Besides the complexity of protein samples, 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. To achieve comprehensive proteome analysis including the identification of low abundance proteins, this project aims to develop and demonstrate a capillary isotachophoresis (CITP)-based proteome platform, capable of providing selective analyte enrichment and extremely high resolving power toward complex protein mixtures. In contrast to universally enriching all proteins by a similar degree, the result of the CITP stacking process is that major components may be diluted, but trace compounds are concentrated.

By employing combined CITP/nano-reversed phase liquid chromatography (nano-RPLC) separations, a total of 6,112 fully tryptic peptides are sequenced by electrospray ionization mass spectrometry (ESI-MS), leading to the identification of 1,479 distinct human SwissProt protein entries from a single proteome sample of whole unstimulated human saliva. By comparing with capillary isoelectric focusing as another electrokinetics-based stacking approach, CITP not only offers a broad field of application, but also is less prone to protein/peptide precipitation during the analysis.

The CITP-based proteome platform is further employed for the analysis of protein expression within synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP separation is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, the collective proteome datasets yield the identification of 2,191 distinct mitochondrial protein entries, corresponding to 76% coverage of the MitoP2-database reference set.

Comparisons among CITP and multidimensional liquid chromatography techniques are conducted using a single processed protein digest from brain cancer stem cells, identical second dimension separation (nano-RPLC) and ESI-MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications, analytical reproducibility of the CITP proteome platform is determined by a Pearson R2 value of 0.98 and a coefficient of variation of 15% across all proteins quantified.