Runsheng Zheng (Germering / DE), Martin Rendl (Germering / DE), Cristina Jacob (San Jose, CA / US), Philip Remes (San Jose, CA / US), Christopher Pynn (Germering / DE), Alec Valenta (Germering / DE), Ece Aydin (Germering / DE), Maksim Daniliuk (Vilnius / LT), Robert van Ling (Breda / NL), Scott Peterman (San Jose, CA / US), Wim Decrop (Germering / DE), Neloni Wijeratne (San Jose, CA / US), Martin Samonig (Germering / DE), Anne Morgenstern (Germering / DE)
Introduction
Pushing the limits of low-flow LC for high-throughput quantitative workflows often comes at the cost of decreasing mass spectrometer utilization, data quality (e.g. proteome depth, column carryover and quantitative accuracy/precision), and method robustness. For fast methods, such trade-offs must be made to maximize acquisition time and minimize method overhead. Here, we present a novel tandem LC workflow for high-throughput quantitative proteomics that eliminates method overhead while improving on traditional column washing and equilibration cycles.
Methods
A Vanquish Neo UHPLC system was configured for the tandem direct injection workflow coupled to either an Orbitrap Exploris 480, TSQ Altis Plus Triple Quadrupole or Stellar mass spectrometer for ultra-fast DDA, DIA and PRM analysis. Human plasma and cell lysates were utilized to demonstrate the performance in bottom-up proteomics at flow rates of 1.5 - 100 µL/min with 150 µm – 1 mm ID columns connecting to only one emitter, offering minimal analytical variation.
Preliminary results
The tandem LC-MS workflow was tested using gradients down to 8 min (corresponding to 180 samples/day) to demonstrate throughput, run-to-run reproducibility, and close to 100% MS utilization for quantitative proteomics. Using the 150 µm x 15 cm column operated at 1.5 µL/min, we reproducibly identified >1,200 protein groups in DDA at 180 samples/day throughput with high quantification accuracy between columns. While moving to the PRM analysis, the tandem direct injection workflow along with the Stellar mass spectrometer directly supports the expansion of large-scale targeted experiments covering > 3500 peptides and >60 samples per day from 300 ng human plasma digest with negligible carryover permitting high-confidence peptide verification. Overall, this new workflow is a promising approach to improving instrument productivity offering high sensitivity, high sample throughput, and maximum quantification confidence.
(For Research Use/Purposes Only – Not for Diagnostic Procedures)