Abstract
To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and α = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available via ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age (p < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn® SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis (p > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.
Highlights
Tissues from biopsies, resections and/or surgery are routinely taken from patients as a treatment option and/or to facilitate more accurate diagnosis
The results indicate that long-term storage of FFPE tissues does not considerably interfere with retrospective proteomic analysis (p > 0.1)
One of the aims of this study was to methodically characterize the effects of storage time on the quality of data produced via label-free LC-MS/MS analysis of FFPE tissue blocks from a resource-limited pathology archive, to dispel any notions that these samples may be inferior for whatever reason so that they can be utilized with confidence in any future studies
Summary
Tissues from biopsies, resections and/or surgery are routinely taken from patients as a treatment option and/or to facilitate more accurate diagnosis. The current universal tissue preservation method of choice is formalin-fixation and paraffin-embedment, to avoid tissue auto-proteolysis and putrefaction, and to allow tissue specimens to be analyzed and examined at a later stage [1,2,3,4]. The formalin-fixed, paraffin-embedded (FFPE) method of tissue preservation allows for the indefinite room temperature storage of FFPE blocks, thereby removing much of the cost and difficulty associated with fresh-cryopreserved tissue storage. The technique involves the immersion and incubation of tissues in formaldehyde solution, which is replaced with alcohol (ethanol) in a dehydration step. Dehydration of the sample is achieved by removing all the water from the sample via ethanol incubation and subsequent alcohol clearing with xylene incubation. The xylene is replaced by molten paraffin, which infiltrates the sample. Samples are stored and archived for future use [1, 3, 4]
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