Enhancing biomarker discovery through the analysis of plasma depleted samples by DIA and PRM strategies using the Orbitrap astral mass spectrometer

Non-infectious chronic diseases are currently the most common cause of disability worldwide, and responsible for 74% of total deaths worldwide. Most chronic disease are underdiagnosed or diagnosed at late stages, primarily due to the late onset of their first clinical symptoms. Thus, the identification of biomarkers in minimally invasive biological fluids with diagnostic ability of these pathologies is mandatory to improve their diagnosis, treatment, and progression.

In this context, proteomics analyses of plasma or serum play a crucial role for the identification of biomarkers that could be implemented in clinical routine. However, mass spectrometry analyses yet face challenges due to the wide dynamic range in protein concentration of these samples. Thus, the depletion of high-abundant proteins, such as albumin or immunoglobulins, is essential to reduce sample complexity and enhance the detection of low-abundant proteins.

We aimed at identifying plasma biomarkers for colorectal cancer (CCR) and Alzheimer" disease (AD), two of the most common chronic diseases worldwide. Plasma samples were depleted using three different strategies to enrich them in low-abundant proteins that might be useful as biomarkers of these diseases: immunoaffinity chromatography, ENRICH-iST kit, and Mag-Net method. Subsequently, the depleted and 1 µL of neat plasma samples were trypsin digested and analyzed using data independent acquisition (DIA) in the Orbitrap Astral and Orbitrap Eclipse Tribrid mass spectrometers, with 15-minutes and 2-hours gradients, respectively. Nearly 4000 plasma proteins were identified in the plasma samples depleted by the non-conventional immunoaffinity methods with the Astral mass spectrometer. Additionally, the efficacy of plasma enrichment methods was assessed using parallel reaction monitoring (PRM) in the Orbitrap Astral and Orbitrap Eclipse Tribrid mass spectrometers, with 15-minutes and 2-hours gradients, respectively, to detect previously described CRC and AD plasma biomarkers. The superior performance of the combination of plasma depletion and the Orbitrap Astral mass spectrometer was further demonstrated for targeted proteomics analyses.

The implementation of plasma protein depletion strategies is crucial for enhancing the sensitivity and depth of mass spectrometry analyses, enabling the identification of a broader range of proteins and potential biomarkers for various diseases. This approach benefits both discovery and targeted proteomic analyses, offering valuable insights into disease mechanisms and potential diagnostic markers.