Direct Analysis and MALDI Imaging of Formalin-Fixed, Paraffin-Embedded Tissue Sections

Abstract

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffinembedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling

“…A decade of developments in instrumentation and chemistry has been required to achieve optimal extraction, detection, and spatial resolution when mapping compounds at the tissue level (Franck et al 2009a, b;Lemaire et al 2006a, b;2007a;van Remoortere et al 2010). These developments, combined with using the appropriate data-processing tools, gave rise to the discovery of biomarkers for diverse pathologies (Bonnel et al 2011;Djidja et al 2010;McCombie et al 2005;Stauber et al 2008), of which cancer is the most studied.…”

Protein unlocking procedures of formalin‐fixed paraffin‐embedded tissues: Application to MALDI‐TOF Imaging MS investigations

“…Lemaire et al [13] showed that a combination of in situ extraction with in situ micro digestion performed on FFPE tissues allows the identification of proteins by nanoLC-nanoESI and MALDI imaging.…”

“…Recently, Schwartz et al [12] obtained good results in the prognosis of brain tumor patient by MALDI-IMS carried out on fresh frozen tissues. Furthermore, Lemaire et al [13] showed that a combination of in situ extraction with in situ microdigestion performed on FFPE tissues allows the identification of proteins by nanoLC-nanoESI and MALDI imaging.…”

In situ molecular imaging of proteins in tissues using mass spectrometry

“…This work demonstrates significant improvements in the sensitivity, the number of observed analytes, the detection of larger proteins, and importantly allows unambiguous MS/MS identification directly from the tissue. Enzymatic digestion prior to IMS has also been applied to the analysis of formalin-fixed paraffin-embedded (FFPE) tissue [11]. FFPE is the predominant method for tissue preservation and storage; however, the procedure cross-links proteins, preventing classical proteomic analysis.…”