Method optimization for the identification of proteins associated with pancreatic ductal adenocarcinoma in plasma using mass spectrometr y
Date
2024
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Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths. The poor survival rate for PDAC is mostly due to the lack of effective biomarkers for early diagnosis. This study aimed to develop a standard operating procedure for a highthroughput mass spectrometry-based workflow that can be used for plasma-based biomarker discovery studies.
Methods: Total protein was extracted from a master pool consisting of 28 PDAC samples (M211158). SDS-PAGE was conducted to visualize the dynamic range of the plasma proteins. Three sample preparation methods were compared, Method 1: plasma diluted 25X with buffer (50mM Tris-HCl pH 8 with 2% SDS), Method 2: plasma diluted 5X with 50mM TEAB buffer, and Method 3: precipitation (using 80% and 100% acetone) followed by resuspension in 50 mM Tris-HCl, 2% SDS. The Hydrophilic Interaction Liquid Chromatography (HILIC) and protein aggregation capture (PAC) based protocols were compared for clean-up and digestion of the proteins. Low pH reverse phase chromatography was applied to separate the peptides and SWATH-MS analysis was conducted for label-free quantification. The methods were compared based on reproducibility, ease of automation and the number of peptides identified. A pilot study including 6 PDAC, 6 Chronic pancreatitis, and 6 healthy individuals was conducted to test the selected optimized method.
Results: The combination of the 25-fold dilution of Method 1 and the 2 hrs HILIC bead-based method (with a Trypsin and LysC combination) were effective in protein sample clean-up and digestion, yielding the highest number of peptides (mean: 2856). The combination of Trypsin and LysC enzymes contributed to the improvements in technical variation and peptide quantitation. The preliminary analysis identified 25 differentially expressed proteins (22 downregulated and 3 upregulated) between the healthy and PDAC patients. Most of these proteins are associated with tumorigenesis.
Conclusion: This study developed a standard operating procedure for processing plasma samples and can be applied in future studies for the identification of potential markers for PDAC.
Description
A research report submitted in partial fulfilment of the requirement for the degree of Master of Science in Medicine to the Faculty of Health Sciences, University of the Witwatersrand, School of Clinical Medicine, Johannesburg, 2023
Keywords
Pancreatic ductal adenocarcinoma (PDAC), Operating procedure, Mass spectrometry