Gas-Phase Deprotonation of the Peptide Backbone for Tripeptides and Their Methyl Esters with Hydrogen and Methyl Side Chains

The gas-phase acidities (GAs) of six tripeptides (GlyGlyGly, GlyAlaGly, AlaGlyAla, AlaAlaAla, AibAibAib, and SarSarSar) and their methyl esters were obtained by proton transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and G3­(MP2) molecular orbital theory calculations. All six peptides have GAs in the range 321.0–323.7 kcal/mol. Their deprotonation to produce [M – H]⁻ occurs at the C-terminal carboxylic acid group. The tripeptides are about 10 kcal/mol more acidic than the amino acids glycine (Gly) and alanine (Ala). This is consistent with the extensive hydrogen bonding that was found in the tripeptide structures. For the methyl esters, deprotonation occurs at the peptide backbone. G3­(MP2) calculations show that the most energetically favored site of deprotonation is an amide nitrogen, with the central amide being generally preferred. Nitrogen deprotonation requires 10–20 kcal/mol less energy than deprotonation at a methylene carbon. Only three of the methyl esters (GlyGlyGly-OMe, GlyAlaGly-OMe, and AlaAlaAla-OMe) deprotonate experimentally by electrospray ionization. Experimental GAs for these esters are in the range of 336.7–338.1 kcal/mol, in excellent agreement with the calculated G3­(MP2) values. Experimental GAs could not be obtained for the other three methyl esters (AlaGlyAla-OMe, AibAibAib-OMe, and SarSarSar-OMe) because they did not produce sufficient deprotonated molecular ions. Trisarcosine methyl ester, SarSarSar-OMe, cannot be deprotonated at a central amide nitrogen because methyl groups are present at these sites; consequently, it has a high G3­(MP2) GA value (less acidic) of 350.6 kcal/mol for deprotonation at the N-terminal nitrogen. For AlaGlyAla-OMe and AibAibAib-OMe, calculations of van der Waals and solvent accessible surfaces reveal that methyl groups are blocking the amide nitrogen sites. Therefore, conformational and steric hindrance effects are limiting the ability of these peptide methyl esters to deprotonate in the mass spectrometer

An Experimental and Computational Investigation into the Gas-Phase Acidities of Tyrosine and Phenylalanine: Three Structures for Deprotonated Tyrosine

Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3­(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3­(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3­(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol

The importance of the backbone and C-terminus to mass spectrometry studies of peptides: gas-phase dissociation and acidity studies

The work described in this dissertation shows the importance of the C-terminus and the backbone in dissociation and deprotonation of peptides. Characteristic dissociative behavior can be extremely valuable in proteomic applications and for mechanistic interpretation of mass spectra. Identification of the deprotonation site of a peptide is also important to the development of mechanisms for mass spectrometry, as dissociation is often charge-directed. Collsion-induced dissociation (CID) and electron transfer dissociation (ETD) have been used to discover distinguishing features of peptide dissociation related to the presence of an amidated C-terminus (-CONH_2) compared with the standard acid C-terminus (-COOH). Protonated peptide acids and amides are found to produce practically identical spectra, except for increased ammonia loss from the precursor for the peptide amide. ETD of multiply-protonated peptide acids and amides also produce similar spectra, although dissociation trends related to the basic amino acid residues (e.g. Arg, His, Lys) are observed for the peptide pairs. Deprotonated peptide acids and amides produced several unique product ions that differentiated the analogs. In CID experiments, abundant c_m-2^- (m = the number of amino acid residues in the peptide) formed for many of the peptide amides and c_m-3^- formed for many of the peptide acids. Supporting computational work by Michele Stover of the Dixon Group shows that the process leading to c_m-2^- from peptide amides is less endothermic than the same process for peptide acids. Gas-phase acidities (GA) have been determined for tyrosine, phenylalanine, their amino acid amides and 4-(4-hydroxyphenyl)-2-butanone using bracketing ion/molecule reactions. Two deprotonated species of tyrosine are observed, corresponding to deprotonation at the carboxylic acid -OH and the phenolic -OH. The two GAs determined for tyrosine are: GA(1) = 332.4 ± 2.2 kcal/mol and GA(2) = 333.5 ± 2.4 kcal/mol. Tyrosine amide has an experimental GA of 336.4 ± 2.7 kcal/mol, phenylalanine has a GA of 332.5 ± 2.2 kcal/mol, phenylalanine amide has a GA of 345.8 ± 3.8 kcal/mol, and 4-(4-hydroxyphenyl)-2-butanone has a GA of 339.6 ± 3.0 kcal/mol. The GAs of six tripeptides (with alkyl or H- side chains) have been determined. All of the experimental GAs fall within a 1.2 kcal/mol range, which is consistent with the C-terminus being the most acidic site on the peptides. The GAs of three methyl esters have been determined, demonstrating the ability of peptides to deprotonate on the backbone. The peptide methyl ester GAs are all very similar and fall within a 1.4 kcal/mol range. Computational results indicate that these methyl esters are deprotonating at the central amide NH. Three other methyl esters could not be deprotonated by ESI, because of conformation and steric hindrance to the deprotonation site. (Published By University of Alabama Libraries

