pr800503u_si_002.xls (1.14 MB)
Evaluation of Archival Time on Shotgun Proteomics of Formalin-Fixed and Paraffin-Embedded Tissues
datasetposted on 2009-02-06, 00:00 authored by Brian M. Balgley, Tong Guo, Kejia Zhao, Xueping Fang, Fattaneh A. Tavassoli, Cheng S. Lee
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 10 archived uterine mesenchymal tumor tissue blocks, including 9 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 1800 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.
10 archived uterine mesenchymal tumor tissue blocksANOVAproteomic measurementsArchival Time80 proteinsFFPE tissues9 uterine leiomyomasstorage periodsVacuolar proton translocating ATPase 116 kDa subunit isoformShotgun Proteomicsproteome analysisIHCtissue block agessubcellular resolutionretrospective translational researchpart sarcomastorage periodtissue proteome analysis28 yearsASPSdigestion proceduresPearson correlation coefficienttissue specimensLow abundance proteinsprotein compositiontissue blockshandling FFPE tissuesOptimized protein extraction