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 characterisation of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient’s metadata (medical history/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, 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 sample preparation methods for label-free LC-MS/MS analysis. The results indicate that the amount of protein extracted (mg/ml) is significantly dependent on block age, with older blocks yielding less protein than newer blocks. This may be due to the effect of formalin-fixation, which is an ongoing process, over long periods of storage. Detergent removal plates were the most efficient and overall reproducible sample preparation method with regard to number of peptide and protein identifications, followed by the SP3/HILIC method, and lastly the APFAR method. Therefore, block age mainly affects initial protein extraction and does not show significant differences/biases with regard to subsequent label-free LC-MS/MS analysis results.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

DISEASE(S): Colon Cancer

SUBMITTER: Catherine Rossouw  

LAB HEAD: Alan Gilbert Christoffels

PROVIDER: PXD017198 | Pride | 2021-04-06

REPOSITORIES: Pride