Selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using transient capillary isotachophoresis/zone electrophoresis coupled with nano-ESI-MS.

ABSTRACT

Besides the complexity in protein samples of biological origin, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. As demonstrated in this work, transient capillary ITP/zone electrophoresis (CITP/CZE) provides selective analyte enrichment through electrokinetic stacking and extremely high resolving power toward protein and peptide mixtures. The result of the CITP process is that major components may be diluted, but trace compounds are concentrated. The on-column transition of CITP to CZE minimizes additional band broadening while providing superior analyte resolution. Online coupling of transient CITP/CZE with nano-ESI-MS allows ultrasensitive detection of trace peptides at levels of subnanomolar concentration or subfemtomole mass in complex peptide mixtures. More importantly, selective enrichment of trace peptides enables the identification and sequence analysis of low-abundance peptides co-migrated with highly abundant species at a concentration ratio of 1500,000. The combined CITP/CZE-nano-ESI-MS system is demonstrated to be at least one to two orders of magnitude more sensitive than that attained in conventional electrophoretic and chromatographic-based proteome technologies over a wide dynamic concentration range, potentially allowing comprehensive analysis of protein profiles within a small cell population and limited tissue samples using conventional mass spectrometers. Furthermore, the speed of CITP/CZE separation and the lack of column equilibration in CITP/CZE not only improve the throughput of proteome analysis, but also facilitate its seamless integration with other separation technologies in a multidimensional protein identification platform.