Significant Fragment Ions Research Articles

Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS.

The utility of matrix-assisted laser desorption time-of-flight (MALD-TOF) mass spectrometry for the analysis of recombinant glycopeptides is discussed and compared to information which may be obtained by fast atom bombardment mass spectrometry (FABMS) and tandem mass spectrometry (MS/MS). MALD-TOF appears to be 10-100 times more sensitive than FAB MS for the analysis of underivatized glycopeptides, providing qualitative site-specific information regarding the carbohydrate microheterogeneity without the extensive isolation and derivatization procedures required to obtain similar information by FAB MS. Analysis of a digest mixture in the positive and negative ion mode of MALD-TOF indicated that, in mixtures, sialylated glycopeptides are preferentially detected in the negative ion mode. The determination of the molecular masses of a glycopeptide with MALD-TOF prior to and after treatment with a variety of specific glycosidases, often without removal of the buffers, coupled to a comparison of molecular mass information available from a carbohydrate database facilitates the assignment of a carbohydrate composition. The vast majority of the molecular ion signal observed in the linear mode for sialylated glycopeptides are metastable ions. Reflector mass spectra reveal a shift to lower mass consistent with the loss of most of the neuraminic acid residues. The loss of Hex and HexNAc residues is also observed. Sequential lowering of the reflector potential reveals structurally significant fragment ions representing the carbohydrate and peptide portions of the molecule.

The dissociation chemistry of permethylated guanosines as articulated by MS/MS

Permethylation of covalently modified guanosines is shown to be an effective means of increasing the abundance of structurally significant fragment ions when protonated base ions from nucleosides are subjected to collision-induced dissociation in MS/MS. The gas-phase chemistry which is observed can be clearly related to the original site of substitution, and provides a valuable means of determining sites of substitution and other modifications in nucleosides related to guanosine.

Molecular surface analysis by TOF-SIMS

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) performs surface sensitive analysis of the elemental and molecular composition of solids. TOFSIMS is a relatively new embodiment of static secondary ion mass spectrometry (SSIMS) in which the dose of primary ions incident on the surface is typically less than 1012 ions/cm2. Since typical solid surfaces have an atomic density of 1015 atoms/cm2, this primary ion dose nominally removes less than 0.1% of a monolayer. Hence, SIMS analyses performed under these static conditions represent near surface analysis in which secondary ions are produced from the top few monolayers of the surface. The actual sampling depth is determined by the primary ion momentum, angle of incidence and chemistry of the surface. Since low dose primary ions cause minimal perturbation of the chemistry of the solid surface, SSIMS analyses often produce molecular or pseudo-molecular ions characteristic of the chemical composition of the surface. Thus, molecular ions or structurally significant fragment ions are often observed in SSIMS analyses of surfaces containing inorganic and organic residues, polymers surfaces, coatings, and biological materials such as tissues and membranes.

Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nonescale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.

Peptide sequence ions produced by postionization of neutral molecules formed during resonant 266-nm laser desorption

A number of examples of peptide mass spectra obtained using 266-nm laser desorption (LD) followed by 255-nm multiphoton ionization (MUPI) of the laser desorbed neutral molecules are presented which show a variety of structurally significant sequence fragment ions. It is believed that most of these sequence fragment ions result from postionization of neutral fragments produced during 266-nm LD. MUPI postionization of the fragment neutrals produced during LD can provide sequence information which is complementary to the sequence information derived from direct ion desorption experiments

Thermodesorption—gas chroamtography—mass spectrometric analysis of biological materials for potential molecular precursors of the constituents of the crude oils

Thermodesorption technique coupled on-line with gas chromatography (GC) and mass spectrometry (MS) has been developed for the determination of hydrocarbons trapped in coal and rock samples. The method can also be applied to the determination of volatile constituent present in biological materials. The desorption step is carried out in the pre-heated injector of a gas chromatograph. The mobilized compounds are transferred without splitting to the initial part of a cold GC column. After 10 min of desorption the GC—MS analysis is started. The identification of individual compouds is achived by GC—MS analysis of related molecular ions or significant fragment ions. When micro-samples (0.25–2.0 mg) of dried plant material are heated to 300°C in a helium stream. compounds such as long-chain fatty acids, carboxylic acid esters, aldehydes and ketones as well as steroids and tocopherols are desorbed from the biological matrix decomposition. In addition, the thermally chlorophylls decompose to yield phytadienes as pyrolysis products of the phytyl moiety. The phytadienes can be used to estimate the original algae and terrestrial plant materials. This ratio partly reflects the pristane:phytane concentration ratio determined in extracts of mature sediments rich in related fossilized biological materials. The analysis also provides chemotaxomic information about the sterol composition of plants.

