Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens

Abstract

Objectives Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins. Methods Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC–MS). Results Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues

“…We postulated that strong acid could enable tissue disruption and nucleic acid hydrolysis without the need for sonication. The use of SDS would further support high protein solubilization, in particular of membrane proteins that are of critical importance in clinical assessments because most biomarkers and drug targets are membrane proteins . The combination with automated single-pot solid-phase-enhanced sample preparation (SP3) would eliminate the necessity for time-consuming and lossy dilution/concentration steps and aid the removal of contaminating molecules.…”

“…The use of SDS would further support high protein solubilization, in particular of membrane proteins that are of critical importance in clinical assessments because most biomarkers and drug targets are membrane proteins. 21 The combination with automated single-pot solid-phase-enhanced sample preparation (SP3) would eliminate the necessity for time-consuming and lossy dilution/concentration steps and aid the removal of contaminating molecules. Based on this hypothesis, we developed the new ASAP workflow and compared the performance with the gold-standard TFE and sonication-based method.…”

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC–MS

“…We compared the performance of the f -SEPOD strategy on targeted protein quantification with two prevalently employed methods for FFPE sample preparation: a Qproteome FFPE tissue kit from Qiagen (i.e., Qproteome method), which has been popularly utilized and has served as a standard method to compare to when a new preparation method is developed; , and a well-recognized, high-yield method involving a high-pH buffer with 2% SDS (i.e., high-pH-SDS method), , which, along with its various minor modifications, has been adopted in many recent applications. , To enable an extensive and well-controlled comparison, a set of 20 tumors from mAb-dosed animals (Figure S1; SET-A ) were cut into 5–8 mm 3 pieces and then homogeneously mixed by pulverizing the tissues to minimize intratissue heterogeneity. Among these, half pieces were subjected to FFPE treatment (Figure S1; SET-A - FFPE ) and the rest of these pieces were flash-frozen for fresh tissue analysis, which served as a control group (Figure S1; SET-A-fresh ).…”

Disease-Specific α-Synuclein Seeding and Structural Properties in Lewy Body Disease and Multiple System Atrophy are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain

“…Different techniques have been reported to remove paraffin residues and reverse formaldehyde crosslinks using various protein extraction buffers [34][35][36][37][38]. Among the different techniques, the application of thermal energy via antigen 3 of 18 retrieval and extraction buffers corrected for ionic strength differences were determined to be the key elements for efficient protein extraction [39][40][41][42].…”