Low-flow liquid chromatography (LC) coupled to electrospray ionization-based mass spectrometry (MS) techniques have the capacity to probe extremely limited sample amounts. The increase in electrospray ionization (ESI) efficiency required is achieved by adopting narrow separation columns and reducing the LC flow rates to the “ultra-nano” (≤ 100nL/min) range.
Several aspects of ultra-low sample quantity analysis must be considered in the creation of robust and reproducible methods for this type of application. First, flow rate must be optimized for both sensitivity and throughput. Second, the LC platform must permit efficient sample analysis without wasting valuable MS acquisition time. Third, MS acquisition parameters must be optimized for the relatively low signal intensity observed from small sample quantities.
Here we describe a standardized LC separation setup together with 5 novel methods for balancing the sensitivity, throughput, and reproducibility required for routine analytics.
Experiments were performed on a Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 mass-spectrometer. HeLa protein digest was separated on a 50 µm I.D. column at a flow rate of 100 nL/min. Contrasting data acquisition strategies, i.e., data-dependent acquisition (DDA) and data-independent acquisition (DIA), were also compared for their impact on method performance.
Overall, we developed five ultra-low nano-flow LC-MS methods with gradients from 10 to 50 min, providing sample throughput of 24, 36, 40, 60, and 72 samples per 24 hours (up to 85% MS utilization). Using 250 pg diluted HeLa digest and the 10-min LC gradient (72 samples/day), ca. 800 protein groups were identified in DDA (Sequest), >1,800 protein groups in DDA (CHIMERYS), and ca. 2,100 – 2,700 protein groups in DIA (SN16 & DIA-NN18).