Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS.
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 predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an 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 5-10-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 (Cancer 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 post-translational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.
Wang
Junhua
J
School of Pharmacy and Department of Chemistry, University of Wisconsin-Madison, 777 Highland Avenue, Madison, Wisconsin 53705-2222, USA.
Ma
Mingming
M
Chen
Ruibing
R
Li
Lingjun
L
D000818:Animals; D003386:Brachyura / Q000502:physiology; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D005583:Fourier Analysis; D019476:Insect Proteins / Q000737:chemistry*; D009479:Neuropeptides / Q000032:analysis*; D010496:Pericardium / Q000166:cytology; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D019476:Insect Proteins; D009479:Neuropeptides; C005062:acetylcellulose; D002482:Cellulose