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.

Introduction

The neuropeptides are a class of important signaling molecules expressed in neurons for intercellular communications. They are consisted of short chains of amino acids and involved in numerous physiological processes. The identification and characterization of these signaling peptides are critical first steps to decipher their functions, however, the study of neuropeptides has long been challenging due to their low abundance (picomolar to nanomolar level), large dynamic range and chemical complexity. The current lack of DNA sequences for many organisms and the post-translational modifications (PTMs) commonly observed in the identified neuropeptides also make it difficult to predict the final products from the genome information. Extensive studies have been performed previously by using Edman degradation and immunocytochemistry [1], [2], [3], [4]. These traditional methods are well established to provide accurate neuropeptide sequences, but they are limited by the need for large amounts of starting materials, extensive purification steps and/or specific antibodies, as discussed previously [5]. In the past two decades, mass spectrometry (MS) has emerged as a central tool for peptide analysis. Taking advantage of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), it has been possible to detect and sequence neuropeptides with high mass accuracy and high sensitivity [6], [7]. Due to the high complexity of tissue extract samples and the need for more in-depth study at the cellular and molecular levels, a number of separation techniques, most commonly including liquid chromatography and capillary electrophoresis, have been coupled with mass spectrometry for the neuropeptide analysis for enhanced MS signals [8], [9]. Several MS-based strategies for neuropeptide analysis have been employed by our previous studies, where sample preparation methods and separation techniques are integrated for separation and purification of samples prior to MS analysis [8].

Among the separation techniques which have been coupled with mass spectrometry, capillary isoelectric focusing (CIEF) is an emerging tool for the separation and focusing of proteins and peptides. Both capillary zone electrophoresis (CZE) and CIEF are considered to have the greatest potential in MS-based proteomics [9]. In both cases, capillary is connected via buffer vials to the high voltage power supply. In classic CZE mode the separation is dependent on the analyte's charge state and molecular size, while it is limited by the loading amount. For CIEF, separation and focusing of a larger amount of samples can be realized simultaneously based on the analyte's pI value, and many applications have been previously reported in proteomics and peptidomics [10], [11], [12], [13], [14], either directly coupled with ESI or MALDI MS [15], [16], [17], or as the first dimension in multi-dimensional separations [18], [19].

Despite the advantages in separation time and efficiency, the conventional CIEF setup suffers from several problems, including sample loss caused by carrier ampholyte band shift, MS signal suppression by carrier ampholyte, disruption of separation by gas bubbles and protein/peptide adsorption or precipitation resulted from unstable wall-coating or long time of focusing [20], [21], [22]. Optimizations have been made to the conventional CIEF, most commonly on capillary coating [23], [24], [25], [26], [27], [28], [29], carrier ampholytes [30], [31], [32], [33], [34], [35], CIEF conditions [36], [37] and sample collection/interface to mass spectrometers [38], [39], [40]. However, to date only a few papers reported the study of complex peptide mixtures by CIEF, coupled with limited types of mass spectrometers. To our knowledge, CIEF has not been previously coupled with MALDI-FTMS, or applied to the study of neuropeptide extracts.

In this paper we developed a modified CIEF system for neuropeptide analysis by adding two membrane-coated joints near both ends of the capillary to prevent carrier ampholytes band shift and interferences from gas bubbles. By coupling to MALDI-FTMS, systematic studies were conducted with BSA tryptic peptides. High separation efficiency and high sensitivity were observed with good reproducibility. This new MA-CIEF/MALDI-FTMS platform has also been applied to the analysis of orcokinin family neuropeptides extracted from crustacean model organisms.