Zichuan Zhang

Recent advances in coupling capillary electrophoresis-based separation techniques to ESI and MALDI-MS.

Coupling CE‐based separation techniques to MS creates a powerful platform for analysis of a wide range of biomolecules from complex samples because it combines the high separation efficiency of CE and the sensitivity and selectivity of MS detection. ESI and MALDI, as the most common soft ionization techniques employed for CE and MS coupling, offer distinct advantages for biomolecular characterization. This review is focused primarily on technological advances in combining CE and chip‐based CE with ESI and MALDI‐MS detection in the past five years. Selected applications in the analyses of metabolites, peptides, and proteins with recently developed CE‐MS platforms are also highlighted.

Combining capillary electrophoresis matrix-assisted laser desorption/ionization mass spectrometry and stable isotopic labeling techniques for comparative crustacean peptidomics.

Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides.

Advancing Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Capillary Electrophoresis Analysis of Peptides

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.

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.

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.

Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging.

Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.

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

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing‐matrix‐assisted laser desorption/ionization mass spectrometry (CIEF‐MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate‐divinylbenzene) (GMA‐DVB)‐based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off‐line coupling of IPG‐CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG‐CIEF. Enhanced neuropeptide detection is also observed after coupling IPG‐CIEF with MALDI MS.

Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures

Herein, we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogeneous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultrahigh mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling a 4- to 6-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

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

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.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

Semi-automated liquid chromatography-mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides.

A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC separation. The LC flow and matrix flow are collected on a commercially available MALDI plate which is mechanically controlled and analyzed with MALDI MSI subsequently. Both tryptic peptides digested from bovine serum albumin (BSA) and endogenous neuropeptides extracted from the blue crab Callinectes sapidus are analyzed with this novel LC-MSI platform. Compared with regular offline LC fractionation coupled with MALDI MS detection, LC-MSI exhibits significantly increased MS signal intensity due to retaining of temporal resolution from separation dimension via continuous sampling, which results in increased number of peptides detected and accurate quantitation. In addition, imaging signals enable improved data analysis based on either mass-to-charge ratio or retention time, which is extremely beneficial for the analysis of complex analytes. These findings have demonstrated the potential of employing LC-MSI platform for enhanced proteomics and peptidomics studies.