Evaluation of new sample preparation methods associated with DIA analysis on Exploris 480 and Tims Tof SCP mass spectrometers to increase proteomic depth on plasma

Human plasma contains biological information from all organs of the human body and therefore offers invaluable possibilities for clinical biomarker detection. However, detecting low-abundant proteins of interest in plasma samples is challenging. The heterogeneity and complexity of plasma, with high dynamic range in protein concentrations, represent strong hurdles for LC-MS-based proteomics, limiting access to full proteome information. Therefore, in recent years, sample preparation methods based on the depletion of highly abundant proteins, on the enrichment of low abundant proteins, or on the selective enrichment of plasma microvesicles, have been reported in order to increase plasma proteomic coverage. In addition, DIA-based methods on modern fast-sequencing mass spectrometers also provide better proteomic depth for such applications.

We used plasma samples obtained from five different patients and evaluated three commercial protocols for human plasma preparation, including:

- depletion columns for single-step removal of 14 high-abundant proteins representing approximately 95% of plasma total protein content (ThermoScientific)

- the ENRICH-iST kit described to enrich low abundant proteins onto paramagnetic beads (Preomics)

- the Mag-Net method based on MagReSyn® strong anion exchange (SAX) magnetic beads (ReSyn Biosciences) to capture extracellular vesicles from plasma

We implemented DIA analysis workflows based either on the Exploris TM 480 from Thermo Fisher Scientific, or on the timsTOF SCP from Bruker, associated with DIA NN 1.8 data processing, and compared the results obtained using these three protocols with the direct analysis of neat plasma. All three methods improved significantly the proteomic characterization of plasma, allowing the identification of 800 to 2900 proteins/run, depending on the protocol and the patient, and giving access to the detection of some very low abundant molecules. The two preparation methods based on enrichment of proteins on magnetic beads (ENRICH-iST and Mag-Net) proved to be more efficient than the depletion approach, and they also appeared to be quite complementary, each of them improving the detection of specific classes of proteins. Altogether, more than 3000 plasmatic proteins could be identified and quantified from one patient using a combination of those sample preparation methods.