Identification of peptide oxidation by tandem mass spectrometry

Oxidative reactions play important roles in a variety of biochemical events ranging from normal metabolism to aging and disease processes. Proteins represent major targets for modification in these reactions, and identification of sites and structures of modifications may lead to mechanistic understanding and approaches for prevention. In this Account, the utility of mass spectrometry and its advantages are described for the identification of oxidative modifications to protein targets. A variety of examples are provided to illustrate how modifications are accurately identified and quantitated using modern methods of ionization coupled with HPLC and tandem mass spectrometry.     

Identification of steroids by chemical ionization mass spectrometry

Chemical ionization (CI) mass spectra of various natural and synthetic steroids have been studied using methane, isobutane, ammonia, trideuterioammonia and hydroxy anion as reagent gases. The CI spectra of steroids give simple and well characterized ions, which provide information about molecular weight as well as functionalities in the molecules. Trideuterioammonia exchanges rapidly with active hydrogens (e.g., OH, SH, COOH, NH₂) in steroid molecules in the CI reaction and thus provides a convenient means of active hydrogen determination by mass spectrometry. Application of various CI processes to the analysis of steroids and conjugates have been made. Low levels of hydroxycholesterols in biological samples and in cholesterol autoxidation products were identified by the 4 ion patterns, [M+NH₄]⁺, [M−OH+NH₃]⁺, [M−OH]⁺ and [M−H₂ O−OH]⁺, in ammonia CI. The position of hydroxy functions in the cholesterol side chain can be identified from the methane CI of hydroxycholesterol trimethylsilyl (TMS) derivatives. Sterol carboxylic esters can be identified as the ammonium adduct ion of the intact molecule, [M+NH₄]⁺, in ammonia CI. Isobutane and hydroxy anion CI spectra of the steroid esters give abundant ion fragments of both steroids and carboxylic acid moieties. Identification of free bile acids and steroid glycosides without derivatization is also feasible with the CI process when ammonia is used as reagent gas.     

Identification and analysis of wax esters by mass spectrometry

Several ions in the mass spectra of wax esters were related to the molecular structures. Assigned structures of ions were confirmed by deuterium labeling. A simple, direct method for quantititive analyses of mixtures was developed. The method involved a comparison of sets of three ions, RCO₂H⁺, RCO₂H₂ ⁺ and [R′−1]⁺ from all compounds in the mixture. The method was found applicable for mixtures of unsaturated wax esters after reduction with tetradeuterio hydrazine. Presented in part before the ISF-AOCS World Congress, Chicago, September 1970. Part III of a series of mass spectrometry of lipids. For VI see Lipids.     

Identification of carrot inositol phospholipids by fats atom bombardment mass spectrometry

Inositol phospholipids from carrot cell membranes grown in suspension cultured were purified by thinlayer chromatography (TLC) or column chromatography and tentatively identified by co-migration on TLC with animal inositol phospholipid standards. For more rigorous chemical characterization, carrot inositol phospholipids were then analyzed by negative ion fast atom bombardment mass spectrometry (FABMS). One phosphatidylinositol (PI), two lysophosphatidylinositols (LPI), and one phosphatidylinositol monophosphate (PIP) were identified in the carrot samples by the observation of ions [M-H]⁻ and numerous fragment ions in the negative FAB mass spectra. MS/MS analysis were carried out to obtain further structural information of these phospholipids using a double-focusing mass spectrometer in which the magnetic sector (B) and the electrostatic analyzer (E) were scanned at a constant ratio (B/E). These B/E linked scans provided fragment ions of selected precursor ions while eliminating matrix and other contaminating ions. No molecular ions were detected for lysophosphatidylinositol monophosphate (LPIP) or phosphatidylinositol bisphosphate (PIP₂), but fragment ions corresponding to these structures were observed. The primary fatty acids present in the carrot inositol phospholipids were linoleic (18∶2) and palmitic (16∶0) acids, whereas animal lipids contained arachidonic (20∶4), stearic (18∶0), linoleic, and palmitic acids. The only phosphatidylinositol found in carrot cells was palmitoyl linoleoyl PI.     

Validation of computational methods for triglyceride composition of fats and oils by liquid chromatography and mass spectrometry

A computer method for predicting triglyceride composition of fats and oils from fatty acid composition is described. The results of the method are compared with results of analyses performed by liquid chromatography (LC) and desorption chemical ionization mass spectrometry. An LC column technology is also described which provides a simple separation of triglycerides according to the equivalent carbon number of the triglyceride.     