COMPARATIVE STUDY OF DESORPTION ANDDIRECT INLET ELECTRON IMPACT ANDCHEMICAL IONIZATION MASS SPECTROMETRYOF SOME 2-CHLOROETHYL PHOSPHONIC ACIDMONO AND DIESTERS

Abstract The application of desorption and direct inlet with electron impact and chemical ionization mass spectrometry of some 2-chloroethyl phosphonates related to etephon is described. Comparison of these methods showed that there are highly useful for obtaining: 1) adduct ions, especially protonated molecular ions: chemical ionization. 2) significant fragment ions: methane and electron impact ionization. Interpretation of spectra, based on the considerable analogy between the different structures of the phosphonates is described in this work. A choice between EI and CI mass spectrometry with either direct inlet or desorption technics, depending on the volatility of the compounds and the purpose of analysis, is done. The low fragmentation obtained by ammonia chemical ionization was used to analyze different mixtures of phosphonates.

Routine liquid charomatography/fast atom bombardment mass spectrometry of peptides and enzymatically digested proteins

AbstractThe development of a high‐performance liquid chromatography (HPLC)/fast atom bombardment (FAB) interface and the subsequent commercial availability of such systems has facilitated the routine application of the technique to problems in pharmaceutical research and development. Although many products are amenable to FAB analysis and hence LC/FAB, the greatest benefit of the interface has been in the field of peptide and protein analysis. It has been found that, even with post‐column matrix addition, chromatographic resolution is maintained and, by plotting mass chromatograms, the resolution may be greater than that achieved by the less specific UV detector. As only 1% matrix is required in the final eluent, the system is stable for extended periods and has been used for 3 h LC/FAB experiments or used continuously for multiple analyses over 8 h periods. In addition to the acquisition of relative molecular mass information, the constant background can be completely subtracted to yield structurally significant fragment ions which may allow sequencing of components from the single LC/FAB experiment. Applications of LC/FAB to date include the characterization of Iys(78)‐plasminogen by the on‐line analysis of complex mixtures of peptides resulting from the various enzymatic digests.

Characterization ofin vitro metabolites of the antipsychotic drug tiospirone by mass spectrometry

Metabolism of the antipsychotic drug tiospirone was studied in vitro with phenobarbital-induced rat liver microsomes. Metabolites were isolated and purified to homogeneity by high-performance liquid chromatography. It was possible to characterize the metabolites as trimethylsilyl (TMS) derivatives by gas chromatography/electron impact mass specrometry (GC/EIMS) so long as the sulfur was present in the reduced form. However, sulfoxide and sulfone analogs of tiospirone underwent reductive decomposition on the GC column. In addition, no molecular ions were observed in the EI spectra of these analogs. Desorption chemical ionization/mass spectrometry (DCI/MS) in the positive ion mode with methane as reagent gas successfully distinguished sulfoxides and sulfones from their parent sulfides. Protonated molecular ions were observed together with structurally significant fragment ions. Five microsomal metabolites of tiospirone were characterized by a combination of GC/EIMS and DCI/MS. From the structures of the metabolites three major pathways of metabolism were identified: N-dealkylation of the butyl side chain at the piperazinyl nitrogen, hydroxylation alpha to the glutarimidyl carbonyl at C-6 on the azaspirodecanedione ring, and sulfoxide formation on the benzisothiazole moiety.

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry ( MS MS ) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H) − together with fragment ions at m z 177 (H 3P 2O 7) −, 176 (H 2P 2O 7) −, 159 (HP 2O 6) −, and 79 (PO 3) − which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.

Characterization of metal complexes of 1,10-phenanthroline, 2,2′-bipyridine, and their derivatives by fast atom bombardment mass spectrometry

A series of 1,10-phenanthroline (phen), 5-nitro-1,10-phenanthroline (5-NO2-phen), 2,9-dimethyl- 1,10-phenanthroline (dmp), 2,2′-bipyridine (bpy), and 4,4′-dimethy-2,2′-bipyridine (dmb) metal complexes has been characterized using fast atom bombardment mass spectrometry (FAB MS). Intact cations are observed in the positive ion FAB mass spectra of all tetracoordinated complexes, [Ag(L)2]NO3, [Cu(L)2]2 SO4•5H2O, [Cu(L)2]SO4, and [Tl(L)2](ClO4), where L=bpy, phen, or dmp. Structurally significant fragment ions (ML2+, ML+, M+, and (L+H)+, where M+=metal ion) are also observed. Species such as Ag[Ag(L)2]NO3+, where L=bpy or phen, and Tl[Tl(phen)2](ClO4)+ are also seen. The hexacoordinate complexes, [Co(phen)3]Cl2, [Ru(bpy)Cl2], [Fe(phen)2(CN)2], [Fe(L)3](ClO4), where L=5-NO2-phen, dmb, or bpy, and [Fe(L)3]SO4, where L=bpy or phen, show intact cations in the positive ion spectra; [Ni(bpy)3]X2, where X=Cl−,Br−, NO3−, ClO4−, SCN−, and tartrate2−, and [Co(bpy)3](ClO4) do not. Generally, fragment ions such as ML3+, ML2X+, ML2+, MLX+, and (L+H)+ are observed, where X=singly charged intact anion or HSO4−. Comparison of FAB results with those of laser mass spectrometry (LMS) show that they are comparable, through reduction processes are more important for FAB. LMS always gave the intact cation while FAB was not consistent. Keywords: FAB mass spectrometry, phenanthroline complexes, bipyridine complexes, laser desorption mass spectrometry.

