An in-depth and high throughput plasma proteomics workflow powered by Orbitrap Exploris 480 mass spectrometer using multi nanoparticle-based workflow

Plasma proteomics using mass spectrometry (MS) remains a promising method to discover disease biomarkers. However, for large-scale plasma proteomics studies, a robust liquid chromatography-mass spectrometry (LC-MS) setup that does not compromise protein identification, sequence coverage, dynamic range, or analytical precision is required. Here we present a high-throughput and maximum identification (Max-ID) workflow on a Thermo ScientificTMOrbitrap ExplorisTM 480 mass spectrometer for in-depth analysis of plasma. The plasma samples were processed with Seer"s Proteograph™ XT Assay utilizing multi-nanoparticles (NPs) for unbiased and deep proteomics enrichment at scale. [SJ1]

Samples were processed using 240 μL aliquots of each plasma sample, mixed with each of the two NP wells included in the Proteograph XT Assay Kit. The enriched protein coronas were reduced, alkylated, and digested with Trypsin/Lys-C to generate tryptic peptides for LC-MS analysis. All steps were performed in a one-pot reaction directly on the NPs. The in-solution digestion mixture was desalted, and all detergents were removed using a mixed-media filter plate and positive pressure (MPE) system. Clean peptides were eluted in a high-organic buffer into a deep-well collection plate. 500 ng of tryptic peptides were analyzed on the Orbitrap Exploris 480 MS with both nanoflow and capillary flow LC methods. For the capillary flow high-throughput method, two individual injections of 24 minutes each were used for a total instrument time of 48 minutes per sample. For the nanoflow Max ID method, the two peptide wells were pooled, and LC-MS analysis was done in a single injection using a 102-minutes total gradient.

The optimized high-throughput DIA method achieved the identification of more than 3,000 protein groups and over 20,000 peptides from the Proteograph Analysis Suite with the match between run (MBR) feature. Of these, 87.2% presented a coefficient of variation (CV) below 20%. For the Max ID method, we successfully identified more than 3,800 protein groups and over 27,000 peptides, with 89.2% of protein groups showing a CV under 20%, highlighting the excellent quantitation performance. In addition, our data revealed that 95% and 99% of protein groups were consistently identified across all replicates from the high-throughput and Max ID methods, respectively. The results underscored the completeness of the data resulting from the LFQ-DIA workflow. Together, we demonstrated a comprehensive plasma proteomics workflow on an Orbitrap Exploris 480 mass spectrometer with Seer"s Proteograph XT suite to offer high-throughput analysis with in-depth proteome coverage and excellent quantitation performance.