Innovations in trap-and-elute LC-MS analysis: ZebraWash and Multidraw

Description:
Low-flow LC-MS is a crucial tool for profiling complex biological samples and quantifying low-abundance targets. Quantifying such targets requires both maximizing the amount of analyte present during analysis and minimizing the residual analyte, or carryover, between runs. While trap-and-elute workflows provide fast sample loading, clean-up, and preconcentration, they also increase the likelihood of carryover and sample breakthrough when injecting large volumes. Consequently, injecting large sample volumes without significant carryover necessitates a thorough understanding of sample injection and separation, trap/analytical column washing and equilibration, and analyte retention. Here we present approaches for large volume injections and minimizing carryover in low-flow LC-MS. Both approaches are investigated using bottom-up proteomics protein digest standards.

All experiments were performed using a Vanquish Neo UHPLC system coupled to an Orbitrap Exploris 480 MS via an EASY-Spray ESI interface. HeLa protein digest peptides were separated on an EASY-Spray PepMap Neo column. Sample injection amounts from 200 ng to 4 µg were used to evaluate the efficiency of the ZebraWash procedure for trap column clean-up, while large volume (500 µL) injections onto the trap column were used to evaluate multi-draw functionality for peptide retention and protein quantification.

We experimentally isolated carryover from two main sources: injection fluidics and the trap/separation columns. With 16 cycles of ZebraWash carryover was reduced to below below 0.2% when overloading the columns with 4 ug HeLa digest. On the other hand, 500 µL sample injections using Multidraw with trifluoroacetic acid in the loading buffer provided close to 100% sample recovery and, thus, suggested a novel way of introducing samples into the LC-MS system for bottom-up proteomics. Overall, both innovations in the trap-and-elute workflow ensure confident results when injecting large sample volumes.

Speaker: Alec Valenta - Thermo Fisher Scientific
Alec is an HPLC product marketing specialist at Thermo Fisher Scientific focused on low-flow LC-MS instrumentation. He received his Ph.D. in Analytical Chemistry from the University of Michigan.