Introduction
Single-shot LC-MS based proteomics has been an essential tool to help researchers unravel the proteome composition of complex biological samples. Even though innovation in LC-MS instrumentation and bioinformatics processing have brought major increases in sensitivity and workflow robustness to the field, there is a growing necessity to identify more proteins in less analysis time. In this contribution, we report on the use of novel HRAM mass spectrometer in combination with a very short pillar array-based separation column to achieve both comprehensive as well as high throughput proteome analysis with a single LC-MS set-up.
Methods
Lyophilized mammalian cell digests were separated on a Thermo Scientific™ Vanquish™ Neo UHPLC system with cycle times of 8, 14.4, and 24 min using a 5.5 cm long µPAC Neo High Throughput column in a trap-and-elute workflow amd data was acquired on a Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer operated in DIA mode. Data processing was performed with Thermo Scientific™ Proteome Discoverer™ 3.1 software.
Preliminary Data
A set of robust LC-MS methods with variable flow rates was developed by combining the extended flow capabilities and minimal delay volumes of the Vanquish Neo UHPLC. A significant increase in instrument productivity was achieved by operating in trap-and-elute configuration and performing column re-equilibration in parallel to sample loading. Utilizing variable flow rates during the gradient formation at flow rates ranging from 2.5 to 0.3 µL/min enabled LC-MS instrument productivity of 68, 79, and 87% could be achieved for respective cycle times of 8, 14.4, and 24 min.
The performance of these different methods on the Orbitrap Astral mass spectrometer was evaluated by injecting 200 ng of HeLa digest sample. Because of the high transmission efficiency and high scan rate, all DIA experiments were performed with the same isolation width as DDA experiments, 2 Th. At a sample throughput of 180 samples per day (8 min method) we were able to identify 7900 protein groups on average from 5 technical replicates (no match-between-runs). Considerable increases in proteome depth could be achieved by extending the gradient length and reducing the active elution flow rate gradually. Within 14.4 and 24 min of total LC-MS time, we identified an average of 8579 and 9167 protein groups, respectively. These preliminary results show that the 5.5 cm µPAC Neo High Throughput column has an extremely high gradient flexibility, and the novel Orbitrap Astral MS is suitable for all high-throughput proteomics methods.