Comparison of electrokinetics-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of human salivary proteins

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of high-abundance proteins such as amylases, mucins, prolinerich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITM/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1 % false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electro-kinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE 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, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.