Evaluation of Archival Time on Shotgun Proteomics of Formalin-Fixed and Paraffin-Embedded Tissues

Abstract

There is increasing acceptance of the critical importance of correlating the morphologic features of tissue with the data obtained from various molecular analytic techniques. Access to archived formalin-fixed and paraffin-embedded (FFPE) tissue specimens via shotgun-based proteomic analyses may, therefore, open new avenues for both prospective and retrospective translational research. However, one of the remaining issues in performing comparative proteomic measurements among FFPE tissues relates to potential variability in protein composition and retrieval based on length of storage periods. Optimized protein extraction and digestion procedures for handling FFPE tissues are coupled with the capillary isotachophoresis-based proteome technology to evaluate the effects of length of storage period on archival tissue proteome analysis across ten archived uterine mesenchymal tumor tissue blocks, including nine uterine leiomyomas dating from 1990 to 2002 and a single case of alveolar soft part sarcoma (ASPS) from 1980. Several statistical measures, including the Pearson correlation coefficient, coefficient of variance, k-means clustering, and ANOVA, are employed to evaluate the possibility of an archival effect on individual proteins or groups of proteins within nine leiomyomas. Low abundance proteins may be more susceptible to the long term storage as these proteins are more difficult to be retrieved and extracted as the tissue block ages in paraffin. Despite using tissue blocks stored for as many as 28 years, high confidence and comparative proteome analysis between the leiomyomas and the sarcoma is achieved. Though sharing over 1,800 common proteins in a core set, a total of 80 proteins unique to the sarcoma are identified distinguishing the ASPS from the leiomyomas. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, mass spectrometry-based proteome profiling enables global identification and quantification of thousands of proteins without a priori knowledge of individual proteins being analyzed or the need of validated antibodies.

Original paper Morphological and immunohistochemical profile of pancreatic neuroendocrine neoplasms

“…We restricted the time period to 10 years in order to avoid loss of immunohistochemical reactivity [122]. Appropriate methods of statistical analysis were selected [30].…”

Antigen Retrieval Immunohistochemistry

“…Mass spectrometry data comparing extracts from fresh tissue and FFPE tissue for the same cell/ tissue sample (Table 2) support the notion that proteins extracted from FFPE tissue using heat-induced AR protocols are suitable for proteomics studies, as do data from protein array techniques (Prieto et al 2005;Rahimi et al 2006;Becker et al 2007;Becker et al 2009). For example, Balgley et al (2009) tested 10 archival FFPE tissue blocks with variable storage periods, as long as 28 years, by "shotgun" proteomics and found that high confidence and comparative proteome analysis were achieved in even the oldest tissue. In this context, AR-based methods, borrowed from the IHC field, are in like manner extending the usefulness and value of archival materials stored as paraffin blocks.…”

Proteomic analysis of FFPE tissue: barriers to clinical impact

“…The few systematic studies addressing these factors have yielded contradictory results. For example, top-down proteomic studies report up to a 50% loss in recoverable protein from FFPE tissue blocks stored for 20 years [8], while bottom-up studies report little effect of storage time on protein identification by mass spectrometry (MS) [9]. One might question the value of considering preanalytical factors for FFPE proteomics because information on sample preparation is rarely available.…”