Carolyn J. Cassady

A comparison of negative and positive ion time-of-flight post-source decay mass spectrometry for peptides containing basic residues

Abstract Nine peptides containing highly basic residues were studied by post-source decay (PSD) in a reflectron time-of-flight (TOF) mass spectrometer. Although these compounds produced more abundant yields of protonated ions, [M+H] + , by matrix-assisted laser desorption ionization (MALDI), deprotonated ions, [MH] − , were formed in sufficient intensities for study by tandem mass spectrometry (MS/MS). PSD was conducted in both the positive and negative ion modes. Peptide backbone cleavage involving the y-ion series is generally seen in both modes and the two Dalton difference in mass between y n ″ + and y n − can be useful in identifying these ions. For negative ions, PSD also generated c n − and (to a lesser extent) a n − , while b n + are produced from positive ions. When a peptide contains a mixture of acidic and basic residues, the negative PSD spectra are more complex and the locations of acidic residues dictate some fragmentations. The most extensive and abundant production of c n − occurs in peptides with no acidic residues. This suggests that the mechanism for c-ion formation does not involve a deprotonated side chain, but may invoke a mobile deprotonation site along the peptide amide backbone or may possibly involve a charge remote cleavage. Negative ion PSD of basic peptides yields structurally informative spectra that complement the positive data. Even highly basic peptides, such as ACTH (11–24), can be studied in the negative ion mode.

Fundamental Thermochemical Properties of Amino Acids: Gas-Phase and Aqueous Acidities and Gas-Phase Heats of Formation

The gas-phase acidities of the 20 L-amino acids have been predicted at the composite G3(MP2) level. A broad range of structures of the neutral and anion were studied to determine the lowest energy conformer. Excellent agreement is found with the available experimental gas-phase deprotonation enthalpies, and the calculated values are within experimental error. We predict that tyrosine is deprotonated at the CO(2)H site. Cysteine is predicted to be deprotonated at the SH but the proton on the CO(2)H is shared with the S(-) site. Self-consistent reaction field (SCRF) calculations with the COSMO parametrization were used to predict the pK(a)s of the non-zwitterion form in aqueous solution. The differences in the non-zwitterion pK(a) values were used to estimate the free energy difference between the zwitterion and nonzwitterion forms in solution. The heats of formation of the neutral compounds were calculated from atomization energies and isodesmic reactions to provide the first reliable set of these values in the gas phase. Further calculations were performed on five rare amino acids to predict their heats of formation, acidities, and pK(a) values.

Characterization of the Organic Component of Low-Molecular-Weight Chromium-Binding Substance and Its Binding of Chromium

Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism.

MALDI MS in-source decay of glycans using a glutathione-capped iron oxide nanoparticle matrix.

A new matrix-assisted laser desorption ionization (MALDI) mass spectrometry matrix is proposed for molecular mass and structural determination of glycans. This matrix contains an iron oxide nanoparticle (NP) core with gluthathione (GSH) molecules covalently bound to the surface. As demonstrated for the monosaccharide glucose and several larger glycans, the mass spectra exhibit good analyte ion intensities and signal-to-noise ratios, as well as an exceptionally clean background in the low mass-to-charge (m/z) region. In addition, abundant in-source decay (ISD) occurs when the laser power is increased above the ionization threshold; this indicates that the matrix provides strong energy transfer to the sample. For five model glycans, ISD produced extensive glycosidic and cross-ring cleavages in the positive ion mode from singly charged precursor ions with bound sodium ions. Linear, branched, and cyclic glycans were employed, and all were found to undergo abundant fragmentation by ISD. (18)O labeling was used to clarify m/z assignment ambiguities and showed that the majority of the fragmentation originates from the nonreducing ends of the glycans. Studies with a peracetylated glycan indicated that abundant ISD fragmentation occurs even in the absence of hydroxyl groups. The ISD product ions generated using this new matrix should prove useful in the sequencing of glycans.

