Setting up the LC-MS/MS approach for plasma proteomics in the DM4You Project

Eduardo da Costa Camilo

Key information

Authors:

Eduardo da Costa Camilo (Eduardo da Costa Camilo)

Supervisors:

Carla Maria Alexandre Pinheiro; Miguel Nobre Parreira Cacho Teixeira (Miguel Nobre Parreira Cacho Teixeira)

Published in

11/20/2024

Abstract

An effective way to assess the nutraceutical impact of diet on health is through the analysis of biomarkers, such as plasma proteins, which are widely used as indicators of various diseases. However, many human biomarkers remain uncharacterised, and the dynamic range of protein abundance in plasma is poorly understood. This study aimed to test procedures to be employed within the DM4You project to minimise post-collection alterations. The inclusion of phosphatase and protease inhibitors, along with standardisation of the time between extraction and centrifugation, demonstrated a one-third preservation in protein concentration, as shown by the Bradford and Kalb and Bernlohr quantification methods. Regarding the reducing potential of the samples, two protocols (Folin-Ciocalteu and TEAC assay) were used, optimisation was achieved despite different results obtained in each assay. For proteomics analysis three preparation protocols were tested to demonstrate the impact of fractionation and enrichment. The cost-time relationship should be considered in future studies, particularly with 80 participants and multiple sample collections. This work provides data for the selection of an experimental protocol for future studies.