Enhanced Neuropeptide Profiling via Capillary Electrophoresis Off-line Coupled with MALDI FTMS

摘要

An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The pre-deposited nanoliter volume DHB spots on Parafilm-coated MALDI sample plate offers improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5 to 10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (C. borealis pericardial organs (n=4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as posttranslational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.