Miniaturization of a Size-Based Separation Device for Top-Down Proteomics Analysis of Low Microgram Samples

摘要

Unambiguous identification of protein sequence can be achieved through top-down proteomics. With over 1000 intact protein identifications, we have recently shown that top-down proteomics can now achieve a level of coverage approaching that of bottom-up proteomics [Tran et al, Nature, 2011]. An enabling aspect to this achievement is the integration of the GELFrEE apparatus [Tran & Doucette, Anal Chem, 2008], a size based separation, into a four-dimensional separation platform. While the level of throughput is encouraging, high loading requirements limited the study to samples, such as those obtained from cell culture, with low milligram quantities of total protein. In order to transition to the analysis of precious clinical samples, significantly lower sample requirements must be possible. Here, we couple a miniaturized GELFrEE apparatus to LC-LTQ-FTMS to resolve protein mass differences down to 700 Da, while achieving similar proteome coverage with 10-20 fold less starting material.