, time courses, and different treatment and allowing acquisition of the necessary numbers of samples to carry out biomarker discovery study. However, the conventional protein analysis approach is not applicable for the analysis of proteins in the formalin-fixed tissue because the formalin fixation process resulted in the cross-linking of proteins, and thus, intact proteins cannot be efficiently extracted. In this study, several protocols were investigated to extract proteins from formalin-fixed mouse liver tissue for shotgun proteome analysis. It was found that incubation of tissue in a lysis buffer containing 6 M guanidine hydrochloride at high temperature led to the highest protein yield and the largest number of proteins identified. The peptides and proteins identified from formalin-fixed tissue were first comprehensively compared with those identified from frozen-fresh tissue. It was found that a majority of peptides identified from fixed tissue were unmodified and proteome coverage for the analysis of fixed tissue was not obviously compromised by the formalin fixation process. Valuable proteome information could be obtained by shotgun proteome analysis of formalin-fixed tissue, which presents a new approach for disease biomarker discovery.
Jiang
Xiaogang
X
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Jiang
Xinning
X
Feng
Shun
S
Tian
Ruijun
R
Ye
Mingliang
M
Zou
Hanfa
H
D000818:Animals; D005557:Formaldehyde; D019791:Guanidine; D006358:Hot Temperature; D008099:Liver; D008722:Methods; D051379:Mice; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D016707:Tissue Fixation
D011506:Proteins; D005557:Formaldehyde; D019791:Guanidine