Sequence analysis of highly sulfated, heparin-derived oligosaccharides using fast atom bombardment mass spectrometry.

Heparin, a polydisperse, sulfated copolymer of 1----4 linked glucosamine and uronic acid residues, has been used clinically as an anticoagulant for half a century. Despite a yearly use of over 50 million doses in the U.S. alone, heparin's exact chemical structure remains unclear. The negative ion fast atom bombardment mass spectrometry (FAB-MS) analysis is presented for a series of enzymatically prepared, homogeneous, structurally characterized, highly sulfated, heparin-derived oligosaccharides using triethanolamine as the FAB matrix. In addition to the clear presence of monoanionic sodiated molecular ions, structurally significant (sequence) fragment ions are observed and characterized with respect to the known structure for five of the heparin-derived oligosaccharides. The structure of a sixth oligosaccharide is predicted by using negative ion FAB-MS and subsequently confirmed by chemical, enzymatic, and NMR spectroscopic methods.

Continuous flow fast atom bombardment mass spectrometry of mononucleotides and their metal complexes

The mass spectra of mononucleotides and their metal adducts Na, K, Mg, Ca, Ni, Co, Cu, and Zn of guanosine 5′-monophosphate (5′-GMP) as well as H, Na, and Mg of adenosine 5′-monophosphate (5′-AMP) and H and Ni of inosine 5′-monophosphate (5′-IMP) have been obtained in low concentrations of matrix in water using continuous-flow fast atom bombardment. The results indicate that this technique is suitable for the analysis of these complexes in aqueous media and yields spectra that are highly characteristic of the compounds analyzed. Parent-molecular ions and structurally significant fragment ions are observed for all compounds studied and the different binding sites for the metal on the nucleotides can be isolated from the fragment ions. Experimental parameters influencing the quality of the spectra such as flow rate, matrix concentration, matrix nature, and analyte concentration have been studied and optimized. For the thirteen compounds studied, it appears that continuous-flow FAB is superior to conventional FAB and that good quality spectra can be obtained with as little as 0.5% of added matrix thus minimizing spectral interferences. Keywords: continuous flow FAB, FAB MS, mass spectrometry, nucleotides, metal-nucleotides.

Atmospheric pressure spray mass spectra of thermolabile and nonvolatile compounds.

Atmospheric pressure spray (APS) mass spectra of thermolabile and nonvolatile compounds such as saccharides, glycosides, peptides, phospholipids and glycolipids are presented. The APS method can produce intense protonated and/or cationized molecules such as (M+H)+, (M+Na)+ and (M+K)+ for these compounds without any buffers or external ion sources, unlike other ionization methods. The APS method combined with a collision-induced dissociation technique can provide significant fragment ions, which are very useful for structural elucidation.

Application of high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry for the analysis of non-volatile compounds

The combination of a high-performance liquid chromatograph and a magnetic type mass spectrometer with an atmospheric ionization source has been applied for analyses of thermolabile compounds, i.e. antibiotics, amino acids and glycosphingolipids. Chromatography was performed on a 4 mm i.d. x 150 mm column packed with 5 μm ODS at a flow rate of 1 ml min−1. The liquid chromatography/atmospheric pressure ionization (LC/API) magnetic-type mass spectrometer provides not only an abundant pseudo-molecular ion but also fragment ions which give a structural information resulting from collision-induced dissociation at the differential pumping region of the liquid chromatograph/mass spectrometer interface. The isomers of kanamycins could be differentiated from each other on the basis of their mass spectra obtained at the relatively higher drift voltage applied. LC/API mass spectra of leucine and isoleucine were very similar, but it was easy to differentiate one from another by checking the existence of the ions at m/z 43 or m/z 57 in their mass spectra. The LC/API mass spectra of glucosylceramides showed [MH]+ ions of good abundance and significant fragment ions useful for chemical characterization at a proper drift voltage setting in a controlled and reproducible manner.

