High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides
2012; Elsevier BV; Volume: 11; Issue: 12 Linguagem: Inglês
10.1074/mcp.m112.020537
ISSN1535-9484
AutoresChenxi Jia, Limei Hui, Weifeng Cao, Christopher B. Lietz, Xiaoyue Jiang, Ruibing Chen, Adam D. Catherman, Paul M. Thomas, Ying Ge, Neil L. Kelleher, Lingjun Li,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoA 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. 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. Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1Li L. Sweedler J.V. Peptides in the brain: mass spectrometry-based measurement approaches and challenges.Annu. Rev. Anal. Chem. 2008; 1: 451-483Crossref PubMed Scopus (112) Google Scholar). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2Morton G.J. Cummings D.E. Baskin D.G. Barsh G.S. Schwartz M.W. Central nervous system control of food intake and body weight.Nature. 2006; 443: 289-295Crossref PubMed Scopus (1900) Google Scholar, 3Fricker L.D. Neuropeptidomics to study peptide processing in animal models of obesity.Endocrinology. 2007; 148: 4185-4190Crossref PubMed Scopus (37) Google Scholar). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4Chen R. Hui L. Cape S.S. Wang J. Li L. Comparative neuropeptidomic analysis of food intake via a multi-faceted mass spectrometric approach.ACS Chem. Neurosci. 2010; 1: 204-214Crossref PubMed Scopus (39) Google Scholar, 5Hui L. Cunningham R. Zhang Z. Cao W. Jia C. Li L. 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.J. Proteome Res. 2011; 10: 4219-4229Crossref PubMed Scopus (25) Google Scholar, 6Hui L. Zhang Y. Wang J. Cook A. Ye H. Nusbaum M.P. Li L. Discovery and functional study of a novel crustacean tachykinin neuropeptide.ACS Chem. Neurosci. 2011; 2: 711-722Crossref PubMed Scopus (22) Google Scholar). Crustacean hyperglycemic hormone (CHH) 1The abbreviations used are:CHHcrustacean hyperglycemic hormoneMIHmolt-inhibiting hormoneMOIHmandibular organ-inhibiting hormoneCPRPCHH precursor related peptidePTMpost-translational modificationIMion mobilityIAAiodoacetamideFTICRFourier transform ion cyclotron resonanceUHRultra-high resolutionLTQlinear trap quadropoleECDelectron capture dissociationETDelectron transfer dissociationHCDhigher-energy collision dissociationDHB2,5-dihydroxybenzoic acidCCScollision cross sectionSGsinus glandPOpericardial organMS/MStandem mass spectrometry. 1The abbreviations used are:CHHcrustacean hyperglycemic hormoneMIHmolt-inhibiting hormoneMOIHmandibular organ-inhibiting hormoneCPRPCHH precursor related peptidePTMpost-translational modificationIMion mobilityIAAiodoacetamideFTICRFourier transform ion cyclotron resonanceUHRultra-high resolutionLTQlinear trap quadropoleECDelectron capture dissociationETDelectron transfer dissociationHCDhigher-energy collision dissociationDHB2,5-dihydroxybenzoic acidCCScollision cross sectionSGsinus glandPOpericardial organMS/MStandem mass spectrometry. family neuropeptides play a central role in energy homeostasis of crustaceans (7Chung J.S. 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Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9Webster S.G. Keller R. Dircksen H. The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction.Gen. Comp. Endocrinol. 2012; 175: 217-233Crossref PubMed Scopus (229) Google Scholar). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis. crustacean hyperglycemic hormone molt-inhibiting hormone mandibular organ-inhibiting hormone CHH precursor related peptide post-translational modification ion mobility iodoacetamide Fourier transform ion cyclotron resonance ultra-high resolution linear trap quadropole electron capture dissociation electron transfer dissociation higher-energy collision dissociation 2,5-dihydroxybenzoic acid collision cross section sinus gland pericardial organ tandem mass spectrometry. crustacean hyperglycemic hormone molt-inhibiting hormone mandibular organ-inhibiting hormone CHH precursor related peptide post-translational modification ion mobility iodoacetamide Fourier transform ion cyclotron resonance ultra-high resolution linear trap quadropole electron capture dissociation electron transfer dissociation higher-energy collision dissociation 2,5-dihydroxybenzoic acid collision cross section sinus gland pericardial organ tandem mass spectrometry. Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7Chung J.S. Zmora N. Katayama H. Tsutsui N. Crustacean hyperglycemic hormone (CHH) neuropeptidesfamily: Functions, titer, and binding to target tissues.Gen. Comp. Endocrinol. 2010; 166: 447-454Crossref PubMed Scopus (159) Google Scholar). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing. MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3Fricker L.D. 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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.J. Proteome Res. 2011; 10: 4219-4229Crossref PubMed Scopus (25) Google Scholar, 6Hui L. Zhang Y. Wang J. Cook A. Ye H. Nusbaum M.P. Li L. Discovery and functional study of a novel crustacean tachykinin neuropeptide.ACS Chem. Neurosci. 2011; 2: 711-722Crossref PubMed Scopus (22) Google Scholar, 25Ma M. Wang J. Chen R. Li L. Expanding the Crustacean neuropeptidome using a multifaceted mass spectrometric approach.J. Proteome Res. 2009; 8: 2426-2437Crossref PubMed Scopus (62) Google Scholar, 26Ma M. Bors E.K. Dickinson E.S. Kwiatkowski M.A. Sousa G.L. Henry R.P. Smith C.M. Towle D.W. Christie A.E. Li L. Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and functional genomics.Gen. Comp. 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Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis.Biochem. Biophys. Res. Commun. 