High-throughput proteome analysis using 50 cm long micro pillar arraycolumns (µPAC)

Introduction : When aiming for comprehensive proteome analysis with deep coverage, relatively long nano LC columns (lengths up to 75 cm) are typically operated with long and shallow solvent gradients, delivering the highest chromatographic performance. However, clinical proteome research demands increased sample throughput, making total analysis times above 120 min undesirable or even impossible. The LC columns typically used for this type of analyses do not exceed 25 cm in length, with a focus on workflow robustness and efficiency rather than on chromatographic performance. In this contribution, we evaluated the potential of micro fabricated column alternatives for use in high througput proteome analysis. Methods : We compared the performance and robustness of 50 cm long PharmaFluidics RP C18 columns to a range of state of-the-art packed bed nano LC columns (packed with sub 2 µm C18 particles and ≤ 25 cm in length) for the analysis of HeLa cell reference, biopharmaceutical and human plasma samples. Different injection methods (direct injection and injection using a novel type of trapping columns) were evaluated in terms of sample throughput, recovery and robustness. A Thermo Ultimate 3000 nano RSLC system coupled to an Orbitrap Thermo Orbitrap Elite mass-spectrometer was used to perform all experiments. Non-linear solvent gradients from 1 - 35% acetonitrile in 0.1% formic and with a maximum duration of 60 min were used for the analytical separation. Preliminary Data : We demonstrate extremely robust and high-throughput proteome analysis using 50 cm long micro pillar array columns at capillary flow rates ranging from 0.5 to 2.0 µl/min. At these flow rates, sample throughput can be increased to 20, 30, 60 and even 100 samples per day with corresponding peak capacity values (nC) of respectively 300, 250, 200 and 150. When comparing the chromatographic performance that could be obtained for single protein, cell lysate and human plasma tryptic digest samples to state-of-the art packed bed nano LC columns, average peptide peak widths could be reduced by a factor of 1.6. For complex samples, this resulted in an increase in peptide and protein group identifications of respectively 60 and 40%. To increase the throughput even further, a micro pillar array based trapping column was implemented into the workflow. By doing so, sample loading times for diluted protein digest samples could be reduced up to a factor of 15 without affecting the chromatographic performance. Novel Aspect : The use of micro fabricated column formats significantly increases reproducibility and chromatographic performance for high throuput proteome analyses.

Jaar van publicatie:2019