Elemental composition from moment analyses of the low‐resolution isotope pattern

AbstractThe basic principles of an approach to mass spectral elemental analysis based on the moments of the low‐resolution isotopic pattern are presented. The moment analyses are faster and more general than conventional methods of isotopic pattern analysis. The procedure is applied to the analysis of compounds which exhibit ‘pure’ molecular ions and the analysis of compounds which produce significant fragment ions at masses overlapping the molecular ion. It appears as though reasonable selectivity can be achieved for low relative molecular mass compounds (relative molecular mass < 400 a) using standard instrumentation and data acquisition softy are. Preliminary studies of instrumental factors affecting the measurement of moments are discussed.

Some general aspects of the chemistry of Organo-alkali metal ions. An overview of recent work

Organo-alkali metal ions of the type [M + xNa - ( x - 1)H] + ( x=1–3) are commonly observed in the desorption ionization mass spectra of polar organic molecules. An overview of our work on the formation and selected applications of organo-alkali metal ions is presented. We interpret our data as evidence that ions of the type [M + xA - ( x - 1)H] + are formed from [M + A(AX) n ] + cluster ions. Another important aspect of this work deals with the dissociation reactions of organo-alkali metal ions. We have found that structurally significant fragment ions are observed in the collision-induced dissociation (CID) spectra of [M + A] + ions that are not observed in the [M + H] + ion CID spectrum. We attribute these differences to functional group basicity and organo-alkali metal ion binding energies. That is, alkali metal ions are bound to specific sites of the organic molecule and the fragment ions formed upon CID are indications of the structure of the ionic organo-alkali metal ion complex.

ChemInform Abstract: Observation of Doubly Charged Ions in Fast Atom Bombardment Mass Spectrometry: Ruthenium(II) Complexes in a Nitrobenzyl Alcohol Matrix

The fast atom bombardment (FAB) mass spectrometric analysis of several series of ruthenium-bidentate ligand complexes such as (RuLL'L'')X/sub 2/, (RuL/sub 2/L')X/sub 2/, and (RuL/sub 2/L')X/sub 2/, and (RuL/sub 3/)X/sub 2/ are discussed, where L, L', and L'' include 2,2'-bipyridine, 1,10-phenanthroline, their derivatives and related compounds. Surprisingly abundant, intact, doubly charged cations (RuL/sub 3/)/sup 2 +/, (RuLL'L'')/sup 2 +/, and (RuL/sub 2/L')/sup 2 +/ are observed for most complexes studied. Structurally significant fragment ions are generally observed for all complexes in addition to singly charged molecular cationic species such as (RuLL'L'')/sup +/ and (RuLL'L'')X/sup +/. The (RuLL'L'')/sup 2 +//(RuLL'L'')/sup +/ ratio, where L = L' = L'', L = L' not equal to L'', or L not equal to L not equal to L'' depending on the series involved, varies from 3.5 to 0.35 over the 22 complexes studied. Reasons for the observation of these unique abundant doubly charged ions in a FAB spectrum, especially in a nitrobenzyl alcohol matrix, are discussed. Ions indicative of ligand exchange reactions between ruthenium centers are not observed for mixed ligand complexes. 23 references, 4 figures, 1 table.

Observation of doubly charged ions in fast atom bombardment mass spectrometry: ruthenium(II) complexes in a nitrobenzyl alcohol matrix

The fast atom bombardment (FAB) mass spectrometric analysis of several series of ruthenium-bidentate ligand complexes such as (RuLL'L'')X/sub 2/, (RuL/sub 2/L')X/sub 2/, and (RuL/sub 2/L')X/sub 2/, and (RuL/sub 3/)X/sub 2/ are discussed, where L, L', and L'' include 2,2'-bipyridine, 1,10-phenanthroline, their derivatives and related compounds. Surprisingly abundant, intact, doubly charged cations (RuL/sub 3/)/sup 2 +/, (RuLL'L'')/sup 2 +/, and (RuL/sub 2/L')/sup 2 +/ are observed for most complexes studied. Structurally significant fragment ions are generally observed for all complexes in addition to singly charged molecular cationic species such as (RuLL'L'')/sup +/ and (RuLL'L'')X/sup +/. The (RuLL'L'')/sup 2 +//(RuLL'L'')/sup +/ ratio, where L = L' = L'', L = L' not equal to L'', or L not equal to L not equal to L'' depending on the series involved, varies from 3.5 to 0.35 over the 22 complexes studied. Reasons for the observation of these unique abundant doubly charged ions in a FAB spectrum, especially in a nitrobenzyl alcohol matrix, are discussed. Ions indicative of ligand exchange reactions between ruthenium centers are not observed for mixed ligand complexes. 23 references, 4 figures, 1 table.

Analysis of some metabolites of t-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography—mass spectrometry

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography—mass spectrometry (GC—MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography—mass spectrometry (HPLC—MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H] + ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate] − cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50–500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.