Chenxi Jia

Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins.

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10(-6)M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.

Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.

Mass spectrometric characterization of the neuropeptidome of the ghost crab Ocypode ceratophthalma (Brachyura, Ocypodidae)

The horn-eyed ghost crab Ocypode ceratophthalma is a terrestrial brachyuran native to the Indo-Pacific region, including the islands of Hawaii. Here, multiple mass spectrometric platforms, including matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS), were used to characterize the neuropeptidome of this species. In total, 156 peptide paracrines/hormones, representing 15 peptide families, were identified from the O. ceratophthalma supraesophageal ganglion (brain), eyestalk ganglia, pericardial organ and/or sinus gland, including 59 neuropeptides de novo sequenced here for the first time. Among the de novo sequenced peptides were isoforms of A-type allatostatin, B-type allatostatin, FMRFamide-like peptide (FLP), orcokinin, orcomyotropin and RYamide. Of particular note, were several novel FLPs including DVRAPALRLRFamide, an isoform of short neuropeptide F, and NRSNLRFamide, the orcokinins NFDEIDRSGYGFV and DFDEIDRSSFGFH, which exhibit novel Y for F and D for N substitutions at positions 10 and 1, respectively, and FDAYTTGFGHS, a member of the orcomyotropin family exhibiting a novel Y for F substitution at position 4. Taken collectively, the set of peptides described here represents the largest number of neuropeptides thus far characterized via mass spectrometry from any single crustacean, and provides a framework for future investigations of the physiological roles played by these molecules in this species.

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

UNLABELLED The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without the assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems. BIOLOGICAL SIGNIFICANCE Mass spectrometry (MS)-based neuropeptidomics aims to completely characterize the neuropeptides in a target organism as an important first step toward a better understanding of the structure and function of these complex signaling molecules. Although liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition is a powerful tool in peptidomic research, it often lacks the capability for de novo sequencing of mid-size and large peptides due to inefficient fragmentation of peptides larger than 4kDa. This study describes a multi-scale strategy for complete and confident sequence elucidation of various sizes of neuropeptides in the crustacean nervous system. The aim is to fill a technical gap where the conventional strategy is inefficient for comprehensive characterization of a complex neuropeptidome without assistance of genomic information. Nine novel neuropeptides in a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ of the spiny lobster, P. interruptus. The resulting molecular information extracted from such multi-scale peptidomic analysis will greatly accelerate functional studies of these novel neuropeptides.

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.

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.

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis

Metabolic reprogramming is a hallmark of cancer. Herein we discover that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator-associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondrial calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol-1,4,5-trisphosphate receptors (InsP3Rs), methylated PKM2 inhibits the influx of calcium from the endoplasmic reticulum to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticles perturbs the metabolic energy balance in cancer cells, leading to a decrease in cell proliferation, migration and metastasis. Collectively, the CARM1–PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to InsP3R-dependent mitochondrial functions.

Quantitative proteomics reveals that long non-coding RNA MALAT1 interacts with DBC1 to regulate p53 acetylation

Abstract Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a broadly expressed lncRNA involved in many aspects of cellular processes. To further delineate the underlying molecular mechanism, we employed a high-throughput strategy to characterize the interacting proteins of MALAT1 by combining RNA pull-down, quantitative proteomics, bioinformatics, and experimental validation. Our approach identified 127 potential MALAT1-interacting proteins and established a highly connected MALAT1 interactome network consisting of 788 connections. Gene ontology annotation and network analysis showed that MALAT1 was highly involved in five biological processes: RNA processing; gene transcription; ribosomal proteins; protein degradation; and metabolism regulation. The interaction between MALAT1 and depleted in breast cancer 1 (DBC1) was validated using RNA pull-down and RNA immunoprecipitation. Further mechanistic studies reveal that MALAT1 binding competes with the interaction between sirtuin1 (SIRT1) and DBC1, which then releases SIRT1 and enhances its deacetylation activity. Consequently, the deacetylation of p53 reduces the transcription of a spectrum of its downstream target genes, promotes cell proliferation and inhibits cell apoptosis. Our results uncover a novel mechanism by which MALAT1 regulates the activity of p53 through the lncRNA–protein interaction.

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.