Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens

Abstract

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3–16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3–16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.

Expansion Microscopy of Lipid Membranes

“…Our new protocol builds on the post-expansion protein-retention ExM (proExM) protocol 3 , as well as tissue proteomics protocols for formalin-fixed paraffin-embedded (FFPE) tissues [26][27][28] . In brief, we heat the sample for half an hour at 100 C and for 2 hours at 80 C, in a "fixation reversal" (FR) buffer 28 containing 0.5% PEG20000, 100mM DTT, 4% SDS, in 100mM Tris pH8 (Supp.…”

Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

“…To gain additional understanding into the mechanism by which melanoma in situ , which is confined to the epidermis, progresses into invasive melanoma at the protein level, label-free MS analysis of FFPE tissue using the FASP methodology was applied in the present study to identify proteins differentially expressed between these two stages of melanoma. The FASP method presents numerous advantages over alternative approaches for analysing FFPE tissues, particularly the ability to buffer-exchange the sample into a more digestion-compatible solution, thus facilitating the classification of large numbers of disease-associated proteins (19). …”

Proteome analysis of tissues by mass spectrometry

“…Molecular weight cut‐off (MWCO) filters have also been described in tissue proteomics, and can be an effective way of contaminant elimination. Wisniewski et al reported use of the FASP method (Wisniewski et al, ; Wiśniewski, ), which was pioneered by Manza et al (Manza et al, ), on several tissue studies (Ostasiewicz et al, ; Wisniewski et al, , ; Shen et al, ; Bennike et al, ; Dowling et al, ; Föll et al, ). Proteins were first solubilized in a 4% SDS solution and with exchange with urea buffer SDS was washed off the sample and proteins were captured on MWCO filter.…”

“…As nonionic detergents, they interrupt protein‐protein interactions, but do not possess a net charge and, therefore, lack electrophoretic mobility. Although they might be less effective in protein extraction than SDS, they can, however, be added to the buffer in order to complement protein extraction (Shen et al, ; Broeckx et al, ). Preference for buffer composition can also be tissue‐dependent due to the diversity of proteins in various tissues.…”