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Biomedical subjects

John F Leite

Publications and source records attributed to John F Leite.

7 recordsLinked to original sources
PubMed2004

Removal of sodium and potassium adducts using a matrix additive during matrix-associated laser desorption/ionization time-of-flight mass spectrometric analysis of peptides.

Monovalent cations often associate with peptides and proteins under mass spectrometry (MS) conditions, resulting in a discernable, but often misleading, adduct cluster pattern. These adduct cluster peaks reduce the signal intensity of specific peptide species by splitting the ion population into multiple mass peaks, suppressing the ionization of neighboring low-abundance peaks, and interfering with identification of post-translational modifications. Further, monovalent contaminants tend to form a distribution of matrix cluster peaks in matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) spectra causing interference and suppression in the mass range below 1400 Da. The most common method for reduction or elimination of adduct clusters is solid-phase extraction via a pipette tip or spin column, which often leads to loss of low-abundance peptide components. In this study we describe the use of a commercially available surfactant blend that markedly reduces the adduction of monovalent cations during peptide analysis by MALDI-TOFMS.

Animals↗
PubMed2004

Cys-loop receptors: new twists and turns.

New hypotheses and predictions have arisen from recent work revealing atomic-scale or near-atomic-scale structures of receptors in the 'Cys-loop' superfamily. How general is the cation-pi interaction between the natural ligand and a tryptophan residue in the aromatic box, and does this interaction extend to other ligands? What is the pathway from the binding site to gating, and what are the conformational changes during gating and desensitization? Is current flow through intracellular 'portals' in the wall of the channel a general feature? This article discusses these and related questions, emphasizing nicotinic ACh receptors and also discussing data from other members of this superfamily.

Animals↗
PubMed2003

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: electrophysiology-coordinated photochemistry and mass spectrometry.

We characterized the differential accessibility of the nicotinic acetylcholine receptor alpha1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or approximately 50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the beta8-beta9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128-142), the cytoplasmic loop (M3-M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane alpha-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3-M4 loop are near to the lipid bilayer.

Amino Acid Sequence↗
PubMed2003

Investigation of apparent mass deviations in electrospray ionization tandem mass spectrometry of a benzophenone-labeled peptide.

In a previous study utilizing benzophenone-based topological probes to study conformationally dependent changes in mouse muscle nicotinic acetylcholine receptor (nAChR) topology, electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis led to a consistent -2.0 Da mass deviation from expected values. In the present study a synthetic peptide, corresponding to nAChR alpha1 subunit residues 130-139, was photolabeled. MS/MS analysis of this peptide using an ion trap confirmed the previously observed mass deviation, associated only with fragment ions that contain the incorporated benzophenone moiety. Analysis of peak profiles for the photolabeled ions does not indicate the typical 'peak fronting' that produces a mass shift when labile ions are prematurely ejected from the ion trap. Rather, hydrogen/deuterium (H/D) exchange experiments support the hypothesis that a chemical rearrangement involving phenyl migration and ketone formation has formed an unexpected oxidized peptide, with molecular mass 2 Da less than that expected, that is isolated for collision-induced dissociation in the ion trap together with the predicted precursor due to the broad ion isolation window specified.

Benzophenones↗
PubMed2002

Probing the topology of the glycine receptor by chemical modification coupled to mass spectrometry.

Tetranitromethane (TNM), a small aqueous reagent that specifically modifies solvent-accessible tyrosine residues to o-nitrotyrosine, was used to probe the topology of the GlyR. Homomers of human alpha1 GlyR were recombinantly expressed via a baculovirus system, affinity-purified, and reconstituted in lipid vesicles of defined composition. The native-like reconstituted receptors were then reacted with TNM, and GlyR reaction products were isolated by SDS-PAGE. After proteolytic digestion, TNM-labeled residues were identified using mass spectrometry by observing the mass shift corresponding to the nitrate moiety. In this manner, we have identified TNM modifications of tyrosine residues at positions 24, 75, 78, 161, 223, and 228 in the receptor. Of significance, nitrations at Tyr 223 and Tyr 228 occur within the first putative transmembrane helix (M1) of the receptor, and their labeling suggests a non-helical secondary structure for M1 for the glycine receptor. In a previously published report [Leite et al. (2000) J. Biol. Chem. 275, 13683], we also identified proteolytic cleavage sites within M1. Taken together, these studies support a topological model where the "historical" M1 segment cannot be entirely alpha-helical and may contain an extramembranous surface loop. Furthermore, we have also identified a tyrosine modification (Tyr 161) within a region of the N-terminal domain critical in agonist and antagonist binding.

Amino Acid Sequence↗
PubMed2002

In vitro interaction of the glycine receptor with the leptin receptor.

The coordination and regulation of electrical signals across excitable cells is a complex, dynamic phenomenon requiring, in part, the interaction of ion channels with cellular constituents. The intracellular loops or domains of many ion channel subunits have been shown to specifically bind other cellular components that act in receptor targeting, localization, regulation, or modulation of function. In this report we describe experiments in which the large intracellular loop of the alpha1 subunit of the glycine receptor (GlyR) was used as "bait" to search a human brain library for proteins that may interact with this receptor. The GlyR is the major inhibitory ligand-gated ion channel in the spinal cord and lower brainstem, and is a member of the nicotinicoid superfamily of receptors. These in vitro studies identified the leptin receptor as a potential binding partner for GlyR, and this interaction was confirmed in binding studies that used the cytoplasmic loop of the GlyR as an affinity ligand for homogenized tissue from rat spinal cords and lower brainstem. Mass spectrometric analyses of eluants showed that the leptin receptor was specifically extracted from the homogenized and solubilized tissue. The long form of the leptin receptor is expressed in the hypothalamus (as is the GlyR) and among its other functions, it quickly evokes a satiation response upon binding leptin. Our in vitro results suggest that this rapid initial response may be mediated through direct interaction of the leptin receptor with GlyR or a related nicotinicoid family member homolog.

Amino Acid Sequence↗