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We analyzed the methanol extracts of six pristine specimens of the Tagish Lake meteorite (TL1, TL4, TL5A, TL6, TL7, and TL10a) and heated and unheated samples of Allende using high‐performance liquid chromatography coupled with high‐resolution, accurate mass–mass spectrometry (HPLC‐HRAM‐MS). All samples contained ppm levels of sulfate and methyl sulfate. The most abundant organosulfur compound in the methanol extracts of the Tagish Lake and Allende samples was methyl sulfate, which was likely formed primarily via an esterification reaction between intrinsic sources of methanol and sulfate. A homologous series of polythionic acids was also observed in the extracts of the Tagish Lake specimens and Allende. The polythionic acids were the most abundant soluble inorganic sulfur species found in the meteorites. Our results were confirmed using retention time, accurate mass, isotope matching, and tandem mass spectrometry (MS/MS). Hydroxymethanesulfonic acid, previously reported in Tagish Lake, was found only in an unheated Allende sample and in low abundance. Here, we propose possible sulfate formation pathways that begin with interstellar dimethyl sulfide, dimethyl disulfide, methyl sulfide, or methanethiol via cold, nebular processes within the interstellar medium and continue via MSA as an intermediary compound ending within planetary bodies with sulfate and methyl sulfate as the final products. [ABSTRACT FROM AUTHOR]

Unusual ionization behavior was observed with novel antineoplastic curcumin analogues during the positive ion mode of matrix-assisted laser desorption ionization (MALDI) and dopant-free atmospheric pressure photoionization (APPI). The tested compounds produced an unusual significant peak designated as [M − H]+ ion along with the expected [M + H]+ species. In contrast, electrospray ionization, atmospheric pressure chemical ionization and the dopant-mediated APPI (dopant-APPI) showed only the expected [M + H]+ peak. The [M − H]+ ion was detected with all evaluated curcumin analogues including phosphoramidates, secondary amines, amides and mixed amines/amides. Our experiments revealed that photon energy triggers the ionization of the curcumin analogues even in the absence of any ionization enhancer such as matrix, solvent or dopant. The possible mechanisms for the formation of both [M − H]+ and [M + H]+ ions are discussed in this paper. In particular, three proposed mechanisms for the formation of [M − H]+ were evaluated. The first mechanism involves the loss of H2 from the protonated [M + H]+ species. The other two mechanisms include hydrogen transfer from the analyte radical cation or hydride abstraction from the neutral analyte molecule. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Unusual ionization behavior was observed with novel antineoplastic curcumin analogues during the positive ion mode of matrix-assisted laser desorption ionization (MALDI) and dopant-free atmospheric pressure photoionization (APPI). The tested compounds produced an unusual significant peak designated as [M - H](+) ion along with the expected [M + H](+) species. In contrast, electrospray ionization, atmospheric pressure chemical ionization and the dopant-mediated APPI (dopant-APPI) showed only the expected [M + H](+) peak. The [M - H](+) ion was detected with all evaluated curcumin analogues including phosphoramidates, secondary amines, amides and mixed amines/amides. Our experiments revealed that photon energy triggers the ionization of the curcumin analogues even in the absence of any ionization enhancer such as matrix, solvent or dopant. The possible mechanisms for the formation of both [M - H](+) and [M + H](+) ions are discussed in this paper. In particular, three proposed mechanisms for the formation of [M - H](+) were evaluated. The first mechanism involves the loss of H2 from the protonated [M + H](+) species. The other two mechanisms include hydrogen transfer from the analyte radical cation or hydride abstraction from the neutral analyte molecule., (Copyright © 2014 John Wiley & Sons, Ltd.)

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane in adrenal and gonadal cells, fostering steroid biosynthesis. MLN64 is a 445-amino acid protein of unknown function. When 218 amino-terminal residues of MLN-64 are deleted, the resulting N-218 MLN64 has 37% amino acid identity with StAR and 50% of StAR's steroidogenic activity in transfected cells. Antiserum to StAR cross-reacts with N-218 MLN64, indicating the presence of similar epitopes in both proteins. Western blotting shows that MLN64 is proteolytically cleaved in the placenta to a size indistinguishable from N-218 MLN64. Bacterially expressed N-218 MLN64 exerts StAR-like activity to promote the transfer of cholesterol from the outer to inner mitochondrial membrane in vitro. CD spectroscopy indicates that N-218 MLN64 is largely alpha-helical and minimally affected by changes in ionic strength or the hydrophobic character of the solvent, although glycerol increases the beta-sheet content. However, decreasing pH diminishes structure, causing aggregation. Limited proteolysis at pH 8.0 shows that the C-terminal domain of N-218 MLN64 is accessible to proteolysis whereas the 244-414 domain is resistant, suggesting it is more compactly folded. The presence of a protease-resistant domain and a protease-sensitive carboxy-terminal domain in N-218 MLN64 is similar to the organization of StAR. However, as MLN64 never enters the mitochondria, the protease-resistant domain of MLN64 cannot be a mitochondrial pause-transfer sequence, as has been proposed for StAR. Thus the protease-resistant domain of N-218 MLN64, and by inference the corresponding domain of StAR, may have direct roles in their action to foster the flux of cholesterol from the outer to the inner mitochondrial membrane.

