Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues

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.

Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa

“…For protein purification before LC-MS/MS analysis, we used the Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) 19 , 20 method, which ensures minimal sample loss during processing and was also found to be highly sensitive, therefore requiring less starting material (tissue). 12 , 19 , 20…”

“…The method used for sample processing, protein extraction and protein yield quantification was modified from the protocols used by Scicchitano 21 and Wiśniewski 22 and previously described in more detail by Rossouw. 12 Both WCPLs and sample pellets were subsequently processed by the hydrophilic interaction liquid chromatography (HILIC)/SP3 magnetic bead digestion method, 19 before LC-MS/MS analysis ( Figure 2 ).…”

“…The method was modified from the protocol used by Hughes 19 and previously described in more detail by Rossouw. 12 …”

“…Mass spectrometry analysis of each sample’s peptides was performed using the Q-Exactive quadrupole-Orbitrap (Thermo Fisher Scientific, Waltham, Massachusetts, United States), which was coupled with a Dionex Ultimate 3000 nano-UPLC system as described before by Rossouw. 12 Using Xcalibur TM (version 4.2) (Thermo Fisher, Waltham, Massachusetts, United States), spectral data was collected in a data-dependent manner and details are shown in Supplementary document – Table S1.…”

“…We have previously studied the effects of FFPE block age on the quality and quantity of protein extracted from FFPE tissues and also evaluated protein purification methods using LC-MS/MS analysis. 12 However, the optimal protein extraction buffer components were not investigated. Therefore, of interest to this study are the effects of polyethylene glycol (PEG), specifically PEG 20 000, on protein extraction efficiency of human FFPE tissues using LC-MS/MS analysis, as there is no current consensus with regard to PEG usage and advantages for human FFPE tissue proteomics.…”