Reversed-phase Liquid Chromatography Mass Spectrometry Research Articles

Kinetics of appearance of hemorphins from bovine hemoglobin peptic hydrolysates by a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry

Reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry was used to improve the preparation of three opioid peptides (Leu–Val–Val–hemorphin-7, Val–Val–hemorphin-7 and Val–Val–hemorphin-4) resulting from bovine hemoglobin peptic hydrolysates. Optimal conditions for the preparation of these peptides were determined thanks to their kinetic studies of appearance in the course of peptic hydrolyses as a function of degree of hydrolysis of hemoglobin. We propose a low degree of hydrolysis (3%) to prepare Leu–Val–Val–hemorphin-7, a mean degree of hydrolysis (11%) to prepare Val–Val–hemorphin-7 and a high degree of hydrolysis (21%) to prepare Val–Val–hemorphin-4.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate

We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification.

Determining affinity-selected ligands and estimating binding affinities by online size exclusion chromatography/liquid chromatography-mass spectrometry.

Size exclusion chromatography (SEC) isolation of affinity-selected ligands combined with reverse phase liquid chromatography-mass spectrometry (LC-MS) is an effective means for identifying members of mixtures which form tightly bound noncovalent complexes with target proteins. A potential liability of the approach is that the SEC isolation is carried out under nonequilibrium conditions favoring protein/ligand complex dissociation. At long SEC isolation times and/or for complexes with fast off-rates the extent of dissociation can jeopardize the ability to detect the affinity-selected components. Additionally, equilibrium binding affinities cannot be exactly determined from the measured distribution of isolated ligands. We present here an online SEC/LC-MS system for determining affinity-selected members of active mixtures which reduces this liability. A kinetic model of the SEC isolation process is developed to determine the practical limits for the application of the method and to extrapolate equilibrium binding affinities from the nonequilibrium data. The utility of online SEC/LC-MS for identifying affinity-selected ligands and for estimating binding affinities is demonstrated for a small molecule mixture of compounds with known binding affinities and for a simple combinatorial mixture.

Development of a method based on liquid chromatography–electrospray mass spectrometry for analyzing imidazolinone herbicides in environmental water at part-per-trillion levels

An evaluation was made of the feasibility of using reversed-phase liquid chromatography–mass spectrometry with an electrospray interface (LC–ESI-MS) to measure traces of imidazolinone herbicides in different natural water samples. The imidazolinones are a significant new class of low-use-rate, reduced-environmental-risk herbicides for the protection of a wide variety of agricultural commodities. The procedure used involved passing 0.5, 1, 2 l of river, ground and drinking water samples, respectively, through a 0.5 g graphitized carbon black (GCB) extraction cartridge. Analytes were eluted from the GCB surface by 8 ml of a methylene chloride–methanol (80:20, v/v) solution acidified with formic acid, 25 m M. Recovery was higher than 89% irrespective of the aqueous matrix in which the analytes were dissolved. A conventional 4.6 mm I.D. reversed-phase LC C 18 column operating with a mobile phase flow-rate of 1 ml/min was used to chromatograph the analytes. A flow of 50 μl/min of the column effluent was diverted to the ESI source. The effects of acid concentration on ESI-MS detector response in the mobile phase were investigated. The effects on the production of diagnostic fragments produced by varying the orifice plate voltage and the response of the MS detector were also evaluated. For the analyte considered, the response of the mass detector was linearly related to the amount of analyte injected between 1 and 50 ng. The limit of detection (signal-to-noise ratio=3) of the method for the pesticides considered in drinking water samples was estimated to be about 2–5 ng/l.

Identification of active-site histidine residues of a self-incompatibility ribonuclease from a wild tomato.

The style component of the self-incompatibility (S) locus of the wild tomato Lycopersicon peruvianum (L.) Mill. is an allelic series of glycoproteins with ribonuclease activity (S-RNases). Treatment of the S3-RNase from L. peruvianum with iodoacetate at pH 6.1 led to a loss of RNase activity. In the presence of a competitive inhibitor, guanosine 3'-monophosphate (3'-GMP), the rate of RNase inactivation by iodoacetate was reduced significantly. Analysis of the tryptic digestion products of the iodoacetate-modified S-RNase by reversed-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry showed that histidine-32 was preferentially modified in the absence of 3'-GMP. Histidine-88 was also modified, but this occurred both in the presence and absence of 3'-GMP, suggesting that this residue is accessible when 3'-GMP is in the active site. Cysteine-150 was modified by iodoacetate in the absence of 3'-GMP and, to a lesser extent, in its presence. The results are discussed with respect to the related fungal RNase T2 family and the mechanism of S-RNase action.