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

The steroidogenic acute regulatory protein (StAR) increases the movement of cholesterol from the outer to the inner membrane of adrenal and gonadal mitochondria, thus providing the substrate for steroid hormone biosynthesis. Deletion of 62 amino-terminal aa produces a cytoplasmic form of StAR (N-62 StAR) that lacks the mitochondrial leader sequence but retains full activity and appears to act at the outer mitochondrial membrane. At neutral pH the native state of bacterially expressed N-62 StAR protein displays cooperative unfolding under the influence of urea with DeltaGH2O = -4.1 kcal/mol, and it remains correctly folded down to pH 4. Limited proteolysis at different pHs shows that the biologically essential C-terminal region is accessible to solvent, and that the N-terminal domain is compact at pH 8 and partially unfolds below pH 4. Secondary structural analysis of CD curves suggests that the unfolding may coincide with an increase in alpha-helical character at pH 3.5. Fluorescence spectroscopy at pH 3-8 and at 0-6 M urea is consistent with two distinct domains, a compact N-terminal domain containing tryptophans 96 and 147 and a more solvent-accessible C-terminal domain containing tryptophans 241 and 250. These observations suggest that StAR forms a molten globule structure at pH 3.5-4.0. As the mitochondrial proton pump results in an electrochemical gradient, and as StAR must unfold during mitochondrial entry, StAR probably undergoes a similar conformational shift to an extended structure while interacting with the mitochondrial outer membrane, allowing this apparent molten globule form to act as an on/off switch for cholesterol entry into the mitochondria.

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.

Although the ionization/desorption mechanisms in matrix-assisted laser desorption/ionization (MALDI) remain poorly understood, there is a clear difference between the energy absorption processes in the ultraviolet (UV) and infrared (IR) modes of operation. UV-MALDI demands an on-resonance electronic transition in the matrix compound, whereas results presented here support earlier work showing that a corresponding resonant vibrational transition is not a requirement for IR-MALDI. In fact, data from the present study suggest that significant absorption of radiant energy by a potential matrix impairs its performance, although this observation is at variance with some other reports. For example, sinapinic acid, with an IR absorption maximum close to the 2.94 micrometer wavelength of the Er-YAG laser, has been little used as an IR-MALDI matrix. By contrast, succinic acid, with much lower IR absorption and no history of use in UV-MALDI as it has no UV absorption at the wavelength of common UV lasers, has become widely recognized as a good general purpose matrix for IR-MALDI. Despite reports by others that glycerol is an effective matrix for IR-MALDI, we find that glycerol, which also absorbs strongly at 2.94 micrometer, is useful only if applied as a very thin film. Thus the cumulative evidence for the role of the matrix in IR-MALDI appears confusing and often contradictory. Water has been postulated to be a major contributor to the absorption of energy in IR-MALDI. Consistent with this, we find that samples dried from D(2)O, which does not absorb at 2.94 micrometer, give spectra of inferior quality compared with the same samples from H(2)O. Similarly, samples dried under vacuum, that probably contain less water than those dried in the open laboratory, give weaker and more erratic spectra. Another potential participant in energy absorption and energy transfer is the surface of the metal support, an alternative mechanism for IR-MALDI, for which some evidence is presented here., (Copyright 1999 John Wiley & Sons, Ltd.)

A nanoliter-chemistry station combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed to characterize proteins at the attomole level. Chemical reactions including protein digestion were carried out in nanoliter or subnanoliter volumes, followed by microspot sample deposition of the digest to a MALDI-TOF mass spectrometer. Accurate mass determination of the peptides from the enzyme digest, in conjunction with protein database searching, allowed the identification of the proteins in the protein database. This method is particularly useful for handling small-volume samples such as in single-cell analysis. The high sensitivity and specificity of this method were demonstrated by peptide mapping and identifying hemoglobin variants of sickle cell disease from a single red blood cell. The approach of combining nanoliter chemistry with highly sensitive mass spectrometric analysis should find general use in characterizing proteins from biological systems where only a limited amount of material is available for interrogation.

Antibodies are important tools to explore receptor-ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the alphaIIb beta3 integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the beta3 subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-A resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CL domains was observed so that the angle of rotation relating the two domains was reduced by 15 degrees. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment., (Copyright 1998 Academic Press.)