Mass Spectrometric and Spectroscopic Studies of the Nutritional Supplement Chromium(III) Nicotinate

Despite chromium nicotinate’s popular use as a chromium nutritional supplement, the structure and composition of chromium nicotinate have only been poorly described. As solid chromium nicotinate is intractable, being insoluble or unstable in common solvents, studies on the solid have been limited, and studies of the solution from which the “compound” precipitates have additionally provided little additional data. The results of mass spectrometric and spectroscopic investigations designed to further elucidate the structure and composition of chromium nicotinate are described. The results demonstrated that the three common methods for producing “chromium nicotinate” all yield different compounds, all of which are polymers of Cr(III), oxygen-bound nicotinate, hydroxide, and water. Implications for interpreting results of nutritional studies of “chromium nicotinate” are discussed.

Negative ion production from peptides and proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M-H](-) ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5-dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H](+) ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1-14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies.

The effects of chromium(III) coordination on the dissociation of acidic peptides.

The complexes formed between chromium(III) and synthetic acidic peptides were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometer equipped with electrospray ionization (ESI). Neutral peptides and peptides containing one, two, and multiple acidic residues were studied. Formation of [M + Cr-2H]+ occurred for all peptides. Three noteworthy features were found in the CID spectra of [M + Cr-2H]+. The first is that fewer fragment ions were produced from [M + Cr-2H]+ than from [M + H]+. The reason may be that multiple coordination between chromium(III) and carboxylate or carbonyl groups hinders the production of fragment ions by continuing to bind pieces of the peptide to chromium(III) after cleavage of bonds within the peptide. The second feature is loss of CO from [M + Cr-2H]+ and [y(n) + Cr-H]+. A mechanism involving coordination of chromium(III) with carboxylate groups is proposed to rationalize elimination of CO. The third feature is that chromium(III) is retained in all fragment ions, indicating strong binding of the metal ion to the peptides. The complex [M + 2Cr-5H]+ is formed as the peptide chain length and number of acidic residues increases. Longer peptides have more sites to coordinate with chromium(III) and more conformational flexibility. In addition, formation of [M + Cr-2H]+ from AGGAAAA-OCH(3), which has no carboxylic acid groups, suggests that chromium(III) can coordinate with sites on the peptide backbone, albeit in low abundance. In the negative mode, [M + Cr-4H](-) was only found for peptides containing four or more carboxylic acid groups. This is consistent with deprotonated carboxylic acid groups being involved in chromium(III) coordination and with chromium existing in the 3 + state in the gas-phase ions.

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 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.

Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines

Transition metal-polyalanine complexes were analyzed in a high-capacity quadrupole ion trap after electrospray ionization. Polyalanines have no polar amino acid side chains to coordinate metal ions, thus allowing the effects metal ion interaction with the peptide backbone to be explored. Positive mode mass spectra produced from peptides mixed with salts of the first row transition metals Cr(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(I), and Cu(II) yield singly and doubly charged metallated ions. These precursor ions undergo collision-induced dissociation (CID) to give almost exclusively metallated N-terminal product ions whose types and relative abundances depend on the identity of the transition metal. For example, Cr(III)-cationized peptides yield CID spectra that are complex and have several neutral losses, whereas Fe(III)-cationized peptides dissociate to give intense non-metallated products. The addition of Cu(II) shows the most promise for sequencing. Spectra obtained from the CID of singly and doubly charged Cu-heptaalanine ions, [M + Cu - H](+) and [M + Cu](2+) , are complimentary and together provide cleavage at every residue and no neutral losses. (This contrasts with [M + H](+) of heptaalanine, where CID does not provide backbone ions to sequence the first three residues.) Transition metal cationization produces abundant metallated a-ions by CID, unlike protonated peptides that produce primarily b- and y-ions. The prominence of metallated a-ions is interesting because they do not always form from b-ions. Tandem mass spectrometry on metallated (Met = metal) a- and b-ions indicate that [b(n)  + Met - H](2+) lose CO to form [a(n)  + Met - H](2+), mimicking protonated structures. In contrast, [a(n)  + Met - H](2+) eliminate an amino acid residue to form [a(n-1)  + Met - H](2+), which may be useful in sequencing.