Characterization of unstable intermediates and oxidized products formed during cyanogen bromide cleavage of peptides and proteins by electrospray mass spectrometry.

Products formed during cyanogen bromide (CNBr) digestion of alpha-endorphin, beta-endorphin, and horse heart myoglobin are examined using reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. It is demonstrated that unstable intermediate reaction products may be formed, as well as oxidized products when the CNBr reaction is performed in 0.1% TFA in water/acetonitrile (6:4 v/v) and that, under other conditions commonly employed for the CNBr cleavage reaction, unstable intermediate products are also generated. The formation of the expected cleavage products is found to be improved by adjusting the hydrolysis conditions. The structure of the intermediate formed from alpha-endorphin is examined using electrospray mass spectrometry in combination with low-energy collision-induced dissociation and tandem mass spectrometry and is shown to have a cyclic hydrated homoserine iminolactone part. The results obtained in this study explain the formation of partially cleaved proteins in the case of Met-Thr-containing sequences, which likely have a cyclic hydrated homoserine iminolactone part instead of the putative homoserine residue.

In vitro metabolism of LVV-hemorphin-7 in human plasma studied by reversed-phase high-performance liquid chromatography and micro-electrospray mass spectrometry

In these experiments we have studied the in vitro metabolism of LVV-hemorphin-7 in human plasma by using reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with micro-electrospray mass spectrometry (micro-ES-MS). Tandem mass spectrometry (MS-MS) was performed in order to verify the structure of the peptide fragments found. Incubations were performed with and without different protease inhibitors. Results showed that LVV-hemorphin-7 was metabolized from the N-terminal end of the peptide, probably by an amastatin-sensitive exopeptidase.

Trace analysis of impurities in 3′-azido-3′-deoxythymidine by reversed-phase high-performance liquid chromatography and thermospray mass spectrometry

An analytical method has been developed for the detection of trace amounts of impurities in 3′-azido-3′-deoxythymidine referred to herein as AZT (Zidovudine). A sample extract was preconcentrated by normal-phase high-performance liquid chromatography (HPLC) with subsequent on-line reversed-phase HPLC-thermospray mass spectrometry (TSP-MS). During the sample extraction and concentration step, carried out by semipreparative normal-phase chromatography, the preliminary separation of the impurities from the AZT takes place. The organic solvent (dichloroethane-acetonitrile, 40:60) is evaporated from the collected fractions and the compounds are redissolved in a smaller volume of the reversed-phase mobile phases for a further degree of concentration. The collected fractions are then subjected to reversed-phase HPLC-TSP-MS. The influence of acetonitrile concentration and pH on the reversed-phase separation together with the sensitivity of the TSP-MS detection have been examined to maximise detection levels. The 3′-azido-3′-deoxy-5′-O-tritylthymidine, triphenyl methanol and 3′-chloro-3′-deoxythymidine, which are route-indicative impurities formed during the synthesis can be detected in the 50–100 ppb (w/w) range.

Separation and characterization of the main methylated nucleobases from nuclear, cytoplasmic and poly (A) + RNA by high-performance liquid chromatography and mass spectrometry

We were able to detect nine methylated nucleobases (3-methyluracil, 1-, 2-, 3- and 7-methylguanine, 1-, 2-, 3- and 6-methyladenine) in RNA from rat and calf liver, baker's yeast, Torula and Euglena cells by using reversed-phase high-performance liquid chromatography and thermospray mass spectrometry. Total cellular, nuclear, cytoplasmic and poly (A) + RNA from rat liver showed marked methylation, mainly of 1- and 3-methylguanine, and 3- and 2-methyladenine. These bases were especially abundant in nuclear RNA and, to a lesser extent, in poly (A) + RNA. In contrast, 7-methylguanine and 6-methyladenine were poorly represented in poly (A) + RNA.

