Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum

Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>10¹⁰ more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.