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.
Highlights
Formalin-fixation and paraffin-embedding (FFPE) enables the preservation and stabilization of tissue morphology, as well as the long-time storage of samples [1]
The numbers of identified proteins were similar among buffers 1, 3, and 4 (Table 1), whereas a much lower number of peptides and proteins were extracted using the buffer from the FFPE-FASP Kit and the ureacontaining buffer
Since the total amount of proteins was relatively low, we used intensity-based absolute quantification (iBAQ) as a quantitative parameter to assess the efficiency of the different extraction buffers
Summary
Formalin-fixation and paraffin-embedding (FFPE) enables the preservation and stabilization of tissue morphology, as well as the long-time storage of samples [1]. FFPE tissues represent a valuable resource for retrospective molecular investigations. The antigen retrieval method proposed in 1991 allows the restoration of antigen immunoreactivity [2], making immunohistochemistry (IHC) an important technique for collecting antibody-based protein information from formalin-fixed samples. Immunological methods rely on the availability and quality of the antibodies, which can be time-consuming and costly. The PLOS ONE | DOI:10.1371/journal.pone.0142650 November 18, 2015
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