High-definition de novo sequencing of Crustacean Hyperglycemic Hormone (CHH)-family neuropeptides

Chenxi Jia, Limei Hui, Weifeng Cao, Christopher B. Lietz, Xiaoyue Jiang, Ruibing Chen, Adam D. Catherman, Paul M. Thomas, Ying Ge, Neil L. Kelleher, Ngjun Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Scopus citations


A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and posttranslational 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 largescale 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 topdown, 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.

Original languageEnglish (US)
Pages (from-to)1951-1964
Number of pages14
JournalMolecular and Cellular Proteomics
Issue number12
StatePublished - Dec 2012

ASJC Scopus subject areas

  • Analytical Chemistry
  • Molecular Biology
  • Biochemistry


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