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Measurement of neuropeptides in crustacean hemolymph via MALDI mass spectrometry

  • Ruibing Chen
  • Mingming Ma
  • Limei Hui
  • Jiang Zhang
  • Lingjun LiEmail author
Articles

Abstract

Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C18 micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were detected, including both known ones and new peptides whose functions remain to be characterized.

Keywords

Collision Induce Dissociation Crustacean Hyperglycemic Hormone Sinus Gland Hemolymph Sample MALDI Mass Spectrometr 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Supplementary material

13361_2011_200400708_MOESM1_ESM.doc (737 kb)
Supplementary material, approximately 755 KB.

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Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • Ruibing Chen
    • 1
  • Mingming Ma
    • 1
  • Limei Hui
    • 1
  • Jiang Zhang
    • 1
  • Lingjun Li
    • 1
    Email author
  1. 1.Department of Chemistry and School of PharmacyUniversity of Wisconsin-MadisonMadisonUSA

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