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Antibody-free, multiple-mechanism enrichment
A novel antibody-free technique, which enables peptide-level enrichment post digestion for high throughput ultra-sensitive quantification of the protein target in biomatrices. The method is based on multi-dimensional orthogonal mechanisms using mixed SCX and RP sorbent to enrich target specifically. We found pH greatly regulating both SCX and RP mechanisms based retention (e.g., pI-2, pI, and pI+2 were selected to enhance selectivity for washing and elution), and organic solvent and ionic strength in buffer displaying exponential and linear effects on desorption isotherms, respectively. A streamlined enrichment procedure was developed and a rapid optimization strategy was established to identify optimal conditions for specific enrichment/cleanup with reduced efforts. With the optimal conditions, by strategically manipulating pH and ionic/solvent strengths, orthogonal multi-dimensional enrichment procedures can selectively enrich the targets while removing non-target matrix peptides, and thus improved the sensitivity significantly.