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, proline-rich 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 CITP/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 electrokinetics-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.