Zhi-Heng Huang

Characterization of diacylglycerylphosphocholine molecular species by FAB-CAD-MS/MS: A general method not sensitive to the nature of the fatty acyl groups

The use of negative ion mode fast-atom bombardment-collision-activated dissociation-tandem mass spectrometry (FAB-CAD-MS/MS) for diacylglycerylphosphocholine molecular species determinations was investigated for 24 naturally occurring and synthetic compounds. The previously proposed method of selecting [M-15]− as the parent ion and using the relative abundance of the carboxylate daughter ions to distinguish the positions of esterification was found to be unreliable in cases where the fatty acyl group at sn1 was much larger than that at sn2. The predicted greater abundance of the sn2 carboxylate daughter, relative to the sn1 carboxylate daughter, was also violated when polyunsaturated fatty acyl groups were esterifred at sn2. In addition, several marginal cases were found where the ratio of intensities of the sn2/sn1 carboxylate daughters followed the expected pattern (sn2 > sn1) initially, but reversed over extended scanning time. The use of an alternative FAB-CAD-MS/MS method is proposed where the [M-B6]− ion is selected as the precursor and the relative intensities of the daughters resulting from loss of the free fatty acids at snl and sn2 are determined. In every case examined to date, the ion formed by loss of the free acid from the sn2 position was always more abundant. Because the parent ion is equivalent to the phosphatidic acid ion, this technique should be equally applicable to all other phospholipid classes where this fragment ion is present in the spectrum.

Comparison of sulfoquinovosyl diacylglycerol from spinach and the purple bacteriumRhodobacter sphaeroides by fast atom bombardment tandem mass spectrometry

Isolated sulfoquinovosyl-diacylglycerol (SQD) from spinach and the purple bacteriumRhodobacter sphaeroides provide two sources of very different molecular species of SQD. We were able to demonstrate by fast atom bombardment-collisionally activated dissociation tandem mass spectrometry in the negative ion mode that the sulfoquinovosyl head group of the plant and bacterial lipids can be characterized by the common fragmentation pattern found in the spectra of both samples. Differences in the acyl functions from the two sources were also identified by this technique. SQD specific fragments are found atm/z 299, 283, 241, 225, 165 and 80 which indicate the presence of the sulfoquinovosyl moiety. The two predominant molecular species found in spinach contain palmitic and linolenic ([M−H]− atm/z 815) or two linolenic acids ([M−H]− atm/z 837) in thesn−1 andsn−2 positions, while the two major species of the bacterial lipid contain palmitic and 18∶1 (vaccenic) cids ([M−H]− atm/z 819) or stearic and 18∶1 (vaccenic) acids, ([M−H]− atm/z 847), respectively.

Characterization of N-ethoxycarbonyl ethyl esters of amino acids by mass spectrometry

Derivatization of amino acids by using ethyl chloroformate-ethanol-pyridine provides volatile products, N-ethoxycarbonyl amino acid ethyl esters (ECEEs), which are easily amenable to GC or GC-MS analysis. MS behavior of these compounds under electron-impact has been studied. The fragments observed in the spectra facilitate recognition of commonly occurring protein amino acids and characterization of unknown analogues.

Analysis of amino acids by gas chromatography—flame ionization detection and gas chromatography—mass spectrometry: Simultaneous derivatization of functional groups by an aqueous-phase chloroformate-mediated reaction

The one-step ethyl chloroformate derivatization of amino acids in an aqueous medium is extended with the use of a variety of alkyl chloroformate reagents. This provides a new and convenient procedure for preparing esters with different alkoxy groups. A new mechanism for esterification during chloroformate derivatization is proposed based on the formation of an intermediate mixed carboxylic-carbonic acid anhydride followed by the exchange with an alcohol. Among the different reagents investigated, isobutyl chloroformate derivatized amino acids were found to provide more sensitivity for analyses by GC-flame ionization detection and GC-MS relative to derivatives prepared by other alkyl chloroformates.

Mercaptobenzothiazoles: A new class of matrices for laser desorption ionization mass spectrometry

We report 2-mercaptobenzothiazole (MBT) and its analogs as a class of new matrices for matrix-assisted laser desorption mass spectrometry (MALDI-MS) at 337 nm. MBT has been used successfully for the desorption of proteins up to 100,000 u. A comparison with sinapinic acid and α-cyano-4-hydroxycinnamic acid indicates that MBT provides the same level of sensitivity and resolution, but offers the advantage of higher tolerance to sample contaminants such as ionic detergents. 5-Chloro-2-mercaptobenzothiazole (CMBT), an analog of MBT, has been found not only effective for the analysis of peptides, low-mass proteins, and glycolipids, but also superior to conventional matrices for the analysis of muropeptides and at least some oligosaccharides. CMBT also exhibits excellent experimental reproducibility of MALDI-MS results owing to the homogeneous crystallization of the analyte/matrix mixture over the entire sample surface area. Finally, all five mercaptobenzothiazoles studied in this work are shown to be well suited for synthetic polymer analysis.

