Versatile and bidirectional use of microfluidic LC columns, optimized configuration for achieving significant increases in proteome coverage at low nanoLC flow rates

LC-MS-based proteomics relies on HRAM mass spectrometers for resolution, speed, and sensitivity, critical for accurate identification and quantification after LC separation. To match the increasing speed of MS instruments, nanoLC column development has focused on reducing dead volume and ensuring optimal elution, particularly at ultra-low flow rates. Pillar array technology, with its precise order and high permeability, demonstrates outstanding performance, although connectivity quality can significantly impact results. An optimal strategy is proposed to enhance connectivity and diminish post-column dead volumes, resulting in notable performance enhancements and enabling more thorough proteomic analysis. Moreover, its unique capabilities facilitate high-throughput, ultra-sensitive analysis by swiftly adapting to programmed flow rates, enabling 100 samples per day at elution rates as low as 100 nL/min.

Utilizing a column heating device directly attached to the ESI source of the mass spectrometer enabled close integration of the nanoLC column, reducing the post-column volume by a factor of 3 to as little as 50 nL. Ensuring a low dead volume connection from the outlet fitting to a suitable nanoelectrospray emitter preserved the "on-chip" separation performance, resulting in significantly improved chromatography and deeper proteome coverage across all tested conditions.

In our initial assessment, we explored the benefits for conventional bottom-up proteomics sample loads (50-500 ng of tryptic digest injected) using customized LC methods. These methods achieved sample throughput rates of 60 and 30 samples per day, respectively, with peptide elution at conventional nanoLC flow rates (250 nL/min), reaching instrument productivity levels of up to 85%. From triplicates at a throughput rate of 60 samples per day, we identified 5970 protein groups, with 94% quantified below a coefficient of variation of 20%. High-resolution LC separation and reproducible elution allowed for quantification of significantly more protein groups (7% below 20% CV and up to 30% below 10% CV) compared to conventional pulled-tip emitter nanoLC column types.

Anticipating greater performance gains at lower flow rates, we conducted benchmarking with sample loads ranging from 50 pg to 10 ng. Methods were optimized for elution at 100 nL/min, achieving sample throughput rates of 96, 72, and 48 samples per day with instrument productivities of 70%, 77.5%, and 85%, respectively. Post-column volume reduction led to a performance increase of up to 25% (reducing average FWHM by 1.5s), increasing proteome coverage by up to 22% on the protein group level and identifying up to 2413 protein groups from as little as 250 pg.