Development of liquid chromatography–mass spectrometry using continuous-flow matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Abstract

The general approach of combining liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry for on-line detection of peptides and proteins based on the use of a continuous-flow (cf) probe is presented. Recent advances in time-of-flight mass spectrometric instrumentation for cf-MALDI are reported. Using 3-nitrobenzyl alcohol (3-NBA) as the liquid matrix, stable flow can be readily achieved and the three-port interface does not introduce chromatographic peak shape degradation for LC–MS. Separation and detection of low picomoles of peptides and proteins can be done with cf-MALDI-LC–MS. Parallel ion extraction and time-lag focusing are shown to provide enhanced performance with regard to mass resolution. However, mass resolution is generally poor for proteins with masses above ∼6000 u. Strong adduct ion formation with the use of 3-NBA as the liquid matrix is believed to be the main cause of this resolution reduction. It is argued that cf-MALDI is a technically viable approach for LC–MS, but the overall performance and wide use of this method depend on the discovery of new liquid matrices that are suitable for continuous flow and provide much enhanced utility for MALDI over 3-NBA, particularly for proteins.