An Ultra-Deep Quantitative Plasma Proteomics Strategy

Blood is considered as the most valuable biofluid for protein biomarker screening as it contains a comprehensive human proteome, including cytokines and tissue leakage proteins that reflect ongoing disease states. Though widely used in large-scale protein quantification, mass spectrometry (MS)-based proteomic strategies still face great challenges in protein biomarker discovery in the blood, due to the particularly large dynamic range of proteins and the strong suppression effect by high-abundant proteins in the blood. As a promising alternative, nanomaterials enrichment-based protein corona strategy is highly efficient in increasing proteome coverage, but with the drawback of potentially disrupting the original protein concentration in the blood. Therefore, we proposed an ultrasensitive strategy that capable of enhancing mass spectrometry signals of the low abundant proteins/peptide to promote quantification of more than 2000 proteins at 288 SPD or 5000 plasma proteins at 36 SPD throughput from neat plasma without enrichment. Our strategy leverages the unique multiplexing properties of TMT labeling to sum the “total” MS1 signal of the same peptide from a boosting channel using nanomaterials enriched protein digests and the study channels using digests of neat plasma to trigger MS/MS fragmentation more efficiently and sensitively for low abundant protein identification, while the reporter ions provide quantitative information. In this way, in depth coverage of low abundant protein in neat plasma can be achieved without the risk of disrupting their original quantification. Accurate and reproducible quantification of plasma proteins with concentration down to low ng/L range was achieved, which is still rate in the field. Sensitive differentiation of plasma proteome characteristics among healthy individuals by this method indicated its potential in disease biomarker screening.