Are Solid Particles Ready for Prime-Time Proteomics?

In this work, we evaluate the performance of nonporous C-18 stationary phases in high-speed proteomics workflows. We employed two commercially available sub-2 µm nonporous particle (NPP) materials, ODSIIIE and SOLAD, to fabricate analytical columns using 150 µm internal diameter (i.d.) fused silica capillaries to ensure compatibility with nano-UHPLC and nano-HPLC pressure regimes. Using long NPP columns (15 – 25 cm) connected to a conventional nano-UHPLC, we found that both materials supported efficient peptide separations within the flow rate and pressure ranges typical of nano-UHPLC systems. Shorter columns (4 – 5 cm), used with the 10- and 16-minute Whisper Zoom 120 and Zoom 80 methods on the Evosep One HPLC, demonstrated competitive separation performance compared to columns packed with fully porous (FPP) and superficially porous particles (SPP), achieving full-width at half maximum (FWHM) values below 2 seconds (Zoom 120) and 3 seconds (Zoom 80). Peak shape remained consistent with sample loads up to 25 ng of a human cell lysate digest, a level well-suited to the sensitivity of modern mass spectrometers. DIA LC-MS/MS analysis of 20 ng from the same digest demonstrated that NPP columns provided comparable or superior performance relative to FPP and SPP materials, identifying approximately 23,000 precursors (~3,000 proteins) with Zoom 120 and ~33,000 precursors (~3,800 proteins) with Zoom 80. These findings establish NPP-based columns as a viable and competitive alternative to FPP and SPP materials, particularly suited for high-throughput and high-sensitivity proteomics applications.