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In this paper, we present a fully integrated valve-free method for the sensitive targeted bottom-up analysis of proteins through on-line aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (AA-SPE-IMER-CE-MS). The method was developed analyzing α-synuclein (α-syn), which is a protein biomarker related to different neurodegenerative disorders, including Parkinson's disease. Under optimized conditions, on-line purification and preconcentration of α-syn, enzymatic digestion, electrophoretic separation, and identification of the tryptic peptides by mass spectrometry was achieved in less than 35 min. The limit of detection was 0.02 μg mL -1 of digested protein (66.7% of coverage, i.e., 8 out of 12 expected tryptic peptides were detected). This value was 125 and 10 times lower than for independent on-line digestion by IMER-CE-MS (2.5 μg mL -1 ) and on-line preconcentration by AA-SPE-CE-MS (0.2 μg mL -1 ). The repeatability of AA-SPE-IMER-CE-MS was adequate (at 0.5 μg mL -1 ,% RSD ranged from 3.7 to 16.9% for peak areas and 3.5 to 7.7% for migration times of the tryptic peptides), and the modified capillary could be reused up to 10 analyses with optimum performance, similarly to IMER-CE-MS. The method was subsequently applied to the analysis of endogenous α-syn from red blood cell lysates. Ten α-syn tryptic peptides were detected (83.3% of coverage), enabling the characterization and localization of post-translational modifications of blood α-syn (i.e., N -terminal acetylation).

analytical chemistry

On-line Coupling of Aptamer Affinity Solid-Phase Extraction and Immobilized Enzyme Microreactor Capillary Electrophoresis-Mass Spectrometry for the Sensitive Targeted Bottom-Up Analysis of Protein Biomarkers