An Experimental and Computational Study of the Gas-Phase Acidities of the Common Amino Acid Amides

Using proton-transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and correlated molecular orbital theory at the G3­(MP2) level, gas-phase acidities (GAs) and the associated structures for amides corresponding to the common amino acids have been determined for the first time. These values are important because amino acid amides are models for residues in peptides and proteins. For compounds whose most acidic site is the C-terminal amide nitrogen, two ions populations were observed experimentally with GAs that differ by 4–7 kcal/mol. The lower energy, more acidic structure accounts for the majority of the ions formed by electrospray ionization. G3­(MP2) calculations predict that the lowest energy anionic conformer has a cis-like orientation of the [−C­(O)­NH]⁻ group whereas the higher energy, less acidic conformer has a trans-like orientation of this group. These two distinct conformers were predicted for compounds with aliphatic, amide, basic, hydroxyl, and thioether side chains. For the most acidic amino acid amides (tyrosine, cysteine, tryptophan, histidine, aspartic acid, and glutamic acid amides) only one conformer was observed experimentally, and its experimental GA correlates with the theoretical GA related to side chain deprotonation

Co-localization of NQO1, Sirt2 and acetyl tubulin in 16HBE cells.

<p>(A) Co-immunostaining for NQO1 (green) and acetyl α-tubulin (red) showing co-localization on mitotic structures. (B) Co-immunostaining for NQO1 (green) and Sirt2 (red) showing co-localization on centrosome(s). Arrows indicate co-localization between high intensity immunostaining for NQO1, acetyl α-tubulin and Sirt2 in different stages of the centriole cycle. (C, centrosome(s); MS, mitotic spindles; MB, midbody).</p

Redox modulation of NQO1

<div><p>NQO1 is a FAD containing NAD(P)H-dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C-terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β-lapachone which caused rapid oxidation of NAD(P)H NQO1 could be readily pulled-down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β-lapachone demonstrating that under non-denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)⁺/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co-localize with acetyl α-tubulin and the NAD⁺-dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle NQO1 may provide NAD⁺ for Sirt2-mediated deacetylation of microtubules. Overall, NQO1 may act as a redox-dependent switch where the protein responds to the NAD(P)⁺/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.</p></div

Co-localization of NQO1 with microtubules/acetylated microtubules in TrHBMEC.

<p>Co-immunostaining for NQO1 (A180) and α-tubulin showing co-localization on microtubules.(A) Co-localization of NQO1 with α-tubulin using fluorescently-labeled secondary antibodies. (B) Co-localization of NQO1 with α-tubulin/acetyl α-tubulin using PLA-based detection. Immunostaining was performed as described in <i>Materials and methods</i>.</p

Intracellular oxidation of pyridine nucleotides induces a conformational change in NQO1.

<p>(A) 16HBE cells were treated with DMSO or β-lapachone (10μM) for 2 h after which NQO1 was immunoprecipitated using anti-NQO1 antibodies. (B) Human cell lines (16HBE, ARPE19, TrHBMEC) were treated with β-lapachone (10μM) for the indicated times after which NQO1 was immunoprecipitated using antibodies that recognize the C-terminal domain of NQO1. (C) Intracellular levels of NADH and NAD⁺ were measured by mass spectrometry in 16HBE cells treated with β-lapachone (10μM) for 2 h in the absence and presence of the PARP inhibitor olaparib (1μM). Results are the mean ± standard deviation, n = 3. (D) Immunoprecipitation of NQO1 from 16HBE cells treated with β-lapachone (10μM) in the absence and presence of olaparib (1μM) for 2 h. Reaction conditions are described in <i>Materials and methods</i>.</p

Increased immunostaining for NQO1 in 16HBE cells following treatment with β-lapachone.

<p>16HBE cells were treated with DMSO or β-lapachone (10μM) for the indicated times then processed for immunocytochemistry and confocal analysis as described in <i>Materials and Methods</i>. Immunostaining for NQO1 was performed under non-denaturing conditions using the A180 antibody combined with DAPI nuclear staining.</p

Reduced pyridine nucleotides and dicumarol induce a conformational change in NQO1.

<p>(A) Comparison of the ability of antibodies which bind to helix 7 (A180) and antibodies which bind to the C-terminal domain (C-Term) to immunoprecipitate rhNQO1 in the absence and presence of NADH. (B, C) Immunoprecipitation of rhNQO1 with the A180 antibody in the absence and presence of NADH or NADPH. (D) Immunoprecipitation of rhNQO1 by C-Term and A180 antibodies in the absence and presence of dicumarol. (E) The effect of NADH and dicumarol on the migration of rhNQO1 in non-denaturing PAGE. Reaction conditions for immunoprecipitation studies and non-denaturing PAGE are described in <i>Materials and methods</i>.</p