Reversed-phase liquid chromatography-mass spectrometry of the uncommon triacylglycerol structures generated by randomization of butteroil

The test triacylglycerols were prepared by randomization of natural butterfat followed by silver ion TLC segregation of the saturates, monoenes and dienes. The molecular species were identified by on-line positive chemical ionization mass spectrometry. Within a series of isologous triacylglycerols, those containing the shortest fatty acid chain were eluted last from a reversed-phase column (XX10 ⪢ XX8 ⪢ XX6 ⪢ XX4, where X = long-chain acid), although common experience would have predicted that the shorter and more polar species would be eluted first. A comparable order of elution was obtained for isologous triacylglycerols containing two short- and one long-chain (X88 ⪢ X86 ⪢ X66 ⪢ X84 ⪢ X64 ⪢ X44) or three short-chain fatty acids per molecule, when compared to triacylglycerols containing medium or long-chain fatty acids in combination with short-chain acids. Since this resolution is similar to that achieved on adsorption chromatography, it is suggested that the C 18 reversed-phase column possesses residual adsorptive activity. No discernible separation was seen among the corresponding positional or rev erse isomers of short-chain triacylglycerols with the present reversed-phase system.

Metabolism of des(64,65)-human proinsulin in the rat. Evidence for the proteolytic processing to insulin.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Reversed-phase liquid chromatography-mass spectrometry of complex mixtures of natural triacylglycerols with chloride-attachment negative chemical ionization

Short chain triacylglycerols of butteroil and long chain triacylglycerols of menhaden oil were resolved by conventional reversed-phase high-performance liquid chromatography (HPLC) using a linear gradient of 10–90% propionitrile in acetonitrile as the mobile phase. The triacylglycerol species were identified by positive chemical ionization mass spectrometry, which provided [MH-RCOOH] + and [MH] + ions and by negative chemical ionization mass spectrometry with chloride attachment, which yielded exclusively the pseudomolecular ions [M + Cl] −. The negative ions were produced by the inclusion of 1% methylene chloride in the HPLC mobile phase, which did not affect the triacylglycerol elution profile. The restriction of ionization to the chloride-attachment pseudomolecular ions increased about 100-fold the sensitivity of detection of all molecular species and facilitated their quantitation by mass spectrometry. By combining the results of positive and negative chemical ionization mass spectrometry of the eluted peaks it was observed that the complex short chain triacylglycerols showed extensive resolution within isologous saturated carbon number while the polyunsaturated fish oil triacylglycerols showed readily detectable resolution within isologous carbon and double bond number☆. These observations permit the identification and quantitation of molecular species of triacylglycerols in such complex mixtures as butterfat and fish oil, which have thus far proved difficult or impossible.

Location of disulfide bonds in antithrombin III

Proteolytic digests of human antithrombin III (ATIII) have been analyzed by a combination of reversed-phase high-performance liquid chromatography and fast atom bombardment (FAB) mass spectrometry for disulfide-containing peptides which are diagnostic for disulfide linkages in ATIII. These results indicate that disulfide bonds join Cys 8 and Cys 128, Cys 21 and Cys 95, and Cys 247 and Cys 430. In addition, these results demonstrate that systematic searches of the amino acid sequence of a large protein for segments with molecular weights matching FAB mass spectral information is a viable method for identifying peptides independent of the specificity of the enzyme used to fragment the protein.

Gaseous acidity and basicity scales as guides to chemical ionization in reversed-phase liquid chromatography-mass spectrometry with direct liquid introduction

During direct liquid introduction (DLI) liquid chromatography-mass spectrometry (LC-MS), the detectability of acetone is shown to be predictable, based on consideration of the gas-phase acidities and basicities of solvents methanol and acetonitrile and modifiers formic acid and ammonium formate. Consequently, ion formation in DLI LC-MS resembles gas-phase chemical ionization processes; since the order of acidities is altered in solution, solution ionization must be much less important than gas-phase. Ion populations could be predicted on the basis of gas-phase proton affinities and acidities: acetone could be detected in all solvent mixture by positive ions, but it was not readily detected by negative ions when acid was also present. In solvents without additives it was always detected.

Quantitative trace analysis by reversed-phase liquid chromatography-mass spectrometry

In order to overcome difficulties in spraying aqueous solvents into the vacuum of a mass spectrometer, an ultrasonic spraying device has been constructed. The vibration is achieved by means of magnetostriction in the nickel inlet tube itself. Applications to aliphatic acid determination in a shale oil process water and to determination of valproic acid, an anti-convulsant, in human serum (SRM 1599) are presented.