High-Throughput Middle-Down Analysis Using an Orbitrap

This report demonstrates the application of a capillary LC-LTQ-orbitrap system to provide automated middle-down analysis of proteolytic peptides in the mass range 3000 to 10 000 Da. The novel workflow combines an underutilized method in the orbitraphigh resolution, mass-accurate product ion measurementswith software tailored to search such data (ProSightPC 2.0) and an Asp-selective chemical cleavage approach that generates peptides across an extended mass range. The strategy using high resolution mass measurements on both precursor and product ions is analogous to that widely used on FT-ICR analyzers. The approach is demonstrated in an analysis of the highly basic ribosomal proteome isolated from human MCF7 cancer cells.