Identification and quantitation of surfactants in consumer products by ion-spray mass spectrometry

Current approaches for identifying and quantitating surfactants in various product matrices require the use of wet chemical, chromatographic, and spectroscopic techniques. The primary objective of this work is to replace current multiple-technique approaches for identifying surfactants and quantitating their homologue distribution with a single rapid technique. To meet this objective, a simple preparation procedure has been developed to prepare consumer product samples for subsequent analysis by ion-spray mass spectrometry. Quantitation of linear alkylbenzene sulfonates in detergents with this technique is demonstrated.     

Identification of conjugated fatty acids by gas chromatography-mass spectrometry of 4-methyl-1,2,4-triazoline-3,5-dione adducts

4-Methyl-1,2,4-triazoline-3,5-dione was reacted with conjugated fatty acid methyl esters to form Diels-Alder cycloaddition products. The electron impact mass spectra of conjugated octadecadienoates and 9,11,13-octadecatrienoate were simple and informative and allowed the positions of the double bonds to be determined. The dienes gave single adducts whereas the triene formed four products that corresponded to two stereoisomers of the adducts of the 9,11-diene system and two of the 11,13-diene system. The method can be used to complement other methods for identifying conjugated fatty acids.     

Characterization of castor bean neutral lipids by mass spectrometry/mass spectrometry

A method has been developed for the characterization of intact neutral lipids isolated from castor bean (Ricinus communis L.) by mass spectrometry/mass spectrometry (MS/MS). The molecular weights of the trimethylsilyl (TMS) derivatives of the neutral lipids are determined by using both electron impact and chemical ionization (ammonia). Collision-induced dissociation daughter spectra of the (M-CH₃)⁺ ions yield fragment ions that allow easy determination of the acyloxy groups present. The chainlength and degree of unsaturation for each acyloxy group are indicated by R in the ion represented by the general formula (RCO + 74)⁺. Other ions of diagnostic value include (M-RCOO)⁺, (M-RCOOH)⁺, [(M-CH₃-RCOOH]⁺ and [(M-RCOOH)-16]⁺. The presence of a TMS group in any of these fragments results in the formation of ions representing the loss of OTMSH. Prior to MS/MS analysis, partial fractionation by high-performance liquid chromatography (according to degree of unsaturation in the neutral lipids) is useful because daughter spectra are generated free of any isotopic contamination, and minor components are concentrated in single fractions, which aids their characterization. By using this method, 11 neutral lipids were characterized in castor bean.     

Identification of milk fat triacylglycerols by capillary supercritical fluid chromatography-atmospheric pressure chemical lonization mass spectrometry

Identification of milk fat triacylglycerols was accomplished by capillary supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. Supercritical carbon dioxide was the carrier fluid in SFC. Ionization was achieved by introducing vapor of ammonia in methanol into the ionization chamber, which resulted in the formation of abundant [M+18]⁺ and [M-RCOO]⁺ ions of triacylglycerols. These ions defined both the molecular weight and the fatty acid constituents of a triacylglycerol, respectively. SFC on a nonpolar stationary phase provided an efficient separation of triacylglycerols according to the combined number of carbon atoms in the acyl chains of a molecule. In addition to the identification of the major chromatographic peaks representing molecules with 26–54 acyl carbons, minor peaks representing triacylglycerols with an odd number of acyl carbons were separated and identified. Furthermore, compositional information on partially separated isobaric triacylglycerols, which differed substantially in the chain length of the fatty acyl residues, was achieved within some of the peaks. A new finding of the present study was the formation of abundant [M+18]⁺ ions of saturated triacylglycerols in addition to diagnostic fragment ions, being of primary importance in structure elucidation. This extends the applicability of capillary SFC-(APCI)MS in the analysis of both saturated and unsaturated triacylglycerols.     

Investigation of protein-ligand interactions by mass spectrometry

The rate of drug discovery is greatly dependent on the development and improvement of rapid and reliable analytical methods that allow screening for protein-ligand interactions. The solution-based methods for investigating protein-ligand interactions by mass spectrometry (MS), which are discussed in this paper, are hydrogen/deuterium exchange of protein backbone amide hydrogens, and photoaffinity labeling. Moreover, MS analysis of intact noncovalent protein-ligand complexes is described. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with its ultra-high resolution and excellent mass accuracy is also considered herein as it is gaining increasing popularity for a mass spectrometric investigation of protein-ligand interactions.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

The analysis of indigoid dyes by mass spectrometry

High–resolution mass spectrometry provides an expeditious technique for positively identifying indigoid dyes of molluscan origin. Indigoids on modern and ancient textile fibres, as well as indigoid mixtures produced by reactions, are discussed. Steam treatment of the fibre or isolation of the dye by solvent extraction permits the analysis by mass spectrometry of dyes which bind more tightly to fibres, such as 6, 6′-dibromoindigotin (C. I. 75800, Natural Vat Dye). The demonstration of the stepwise photolytic denomination of the leuco base of 6, 6′-dibromoindigotin may be of more far-reaching significance in distinguishing between natural and intentional mixtures of indigotin and its dibromo derivative.     

