High-energy Collision-induced Dissociation Tandem Mass Research Articles

High-energy collision-induced dissociation tandem mass spectrometry of regioisomeric lactose palmitic acid monoesters using matrix-assisted laser desorption/ionization

Structural characterization and differentiation of three newly synthesized lactose monopalmitate regioisomers at positions O-3, O-3' and O-6' were realized by single-stage matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) in the positive ion mode and by high-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS). A MALDI-TOF/TOF analyzer was utilized for the analysis of these isobaric lactose monopalmitate regioisomers. The CID-MS/MS spectra were acquired using high-energy cid with a 2 kV potential difference between the source acceleration voltage and the collision cell. High-energy (CID) tandem mass spectrometry (MS/MS) analyses of the sodiated molecules, [M + Na](+), showed distinguishing cross-ring product ions and characteristic fingerprint product ions, which allowed the straight-forward mass spectrometric characterization of these different regiosiomers. This investigation allowed us to unravel the novel fragmentation behavior of the sodiated regioisoimer molecules obtained from the mono-substituted D-lactose fatty acid esters using high-energy CID-TOF/TOF-MS/MS analyses. The high-energy CID of the [M + Na](+) ions from the isobaric lactose monopalmitate regioiosmers promoted the formation of characteristic (0,2) A2 cross-ring cleavage product ions.

Alterations in Fatty Acid Utilization and an Impaired Antioxidant Defense Mechanism Are Early Events in Podocyte Injury: A Proteomic Analysis

Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.

Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase

We have recently detected that the lipase from Streptomyces rimosus belongs to a large but poorly characterised family of SGNH hydrolases having the αβα-fold. Our biochemical characterisation relates to the specific inhibition of an extracellular lipase from Streptomyces rimosus (SRL, 24.2 kDa, Q93MW7) by the preincubation method with tetrahydrolipstatin (THL). In high molar excess (THL/SRL = 590 at 25 °C, pH = 7.0) and after 2 h of incubation in an aqueous system, 56% of the enzyme inhibition was reached. Under the same conditions and in the presence of 50% (v/v) 2-propanol/water, 71% enzyme inhibition was obtained. Kinetic measurements are in agreement with pseudo-first-order kinetics. The nucleophilic attack of the catalytic serine residue 10 of SRL occurs via an opening of the β-lactone ring of tetrahydrolipstatin and formation of a covalent ester bond. The intact covalent complex of SRL-inhibitor was analysed by ESI and vacuum MALDI mass spectrometry and, furthermore, the exact covalent THL linkage was determined by vacuum MALDI high-energy collision-induced dissociation tandem mass spectrometry.

Structural analysis of polymer end groups by electrospray ionization high-energy collision-induced dissociation tandem mass spectrometry

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.

Structure elucidation of annoheptocins, two new heptahydroxylated C37 acetogenins by high-energy collision-induced dissociation tandem mass spectrometry

Collision-induced dissociation (CID) of lithiated molecules generated by liquid SIMS (LSIMS) from two new natural compounds, annoheptocin A and B, led to their structural elucidation. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) investigations of their acetonide derivatives displayed obvious differences of reactivity of the lithiated dioxane and dioxolane rings towards CID. The 1,3-dioxane (six-membered) ring could be the target of hydrogen rearrangement processes, leading to the formation of a tetrahydrofuran ring through the loss of a molecule of acetone. Copyright © 1998 John Wiley & Sons, Ltd.

Investigation of beam-induced reactions occurring under fast-atom bombardment conditions between triethanolamine and various phospholipids

The generation of adduct ions when using a triethanolamine (TEA) matrix under conditions of fast-atom bombardment has been investigated. In particular, phospholipids containing a free amino group react in the TEA matrix to form abundant adduct ions 26 and 42 mass units higher than the analyte pseudomolecular ions. The identity of these adduct ions was investigated using specific phosphatidylethanolamines and phosphatidylserine as well as 2-aminoethyldihydrogenphosphate and 2-aminoethylmonomethylphosphate as model compounds. Evidence obtained from positive- and negative-ion high-energy collision-induced dissociation tandem mass spectra, high resolution mass spectra, deuterium labelling and experiments using diethanolamine/TEA mixtures revealed that the intensities of the adduct ions are dependent on the percentage of TEA in the matrix and on the polarity of the analyte. The nature of the adduct ions is best reflected by a structure in which a hydrogen atom of the free amino group is substituted by a vinyl (+26) or by an acetyl (+42) group. A mechanism for the adduct ion formation is proposed. © 1997 John Wiley & Sons, Ltd.

