We report the identification of a photo-cleavable anionic surfactant, 4-hexylphenylazosulfonate (Azo) that can be rapidly degraded upon UV irradiation, for top-down proteomics. Azo can effectively solubilize proteins with performance comparable to SDS and is mass spectrometry (MS)-compatible. Importantly, Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of post-translational modifications. Moreover, Azo is simple to synthesize and can be used as a general SDS replacement in SDS-PAGE.

Photoactivation of “snap-top” stoppers over the pore openings of mesoporous silica nanoparticles releases intact cargo molecules from the pores. The on-command release can be stimulated by either one UV photon or two coherent near-IR photons. Two-photon activation is particularly desirable for use in biological systems because it enables good tissue penetration and precise spatial control. Stoppers were assembled by first binding photolabile coumarin-based molecules to the nanoparticle surface. Then, after loading the particles with cargo, bulky β-cyclodextrin molecules were noncovalently associated with the substituted coumarin molecule, blocking the pores and preventing the cargo from escaping. One-photon excitation at 376 nm or two-photon excitation at 800 nm cleaves the bond holding the coumarin to the nanopore, releasing both the cyclodextrin cap and the cargo. The dynamics of both the cleavage of the cap and the cargo release was monitored using fluorescence spectroscopy. This system traps intact cargo molecules without the necessity of chemical modification, releases them with tissue penetrating near-IR light and have possible applications in photo-stimulated drug delivery.

A therapy of cancer cells: Two-photon-triggered camptothecin delivery (see picture) with nanoimpellers was studied in MCF-7 breast cancer cells. A fluorophore with a high two-photon absorption cross-section was first incorporated in the nanoimpellers. Fluorescence resonance energy transfer (FRET) from the fluorophore to the azobenzene moiety was demonstrated.

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 (>10 10 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide highresolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibodyfree strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.