In this article, a novel and sensitive analytical strategy for direct characterization of neuropeptides from the X-Organ-sinus gland neurosecretory system of the crayfish Orconectes limosus is presented. A desalted extract corresponding to 0.5 sinus gland equivalents was analyzed in a nanoflow liquid chromatography system coupled to quadrupole time-of-flight tandem mass spectrometry (nanoLC-QTOF MS/MS). The existence and structural identity of four crustacean hyperglycemic hormone precursor-related peptide variants and two new genetic variants of the pigment-dispersing hormone, not detected by conventional chromatographic systems, molecular cloning, or immunochemical methods before, was revealed. Studies in crustaceans have demonstrated the X-Organsinus gland neurosecretory system (XO-SG) 1 as a source of various neuropeptides (1, 2). The most relevant progress has been achieved in structure elucidation of a novel family of large peptides from the sinus gland (SG), which includes, according to their first-discovered biological activities, crustacean hyperglycaemic (CHH), moult-inhibiting (MIH) and vitellogenesis/gonad-inhibiting (VIH/GIH) hormones (3, 4). Two subfamilies may be distinguished at the preprohormone level, either containing an associated CHH precursor-related peptide sequence (CPRP), located between the signal peptide and the native hormone sequence, characteristic for the CHH subfamily, or without this CPRP sequence, as determined for the MIH/VIH/GIH subfamily. CPRPs have also been identified in other invertebrate groups such as insects and nematodes (4, 5). These neurohormones are classically considered as major physiological regulators involved in the control of carbohydrate metabolism and in various functions in development such as moult and reproduction (3, 6, 7). Additionally, the chromatophore-regulating hormones, like red pigmentconcentrating hormone (RPCH) and pigment-dispersing hormone (PDH), have been found in the XO-SG system of different crustacean species (8 -11).Different approaches, such as conventional chromatography, molecular cloning, and immunochemical methods, were used previously to investigate sinus gland components (12)(13)(14)(15)(16)(17). The limitations of these established methods to identify and characterize new peptides are the relatively low sensitivity in the case of conventional chromatography, the necessity of sufficient sequence homology in the case of molecular cloning, and the availability of specific antibodies in the case of immunochemical methods. In contrast to this, the combination of nanoscale liquid chromatography and mass spectrometry (MS) capable of tandem MS analysis (nanoLC-MS/MS) is designed for detection and sequencing of known and unknown peptides independent of their sequence homology. This is the first report on the direct analysis of neuropeptides from the neurosecretory XO-SG system using nanoLC-MS/MS.
