Development of a multi-matrix LC-MS/MS method for urea quantitation and its application in human respiratory disease studies.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

Mass spectrometric imaging of neuropeptides in decapod crustacean neuronal tissues.

The emerging technology mass spectrometric imaging (MSI) provides an attractive opportunity to detect and probe the molecular content of tissues in an anatomical context. This powerful methodology has been applied extensively to the localization of proteins, peptides, pharmaceuticals, metabolites, lipids, and other biological and chemical compounds in tissues. Herein, we present a method developed specifically for mapping neuropeptides in crustacean neuronal tissues. Both cryostat tissue sectioning and whole-mount tissue blotting techniques are highlighted. Careful sample preparation is essential for obtaining sufficient analyte/matrix mixing while retaining the spatial localization of the neuropeptides. Several matrix application apparatus and techniques are described and compared. Furthermore, three-dimensional (3D) imaging has been developed to provide detailed information about the distribution of neuropeptides within 3D structure of a crustacean brain.

Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach.

Feeding behavior is a fundamental aspect of energy homeostasis and is crucial for animal survival. This process is regulated by a multitude of neurotransmitters including neuropeptides within a complex neuroendocrine system. Given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. Here we report comparative neuropeptidomic analysis of brain and major neuroendocrine organ in a crustacean model organism in response to feeding. A multi-faceted approach employing direct tissue matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), stable isotopic labeling of neuropeptide extracts for quantitation, and mass spectrometric imaging (MSI) has been employed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and the pericardial organ (PO) in the crab Cancer borealis. Multiple neuropeptides exhibited changes in abundance after feeding, including RFamides, Cancer borealis tachykinin related peptides (CabTRPs), RYamides, and pyrokinins. By combining quantitative analysis of neuropeptide changes via isotopic labeling of brain extract and MSI mapping of neuropeptides of brain slices, we identified the boundary of olfactory lobe (ON) and median protocerebrum (MPC) area as two potential feeding centers in the crab brain.

Mass spectrometric characterization of neuropeptides.

Because of their great biological significance, neuropeptides are the subject of intensive research. Mass spectrometry (MS) is a highly informative and sensitive method used for detecting and characterizing these compounds. Successful MS analysis of neuropeptides is dependent on careful sample preparation. Herein, we present two common sample preparation strategies: direct tissue analysis and pooled tissue extraction coupled with fractionation.

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus.

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val(1)]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.