The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations.
Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.
Stemmler
Elizabeth A
EA
Department of Chemistry, Bowdoin College, Brunswick, ME 04011, USA. estemmle@bowdoin.edu
Gardner
Noah P
NP
Guiney
Maureen E
ME
Bruns
Emily A
EA
Dickinson
Patsy S
PS
D000818:Animals; D003386:Brachyura / Q000737:chemistry*; D002412:Cations / Q000032:analysis / Q000737:chemistry; D005123:Eye / Q000737:chemistry*; D005583:Fourier Analysis; D008672:Metals, Alkali / Q000737:chemistry; D008121:Nephropidae / Q000737:chemistry*; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D011522:Protons; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002412:Cations; D008672:Metals, Alkali; D009842:Oligopeptides; D011522:Protons; C022059:red pigment-concentrating hormone; D011761:Pyrrolidonecarboxylic Acid