Quantitative determination of nitric oxide from tissue samples using liquid chromatography-Mass spectrometry.

Ever since it was found to mediate the endothelium-dependent dilation of blood vessels, nitric oxide (NO) has generated enormous research interest throughout the biological sciences. Over thirty years of research has identified NO as a ubiquitous and versatile regulatory factor utilized by both vertebrates and invertebrates. The short lifetime and low concentration of NO make quantitation difficult. Here we report a method for measuring NO using the selective reaction with 2-​(4-​carboxyphenyl)-​4,​5-​dihydro-​4,​4,​5,​5-​tetramethyl-1H-​imidazolyl-​1-​oxy-​3-​oxide (carboxy-PTIO) to form carboxy-PTI. We used tandem mass spectrometry to verify the validity of this reaction, and liquid chromatography - mass spectrometry to quantitate the amount of carboxy-PTI formed. Using diethylamine nonoate as a NO donor we demonstrate this method can quantitate NO concentrations with a detection limit of 5 nM. We successfully determined the amount of NO generated endogenously by frog heart/aorta when stimulated by carbachol, a non-selective acetylcholine receptor agonist. Based on these results, we suggest that this technique can be useful for the quantitative determination of NO in biological samples.•We report a method to measure NO by reacting it with carboxy-PTIO to form carboxy-PTI.•The carboxy-PTI is quantified by liquid chromatography mass spectrometry (LCMS).•This method can quantitate NO concentrations ranging from 5 nM to 1 µM.

Evidence of NAAG-family tripeptide NAAG2 in the Drosophila nervous system.

N-acetylaspartylglutamate (NAAG) is a common neurotransmitter in the mammalian nervous system; however, it has never been reported in the nervous system of the fruit fly, Drosophila melanogaster. Using antiserum against NAAG, we localized NAAG-like immunoreactivity to neurons in the ventral nerve cord and to type Is glutamatergic nerve terminals in larval neuromuscular junctions. Using liquid chromatography tandem mass spectrometry (LC-MS), we failed to find NAAG but found the related peptide N-acetylaspartylglutamylglutamate (NAAG2 ) in Drosophila CNS and body wall tissue. This is the first report of any NAAG-family peptide in the nervous system of Drosophila and is also the first report of NAAG2 being present in a much higher concentration than NAAG in the nervous system of any species. Thus, the larval fruit fly presents an interesting model for the study of the functional role of NAAG2 of which very little is known-especially in the absence of an abundance of NAAG.

Native Mass Spectrometry of Antimicrobial Peptides in Lipid Nanodiscs Elucidates Complex Assembly.

Antimicrobial peptides (AMPs) are generally cationic and amphipathic peptides that show potential applications to combat the growing threat of antibiotic resistant infections. AMPs are known to interact with bacterial membranes, but their mechanisms of toxicity and selectivity are poorly understood, in part because it is challenging to characterize AMP oligomeric complexes within lipid bilayers. Here, we used native mass spectrometry to measure the stoichiometry of AMPs inserted into lipoprotein nanodiscs with different lipid components. Titrations of increasing peptide concentration and collisional activation experiments reveal that AMPs can exhibit a range of behaviors from nonspecific incorporation into the nanodisc to formation of specific complexes. This new approach to characterizing formation of AMP complexes within lipid membranes will provide unique insights into AMP mechanisms.

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry.

Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion.

Gas phase hydrogen/deuterium exchange of arginine and arginine dipeptides complexed with alkali metals.

The hydrogen/deuterium (H/D) exchange of protonated and alkali-metal cationized Arg-Gly and Gly-Arg peptides with D(2)O in the gas phase was studied using electrospray ionization quadropole ion trap mass spectrometry. The Arg-Gly and Gly-Arg alkali metal complexes exchange significantly more hydrogens than protonated Arg-Gly and Gly-Arg. We propose a mechanism where the peptide shifts between a zwitterionic salt bridge and nonzwitterionic charge solvated conformations. The increased rate of H/D exchange of the alkali metal complexes is attributed to the peptide metal complexes' small energy difference between the salt-bridge conformation and the nonzwitterionic charge-solvated conformation. Implications for the applicability of this mechanism to other zwitterionic systems are discussed.

Infrared spectroscopy and structure of photochemically protonated biomolecules in the gas phase: a noradrenaline analogue, lysine and alanyl alanine.

A novel photochemical technique combined with mass spectrometry and resonant infrared multiphoton dissociation spectroscopy (R-IRMPD) has been used to record infrared vibrational spectra of the free protonated noradrenaline analogue, 2-amino-1-phenylethanol (APE-H(+)), the amino acid, lysine (Lys-H(+)), and the dipeptide, alanyl alanine (Ala-Ala-H(+)) in the gas phase. Coupling their spectra, obtained in the OH, NH and CH stretch regions, with ab initio calculations has allowed assignment of their preferred protonation sites and conformations. This simple technique will have wide applicability in future investigations of protonated biomolecular structure and conformation.

Adding water to sugar: a spectroscopic and computational study of alpha- and beta-phenylxyloside in the gas phase.

The gas phase structures of phenyl alpha- and beta-d-xylopyranoside (alpha- and beta-pXyl) and their mono-hydrates have been investigated using a combination of resonant two-photon ionization (R2PI), ultra-violet hole-burning and resonant infrared ion dip spectroscopy, coupled with density functional theory (DFT) and ab initio computation. The hole-burning experiments indicate the population of a single conformer only, in each of the two anomers. Their experimental and calculated infrared spectra are both consistent with a conformational assignment corresponding to the computed global minimum configuration. All three OH groups are oriented towards the oxygen atom (O1) on the anomeric carbon atom to form an all trans(ttt) counter-clockwise chain of hydrogen bonds. The mono-hydrates, alpha- and beta-pXyl(H(2)O) each populate two distinct structures in the molecular beam environment, with the water molecule inserted between OH4 and OH3 or between OH3 and OH2 in alpha-pXyl(H2O), and between OH2 and O1 in either of two alternative orientations, in beta-pXyl(H2O). In all of the mono-hydrated xyloside complexes, the water molecule inserts into the weakest link of the sugar molecules' hydrogen-bonded chain of hydroxy groups, creating a single extended chain, strengthened by co-operativity. The all-trans configuration of the xylose moiety is retained and the mono-hydrate structures correspond to those calculated to lie at the lowest relative energies.