Extraction of proteins from formalin-fixed, paraffin-embedded tissue using the Qproteome extraction technique and preparation of tryptic peptides for liquid chromatography/mass spectrometry analysis.
This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue.
Geoui
Thibault
T
QIAGEN GmbH, Hilden, Germany.
Urlaub
Henning
H
Plessmann
Uwe
U
Porschewski
Peter
P
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020543:Proteome / Q000032:analysis; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D005404:Fixatives; D010455:Peptides; D011506:Proteins; D020543:Proteome; D005557:Formaldehyde; D014357:Trypsin