title: High-definition de novo sequencing of crustacean hyperglycemic hormone (CHH)-family neuropeptides.
abstract: 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.
authors.affiliation: School of Pharmacy and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
authors.forename: Chenxi
authors.initials: C
authors.lastname: Jia
authors.forename: Limei
authors.initials: L
authors.lastname: Hui
authors.forename: Weifeng
authors.initials: W
authors.lastname: Cao
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Lietz
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Jiang
authors.forename: Ruibing
authors.initials: R
authors.lastname: Chen
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Lingjun
authors.initials: L
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry*; D003386:Brachyura; D007447:Invertebrate Hormones / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; C015947:hyperglycemic hormone, crustacean