Membrane-assisted capillary isoelectric focusing coupling with matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry for neuropeptide analysis

Abstract

Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis.

Discovery and Characterization of the Crustacean Hyperglycemic Hormone Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniques

“…MA-CIEF is a modified CIEF system developed in our lab, which provides great potential in MS-based proteomics 53 and neuropeptide analysis. 54 Compared with regular CIEF, the MA-CIEF method offers much faster separation and higher peak intensities. Orcokinin family neuropeptides feature unique p I values that are different from other neuropeptide families.…”

“…It was found that with the MA-CIEF/MALDI-FTMS based platform, MS signals were significantly improved, and more orcokinins were detected compared with those by direct MALDI-FTMS profiling without any separation. 54 . As shown in Figure 6(a), only 11 orcokinins including FDEIDRSSFA ( m/z 1186.59), NFDEIDRSGF ( m/z 1199.65), NFDEIDRSGFG ( m/z 1256.63), NFDEIDRSGFA ( m/z 1270.65), NFDEIDRSSFG ( m/z 1286.63), NFDEIDRSGFGFA ( m/z 1474.75), DFDEIDRSGFGFA ( m/z 1475.85), NFDEIDRSGFGFV ( m/z 1502.79), NFDEIDRSSFGFA ( m/z 1504.75), NFDEIDRSSFGFV ( m/z 1532.81) and NFDEIDRSSFGFN ( m/z 1547.74) were identified from 0.3 μL tissue extraction of SG using MALDI-FTMS with relatively low intensity.…”

Poly(glycidyl methacrylate‐divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis

“…Applications have been reported including coupling CIEF to both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS [8–12]. We have previously reported a membrane-assisted CIEF (MA-CIEF) system which can be coupled with MALDI-Fourier transform mass spectrometry (FTMS) for the analysis of neuropeptides and other complex peptide mixtures with enhanced efficiencies [13]. Acidic peptides are found to be more favored in the CIEF system; however, the p I values for most crustacean neuropeptide families are in the basic range of 13 to 14, which cannot be separated or detected.…”

“…Profiling of peptides in the range of 500–3000 Da was performed using an autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam 2 laser and a ground MALDI target. Detailed information on the instrument settings have been described in a previous report [13]. …”

“…A previously developed regular CIEF [13] was employed here to compare its separation efficiency and sensitivity to the IPG-CIEF. Similar to our previous report, a regular CIEF separated the BSA tryptic peptides into fractions in accordance with their p I values.…”