Microheterogeneity characterization of a paracelsin mixture from Trichoderma reesei using high-energy collision-induced dissociation tandem mass spectrometry.

The microheterogeneity of the paracelsin mixture broth of Trichoderma reesei was analysed using mass spectrometric methods, in particular high-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). Based on the liquid secondary ion mass spectrum of the mixture, there are three main components, with molecular masses M and (M +/- 14), together with two minor components of molecular weight (M +/- 28). The high-energy CID tandem mass spectra of both the protonated and sodiated molecules yielded abundant and characteristic fragment ions, but of very different types. It was found that a paracelsin peptaibol in a mixture could be successfully sequenced based on the tandem mass spectra of its protonated and sodiated molecules or, alternatively, on the tandem mass spectra of its y7 and b13 fragment ions. A terminology for indicating these sequential peptide fragments is proposed. To determine the sequence of new analogues, tandem mass spectra of the y7, (y7 +/- 14), b13, (b13 +/- 14) and (MH +/- 14) positive ions were also taken. Based on these experiments, four new paracelsin components (PA-F, PA-G, PA-H and PA-I) were sequenced successfully. The microheterogeneity of the mixture was found to be more pronounced than had been assumed previously. In these new analogues, besides positions 6 and 9, position 17 is also involved in the exchange. MS/MS studies on minor fragment ions, such as (b13-28) and (b8-14) show further microheterogeneity at positions 3, 5, 10 and 12, which increase the number of possible minor components.

Comparison of Low- and High-energy Collision-induced Dissociation Tandem Mass Spectrometry in the Analysis of Ricinoleic and Ricinelaidic Acid

Ricinoleic acid and itstransisomer ricinelaidic acid, and their methyl esters, were analyzed by low- and high-energy collision-induced dissociation (CID) tandem mass spectrometry. It is shown that a stable charge centre is required to observe the rearrangement, which occurs in the β-hydroxyalkene part and results in the loss of heptaldehyde, and that this rearrangement corresponds to a low-energy CID reaction. Differences between the CID behaviour of ricinoleic acid and that of itstransisomer are related to the loss of water, which can also be regarded as a low-energy rearrangement reaction. Comparison of low- and high-energy CID spectra further revealed that high-energy CID gives rise to loss of a H•radical and H2 together with low-energy fragmentation. Examination of different molecule ions, including [M-H]-, [M+Li]+ and [M-H+2Li]+ ions of free fatty acids and [M+Li]+ ions of the methyl esters shows that charge-remote homolytic fragmentation is most pronounced for the [M+Li]+ ions of the methyl esters. © 1997 by John Wiley & Sons, Ltd.

Characterization of Ozonated C60 and C70 by High Performance Liquid Chromatography and Low- and High-Energy Collision-induced Dissociation Tandem Mass Spectrometry

The formation of C60 and C70 oxides by reaction with ozone has been examined using reversed-phase high-performance liquid chromatography and mass spectrometry including liquid secondary ion mass spectrometry, collision-induced dissociation (CID) and tandem mass spectrometry. C60 and C70 oxides containing up to four oxygen atoms could be detected and were found to be chemically unstable. The mass spectral results show that low-energy CID of molecular anions of the higher C60 and C70 oxides results in the loss of one CO molecule, whereas molecular cations eliminate two molecules of CO. In both ion modes the formation of odd-numbered fullerenes was noticed. In contrast, high-energy CID results in the loss of O and O2, followed by sequential C2 losses. For some product ions the formation of fullerene–helium adduct ions was observed.

Use of low-and high-energy collision-induced dissociation tandem mass spectrometry in the identification of an unusual amino acid in a semisynthetic polypeptide

During production of the semisynthetic eicosapeptide Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, two minor impurities were isolated in which Ile-15 had been replaced by other amino acids. The molecular weights of the two peptide minor products were 14 u lower and 14 u higher, respectively, than the major product with the foregoing amino acid sequence. It was determined that the first, lower molecular weight, impurity contained Val instead of Ile at position 15 of the sequence, whereas the second, higher molecular weight, impurity contained an unusual amino acid at the same position. This amino acid, which was characterized from the low-and high-energy collision-induced dissociation product ion tandem mass spectra of the second peptide impurity, is an α-amino acid with an asymmetric side chain branched at the β-carbon.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.