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.

基于唾液的诊断技术的核心基础，是建立唾液中可检测分泌蛋白的完整目录。除蛋白质组固有的复杂性外，唾液样品全面分析所面临的最大生物分析挑战，在于蛋白质相对丰度存在显著差异，其中包含淀粉酶、黏蛋白、富脯氨酸蛋白（PRPs）以及分泌型免疫球蛋白A复合物等多种高丰度蛋白。在众多电动分离技术中，瞬态毛细管等速电泳/毛细管区带电泳（transient capillary isotachophoresis/capillary zone electrophoresis，CITP/CZE）可特异性靶向痕量蛋白质，从而缩小蛋白质相对丰度的分布范围，为低丰度蛋白质的鉴定提供无与伦比的技术优势。通过采用基于CITP/CZE的多维分离平台并联用电喷雾电离串联质谱（ESI-tandem MS），本研究在1%错误发现率（FDR）下共鉴定得到6112条完整胰蛋白酶肽段，进而识别出1479个独特的人类SwissProt蛋白质条目。将其与另一种基于电动富集的分离方法——毛细管等电聚焦（CIEF）进行对比，CITP/CZE不仅应用场景更为广泛，且在分析过程中更不易发生蛋白质或肽段沉淀。CITP/CZE的超高分辨能力可通过两点得到证实：其一，每个CITP组分均可鉴定出大量独特肽段；其二，已鉴定肽段间的组分重叠率极低。此外，通过比对匹配至每个鉴定蛋白的独特肽段数量以评估蛋白质序列覆盖度，基于CITP的蛋白质组技术同样展现出优异性能：其中674种蛋白（占比46%）匹配到3条及以上独特肽段，288种（占比19%）匹配到2条独特肽段，另有517种（占比35%）仅匹配到1条独特肽段。