2009; 390: 325-330Crossref PubMed Scopus (12) Google Scholar, 31Ma M. Szabo T.M. Jia C. Marder E. Li L. Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins.Peptides. 2009; 30: 1660-1668Crossref PubMed Scopus (54) Google Scholar, 34Ma M. Chen R. Ge Y. He H. Marshall A.G. Li L. Combining bottom-up and top-down mass spectrometric strategies for de novo sequencing of the crustacean hyperglycemic hormone from Cancer borealis.Anal. Chem. 2009; 81: 240-247Crossref PubMed Scopus (28) Google Scholar). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. 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A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9507-9512Crossref PubMed Scopus (95) Google Scholar, 40Nässel D.R. Wegener C. A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling?.Peptides. 2011; 32: 1335-1355Crossref PubMed Scopus (218) Google Scholar). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties. All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Optima grade formic acid, ACN, water, and methanol were purchased from Fisher Scientific (Pittsburgh, PA). Blue crabs Callinectes sapidus and Jonah crabs Cancer borealis were shipped from the Fresh Lobster Company (Gloucester, MA), and then maintained in artificial seawater. The animals were anesthetized in ice, and the sinus glands (SGs) and pericardial organs (POs) were dissected and collected in acidified methanol. The tissue was then homogenized and extracted with acidified methanol. After centrifugation, supernatant fractions were combined and concentrated to dryness. The sample was re-suspended in 100 μl of water for further analysis (5Hui L. Cunningham R. Zhang Z. Cao W. Jia C. Li L. 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.J. Proteome Res. 2011; 10: 4219-4229Crossref PubMed Scopus (25) Google Scholar). The detailed protocol is described in Supplementary Materials. HPLC separations were performed with a Waters Alliance HPLC system (Milford, MA). The mobile phases included solution A (water containing 0.1% formic acid) and solution B (acetonitrile (ACN) containing 0.1% formic acid). Approximately 50 μl of extract was injected onto a Phenomenex Gemini C18 column (2.1 mm i.d., 150 mm length, 5 μm particle size; Torrance, CA). The separations consisted of a 120 min gradient of 5–95% solution B. The flow rate was 0.2 ml/min. Fractions were automatically collected every 2 min with a Rainin Dynamax FC-4 fraction collector, followed by lyophilized, re-suspended in 20 μl water, and stored in −80 °C. A model 4800 MALDI-TOF/TOF analyzer (Applied Biosystems, Framingham, MA) equipped with a 200 Hz, 355 nm Nd:YAG laser was used. Acquisitions were performed in positive ion reflectron mode. Instrument parameters were set using the 4000 Series Explorer software (Applied Biosystems). Mass spectra were obtained by averaging 1000 laser shots covering mass range m/z 500–4000 in reflectron mode and m/z 2000–10000 in linear mode. MS/MS was achieved by 1 kV CID. For sample analysis, 0.4 μl of sample was spotted on MALDI plate first and allowed to dry followed by the addition of 0.4 μl 2,5-dihydroxybenzoic acid (DHB) matrix (4Chen R. Hui L. Cape S.S. Wang J. Li L. Comparative neuropeptidomic analysis of food intake via a multi-faceted mass spectrometric approach.ACS Chem. Neurosci. 2010; 1: 204-214Crossref PubMed Scopus (39) Google Scholar). An aliquot of 3 μl peptide fraction was reduced and alkylated by dithiothreitol (DTT) and iodoacetamide (IAA), followed by digestion with trypsin, Glu-C and Lys-C (41Swaney D.L. Wenger C.D. Coon J.J. Value of using multiple proteases for large-scale mass spectrometry-based proteomics.J Proteome Res. 2010; 9: 1323-1329Crossref PubMed Scopus (331) Google Scholar) (see Supplemental materials for details). Nano-LC-ESI-QTOF MS/MS was performed using a Waters nanoAcquity UPLC system coupled to a QTOF Micro mass spectrometer (Waters, Milford, MA) as described previously (5Hui L. Cunningham R. Zhang Z. Cao W. Jia C. Li L. 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.J. Proteome Res. 2011; 10: 4219-4229Crossref PubMed Scopus (25) Google Scholar). The MS/MS raw data were converted to peak list (.pkl) files using ProteinLynx software 2.4 (Waters) (5Hui L. Cunningham R. Zhang Z. Cao W. Jia C. Li L. 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.J. Proteome Res. 2011; 10: 4219-4229Crossref PubMed Scopus (25) Google Scholar). Peptides were identified by searching against an NCBInr 20090726 protein database (9330197 sequences; 3196564765 residues) using the Mascot v2.1 search engine. Trypsin or Glu-C was selected as enzyme allowing up to two missed cleavages. Carboxylmethyl cysteine was specified as fixed modifications, and methionine oxidation and pyro-Glu as variable modifications. Precursor and MS/MS tolerances were set within 30 ppm and 0.6 Da for monoisotopic mass, respectively. Peptide charge states include 1+, 2+, and 3+ charged peptides. A 0.5 μl of peptide fraction was reduced by incubation in 2.5 mm DTT for 1 h at 37 °C and desalted by C18 ZipTip and resuspended in 10 μl of 50% ACN containing 2% formic acid. The sample was analyzed using a 7T linear trap quadrupole (LTQ)/Fourier transform ion cyclotron resonance (FTICR) (LTQ-FT Ultra) hybrid mass spectrometer (Thermo Scientific Inc., Bremen, Germany) equipped with an automated chip-based nano-ESI source (Triversa NanoMate; Advion BioSciences, Ithaca, NY) as described previously (42Ge Y. Rybakova I.N. Xu Q. Moss R.L. Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12658-12663Crossref PubMed Scopus (130) Google Scholar). For CID and ECD fragmentation, individual charge states of peptide molecular ions were first isolated and then dissociated using 22–28% of n
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