Deja vu LC-MS/MS: How to RePlay~(TM) an LC-MS/MS Chromatogram Without Injecting the Sample Again

摘要

Old style re-injection strategies do NOT increase random information gain when dilution is taken into account (medium complex sample investigated). Instead they decrease sample throughput significantly. RePlay technology: (1) splits nLC flow of analytes into two streams after the column, enabling a second MS analysis of all analytes without affecting peak shape. (2) comes with no cost in sensitivity (concentration dependent ESI-MS). (3) enables a second pass of analytes towards the MS, which equals a second injection in terms of random information gain, but comes without the sample dilution required in old style re-injection strategies. (4) therefore always increases sensitivity of any nLC analytical approach, the time advantage (column washing and equilibration phase used to flush out captured chromatogram) is approach-dependent and can be up to 100percent. RePlay increased the information gain of a Trap and Elute strategy by up to 60percent using only one thrid of the time of a typical nLC experiment. Overall sensitivity of this high throughput approach for protein identification is now close to that of standard nLC-MS/MS. Identification rate of E. coli 2D gel separated proteins was increased up to 100percent using the Trap and Elute strategy in combination with RePlay. Sensitivity of the Trap and Elute strategy with RePlay is now sufficient for high throughout analysis of most stained gels, only the least intense spots on a Krypton stained gel (detection limit ca. 0.25 ng) were not identified at a 100percent level. Trap and Elute with RePlay is an LC-MS/MS based high throughput alternative to rapid protein identifications based on Infusion-MS/MS and MALDI-MS/MS.