2-D PAGE and MS analysis of proteins from formalin-fixed, paraffin-embedded tissues.
In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues. However, 2-D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2-D PAGE separation and MS identification of full-length proteins extracted from FFPE skeletal muscle tissue. The 2-D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2-D maps of proteins from FFPE tissue following standard mass-compatible silver staining. Protein spots from both FFPE and frozen tissue 2-D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2-D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh-frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full-length proteins from FFPE tissues might be suitable to 2-D PAGE-MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological-fixed tissues.
Addis
Maria Filippa
MF
PortoConteRicerche Srl, Tramariglio, Alghero, Sassari, Italy. addis@portocontericerche.it
Tanca
Alessandro
A
Pagnozzi
Daniela
D
Rocca
Stefano
S
Uzzau
Sergio
S
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005557:Formaldehyde / Q000737:chemistry; D006868:Hydrolysis; D015151:Immunoblotting; D007526:Isoelectric Point; D013058:Mass Spectrometry / Q000379:methods*; D009124:Muscle Proteins / Q000032:analysis; D018482:Muscle, Skeletal / Q000737:chemistry; D016612:Paraffin Embedding / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D012756:Sheep; D016622:Silver Staining; D013048:Specimen Handling / Q000379:methods*
D009124:Muscle Proteins; D011506:Proteins; D005557:Formaldehyde