Rapid qualitative identification of sulphonamides by mass spectrometry

Rapid qualitative identification of the sulphonamides present in pharmaceutical preparations is conveniently carried out using mass spectrometry. Direct introduction of a small quantity of the powdered tablet into the mass spectrometer results in the production of characteristic ions even in preparations containing a mixture of therapeutically active constituents and without prior separation of the various constituents.     

Identification of acidic species extracted from heavy crude oil by fourier transform ion cyclotron resonance mass spectrometry

Acidic species were extracted from heavy crude oil by alcohol-alkali solution and ion exchange resin. The acidic species in the obtained extracts and extracted crude oil were characterized by negative-ion ESI FT-ICR MS. The analytical results indicated that some class species, O₂, O₁, N₁, N₁O₁, N₁O₂, N₁S₁ and O₂S₁ etc., were identified in the negative-ion spectrum, in which O₂, O₁ and N₁ class showed much higher abundance than others. Compared to O₁ and N₁ class, O₂ class could be extracted from heavy crude oil by using the two methods more efficiently, which means that a great majority of O₂ class can be extracted; meanwhile, almost half of O₁ and N₁ class still remained in residual phase. Detailed analysis demonstrated that alcohol-alkali method was effective for extracting O₂, O₁ and N₁ class with lower molecular weight; ion exchange method, however, was helpful to extract higher molecular weight O₁ and N₁ class and showed almost equal extraction selectivity to all kind of O₂ class.     

Thermal characterisation of polymeric systems by mass spectrometry

The utility of mass spectrometry as a technique for the characterisation of polymeric formulations is discussed in general terms. The use of thermal techniques in which sufficient energy is given to the system to liberate volatile components of the formulation and, in some cases, to induce small amounts of polymer degradation, is described. Results obtained using this technique for a variety of polymer systems are discussed and the advantages of using this approach outlined. Recent developments in field desorption-mass spectrometry for the direct characterisation of modern polymer systems are discussed and some early results presented.     

高效液相色谱质谱联用鉴别口服疫苗中硫柳汞降解产物

目的采用高效液相色谱质谱联用的方法鉴别口服疫苗中硫柳汞的降解产物。方法采用C18色谱柱,甲醇-醋酸铵缓冲溶液(pH 4.5)梯度洗脱,以紫外和质谱检测器进行检测,对某口服疫苗中的未知色谱峰进行鉴别。结果疫苗样品中两个色谱峰的保留时间及其一级二级质谱图与硫柳汞的降解产物硫代水杨酸以及2,2’-二硫代二苯甲酸基本一致。结论高效液相色谱质谱联用可鉴别口服疫苗中的硫柳汞降解产物。     

The characterization of protein post-translational modifications by mass spectrometry

Most biological processes are regulated by post-translational modifications of proteins, and conditions that disrupt the regulation of such events can lead to disease. In the past decade, the identification and characterization of covalent modifications have been driven by advances in mass spectrometry. Here, we discuss current mass spectrometric and proteomic approaches for the identification of proteins and their covalent modifications, and we highlight high-throughput strategies for comprehensive analysis of cell proteomes.     

Headspace gas analysis of salad oils by mass spectrometry

A mass spectrometer was used to analyze the content of hydrogen, nitrogen, oxygen, carbon dioxide and argon in headspace gas of commercially packaged soybean, cottonseed and corn salad oils. A leakproof sampling system was designed to avoid air contamination and obtain a representative headspace gas sample. Some edible oils are packaged under pure nitrogen, whereas other samples contained various amounts of oxygen in the headspace gas. The presence or absence of argon in the headspace gas indicates that some oils are packaged with pure nitrogen and others with nitrogen obtained by controlled burning of hydrocarbons to remove all the oxygen in air. The presence of hydrogen in some samples where argon was also present suggested that catalytic purifiers were used to remove the last traces of oxygen and to ensure pure nitrogen for packaging oils. The decrease in oxygen of oils bottled in air was followed during storage at room and at elevated temperatures. No. Market. and Nutr. Res. Div., ARS, USDA.