We report a polymer characterization study by matrix-assisted laser description/ionization (MALDI) on a linear time-of-flight instrument equipped with pulsed ion extraction for time-lag focusing. It is demonstrated that time-lag focusing MALDI provides improved mass resolution and mass accuracy over continuous extraction instruments. Oligomer resolution is extended to a much higher mass range than that observed even by continuous extraction reflectron systems. This allows new opportunities to study the chemical composition and determine the molecular weight of individual components in a mixture of higher molecular weight polymers. It is shown that oligomer resolution can be obtained for poly(ethylene glycol) (repeat unit mass of 44) of mass up to 25,000 u and poly(styrene) (repeat unit mass of 104) up to 55,000 u. Mass measurement accuracy of 80 ppm or better is demonstrated, and the relevance to end-group analysis is shown for two derivative of poly(ethylene glycol) used as slow-release drugs. The analysis of the molecular weight distribution was investigated at several extraction pulse potentials to determine if there was an effect on the relative peak area. We found that the values of the number-average molecular weight (Mn and the weight-average molecular weight (Mw) do not change significantly for a poly(styrene) blend with oligomer masses between 2,000 and 15,000 u and a polydispersity of 1.155. The values are within the 1.6% standard deviation observed for repeat analyses at the same extraction pulse.

A strength of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is its ability to analyze mixtures without separation. MALDI mass spectrometers capable of providing a linear mass calibration over a broad mass range should find wide use in these applications. This work addresses issues pertinent to mass measurement accuracy of a time-lag focusing MALDI time-of-flight instrument and presents a new approach to improving mass accuracy by using a functional wave extraction pulse, instead of a square wave, for time-lag focusing. A model is described of an ideal extraction pulse shape that provides constant total kinetic energy for all ions. If total kinetic energy is constant, then there is an exact linear correlation between ion mass and flight time raised to the second power. Using a descending wave extraction pulse, it is demonstrated that mass accuracy of better than 30 ppm using two internal calibrants and better than 70 ppm using external calibrants can be obtained over a 25 ku mass range. The practical aspects of an instrument needed to obtain consistent mass accuracy is discussed. It is found that ion flight time shows a small dependence upon laser flux; flight times increase slightly as the flux increases. But this dependence is much smaller than is observed in continuous-extraction MALDI.

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is strongly influenced by the method of analyte and matrix preparation. We report a nonintrusive method based on laser confocal microscopic imaging technology to examine the MALDI samples prepared by various protocols. In this method, the analyte is tagged with a fluorescent group. The matrix and analyte are prepared under the same conditions as those used in conventional MALDI experiments. It is demonstrated that confocal microscopy can provide clear, three-dimensional images of sample crystals as well as the analyte distribution within the crystals. It is shown that the analyte is incorporated into the matrix crystals for all the sample preparation protocols examined. Moreover, the confocal microscopic images reveal that, with the use of a dried-droplet method for sample/matrix preparation, the analyte is not uniformly distributed within the matrix crystals. In some crystals, no analyte is incorporated. In addition, it is found that large crystals formed using a slow growth process display a more uniform analyte distribution. Relatively more uniform analyte distribution is observed for samples prepared with the formation of microcrystals. The possible correlation between the ion signal variations observed in MALDI and the uniformity of the analyte distribution obtained by the confocal microscopic imaging method is discussed. Finally, a double-imaging method involving the use of two analytes with different labeling groups is demonstrated. It is found that different analytes are not coherently distributed in the matrix crystals.

A method for accurate mass measurement of oligonucleotides up to a DNA 35-mer based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is described. In this method, a time-lag focusing time-of-flight mass spectrometer is used to achieve high mass resolution to resolve adduct ions that often complicate the mass analysis of oligonucleotides. Mass resolutions between 1170 and 1300 (full width at half maximum) for a 17-mer, 23-mer, and 35-mer are obtained using a 1 m linear time-of-flight instrument with a total sample loading of less than 10 pmol. The effects of sample preparation, type of calibrant and matrix used on the accuracy of mass measurement, based on external calibration, are discussed. A sample preparation protocol that forms a thin film of matrix and sample crystals on a MALDI probe is described. It is shown that mass measurement error less than 100 ppm with reproducibility better than +/-60 ppm can be obtained with either proteins or DNA fragments as external calibrants. Accurate mass measurement for a mixture of DNA fragments is also illustrated.

Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve both the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharide alpha Fuc(1-->2)beta Gal-TMR to the blood group B active trisaccharide alpha Fuc(1-->2)[alpha Gal(1-->3)]beta Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.

A time-lag focusing method is developed for the improvement of mass resolution in a linear time-of-flight mass spectrometer for matrix-assisted laser desorption/ionization (MALDI). In this technique, the ions generated by the MALDI process are extracted by a pulsed voltage. A short time delay (280 ns) is inserted in between the laser desorption/ionization event and the ion extraction. The region between the repeller and extraction grid is field-free during the delay. The time-lag extraction allows the ions generated in the region between the repeller and the extraction grid to separate according to their velocity (energy). Application, to the repeller, of the appropriate pulse voltage provides the energy correction necessary to simultaneously detect all ions of the same mass/charge regardless of their initial energy, resulting in improved mass resolution. It is demonstrated that mass resolution in the range of 3000-6000 fwhm can be obtained. With this mass resolution, isotopically resolved mass spectra are observed for peptides with masses up to 3000 Da. For proteins, such as bovine insulin, cytochrome c, and apomyoglobin, resolution in the range of 800-1000 fwhm is observed with a mass measurement accuracy better than 0.01%.