Extreme Pressure Colloidal NanoLC-MS/MS for Ultra-fast In-depth Proteomics and Low-input Sample

Proteomics is increasingly contributing to the development of quantitative biology, precision medicine and public health. It now faces tremendous number of samples from different species, human population, and single cells from
tissues and organs. This demands a throughput on the scale of thousands, even millions of samples per day to provide sufficient data for answering specific biological questions. However, current throughput of fast in-depth proteomics
is limited to 50 – 120 samples per day on single platform, constrained by the separation speed of nanoflow liquid chromatography (nanoLC) and acquisition speed of MS instrument. To increasing the analysis throughput, faster
chromatography and mass spectrometry are needed, along with automated sample preparation and data analysis.
As the rate-determining step, nanoLC separation must be completed within a few minutes, or even below one minute, to enable over 1000 samples per day on single nanoLC-MS/MS platform, which presents a significant challenge. One promising approach is applying nanoparticle material in a chromatography column. According to kinetic performance limit (KPL) calculation, we found that, by using a 1500 bar nanoLC instrument, the analysis time could be reduced to 1/6 with same separation efficiency by changing the particle size from 1.5 µm to 500 nm. With an extreme pressure instrument (with maximum pressure of 3000 bar), the analysis time can reduce to 1/10, potentially support 600-1000 samples per day on single platform. When self-assembly of nanoparticle is
considered, ordered column bed can further reduce the analysis time compared to a random packed column, even when accounting for the drastic increase of flow resistance. In addition to fast analysis, the reduced surface area
and stationary phase diffusion makes the non-porous nanoparticle-based column suitable for high-recovery, high resolution, low-input analysis of rare and small amount sample, such as phosphoproteomics, glycomics and singlecell studies.
Using the single particle frit and convection-driven assemble of stabilized silica colloid, we successfully assembled silica nanoparticle materials inside fused silica capillary. The flexibility of single particle frit and stability of silica colloid (stable for over 12 h) support robust assemble of various nanoparticle sizes (200 – 750 nm) into capillaries of different inner diameter (25 – 200 µm) and length up to 300 mm. This fabrication capability can cover all column formats possibly required in ultra-fast nanoLC. During the assembling, we assumed that nanoparticle will be guided to the favored position inside capillary by convection through the formed column bed channel and weak interaction between particles. We studied the impact of convection speed, nanoparticle concentration and aspect ratio, showing the potential to fabricate ‘perfectly’ packed chromatography columns via colloidal assembly. Preliminary peptide mapping and DDA/DIA proteomics by C18-grafted nanoparticle column was performed on 1500 bar VanquishtimsTOF platform and compared with conventional sub-2 µm nanocolumn under fast and low-input proeomics, demonstrating promising future of nanoparticle column for ultra-fast proteomics on a 3000 bar extreme pressure platform