Charge remote fragmentation of peptides following attachment of a fixed positive charge: A matrix-assisted laser desorption/ionization postsource decay study

A fixed positive charge can be placed at the N-terminus of a peptide by addition of a tris[(2,4,6-trimethoxyphenyl)phosphonium]acetyl group. The usefulness of these charged derivatives has been demonstrated in fast atom bombardment mass spectrometry and in matrix-assisted laser desorption/ionization mass spectrometry. After ion formation and acceleration, these derivatized peptide ions dissociate and their fragment ions can be analyzed in a postsource decay experiment by using a time-of-flight mass spectrometer. The matrix-assisted laser desorption/ionization-postsource decay spectra are very different from what may be expected based on fragment ions observed from protonated peptide molecules. Cleavage of CHR-C(O) bonds dominates to form a series of a type ions. Mechanistic possibilities are evaluated. When aspartic acid residues are encountered, the chemistry radically changes. This appears to be due to the formation of geometrically accessible intermediates that can dissociate via low energy processes. This chargeremote chemistry parallels that for much simpler systems, resulting in spectra that are very easy to interpret.

N-Acylphosphatidylethanolamine in Dry and Imbibing Cottonseeds (Amounts, Molecular Species, and Enzymatic Synthesis)

N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), was recently shown to be synthesized from PE and free fatty acids in cotyledons of cotton (Gossypium hirsutum L.) seedlings (K.D. Chapman, T.S. Moore [1993] Plant Physiol 102: 761–769). Here we report that NAPE is present in dry seeds of cotton and increases with time of imbibition from 2.31 nmol/seed in dry seeds to 4.26 nmol/seed in 4-h-soaked seeds. Total phospholipid/seed also increased such that the relative percentage of NAPE was similar in dry and soaked seeds (2.3 mol% compared to 2.6 mol%, respectively). The major molecular species of NAPE were identified in both dry and soaked seeds by fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry as 16:0/18:2-PE(N-palmitoyl), 16:0/18:2-PE(N-linoleoyl), and 18:2/18:2-PE(N-palmitoyl). The specific activity of NAPE synthase in seed extracts increased with increasing time of imbibition from 35 pmol h-1 mg-1 protein in dry seeds to 129 pmol h-1 mg-1 protein in 4-h-soaked seeds. Collectively, our results indicate that NAPE is present in dry cottonseeds and synthesized during imbibition. The biosynthesis of NAPE provides a mechanism for maintaining membrane integrity during seed rehydration and may indicate that NAPE plays a protective role in intracellular membranes of plant tissues, as has been suggested for intracellular membranes of animal tissues.

Analysis of acylcarnitines as their N-demethylated ester derivatives by gas chromatography-chemical ionization mass spectrometry

A novel approach to the analysis of acylcarnitines has been developed. It involves a direct esterification using propyl chloroformate in aqueous propanol followed by ion-pair extraction with potassium iodide into chloroform and subsequent on-column N-demethylation of the resulting acylcarnitine propyl ester iodides. The products, acyl N-demethylcarnitine propyl esters, are volatile and are easily analyzed by gas chromatography-chemical ionization mass spectrometry. For medium-chain-length (C4-C12) acylcarnitine standards, detection limits are demonstrated to be well below 1 ng starting material using selected ion monitoring. Well-separated gas chromatographic peaks and structure-specific mass spectra are obtained with samples of synthetic and biological origin. Seven acylcarnitines have been characterized in the urine of a patient suffering from medium-chain acyl-CoA dehydrogenase deficiency.

Gas chromatographic determination of amines, aminoalcohols and acids after treatment with alkyl chloroformates

Abstract Amines such as tyramine, tryptamine, spermine, and spermidine; aminoalcohols (catecholamines and related compounds); and their acidic metabolites were treated with methyl or ethyl chloroformate in either sodium bicarbonate solution (10 min at 20°C) or in a medium of aqueous acetonitrile containing methanol (or ethanol) and pyridine for instantaneous derivatization. Gas chromatographic (GC) analysis revealed that aminoalcohols with a free alcohol group can be analyzed by GC with a fused-silica capillary column and that the derivatization yields by both procedures were comparable. Complete derivatization of the acidic metabolites, however, was possible only by the latter procedure. Mass spectra of some of the derivatized compounds are included.

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix‐assisted laser desorption/ionization post‐source decay mass spectrometry

A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.