Comparison of multidimensional shotgun technologies targeting tissue proteomics

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Abstract
A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)‐based multidimensional separations and multidimensional LC system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme‐derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second‐dimension separation (RPLC) and 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 coupled with the spectral counting approach are determined by a Pearson R2 value of 0.98 and a CV of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional LC separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.