THERMO Scientific | LC-MS high throughput Proteome profiling

Historically, discovery proteomics laboratories have achieved deep proteome profiling by using long columns (≥25 cm length) with 75 μm I.D. operated at nano-flow rates (approx. 300 nL/min). Such separation conditions provide high chromatographic resolution but result in long runtimes and limit LC-MS throughput to several samples per day. While this remains the best route for maximizing protein coverage, there are two main disadvantages: (i) analyzing a sample cohort of sufficient size to generate impactful data is very time-consuming; (ii) actual mass spectrometer utilization is very low in traditional nano-flow applications because of the time required for sample loading, column washing and column equilibration at nano-flow rates.
Throughput limitations often preclude the adoption of nanoLC methods for translational proteomics applications such as biomarker validation and precision medicine study because a statistically significant number of samples must be analyzed to compare the biological variation and cases related variation. Although long capillary columns yield improved peak capacities, the pressure limitations of most commercial low-flow UHPLC systems lead to impractical total cycle times when using columns beyond 15 cm length. Moreover, method overhead times can account for up to 50% of the total analysis time, leaving the MS idle for all but a short elution window.
Recently, high-throughput capillary- and micro-flow LC-MS methods have gained traction for fast proteome profiling due to improved sample throughput and method robustness over traditional nano-flow LCMS approaches. The benefits of operating nanoLC columns packed with 2 µm particles at higher flow rates have been, until now, unavailable due to the pressure and workflow limitations of existing low-flow UHPLC systems. Another aspect of developing high-performance, high-throughput LC-MS methods is MS utilization. The impact of sample pickup and loading, column washing, and column equilibration on the cycle times can be reduced by utilizing ultra-high-pressure capabilities and intelligent method execution.
Here, we present the Vanquish Neo UHPLC system for high throughput, bottom-up proteome profiling using elevated flow rates on 75 µm I.D. columns. The 1500 bar pressure limit for the UHPLC system and 75 µm I.D. × 15 cm, 2 µm EASY-Spray PepMap Neo columns provides the flexibility to run methods with throughputs from 24 to 180 samples per day without any changes to fluidics or hardware. Optimized sample pickup, sample loading, column washing, and column equilibration at increased flow rates enable up to 95% MS utilization. The described methods also provide an excellent level of proteome coverage, robustness, and reproducibility between multiple laboratories.

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