On-line Electrospray Mass Research Articles

Analysis of nonderivatized neutral and sialylated oligosaccharides by electrospray mass spectrometry.

An HPLC/MS method has been developed that allows rapid, direct analysis of underivatized sialylated as well as neutral oligosaccharides. The method involves the separation of oligosaccharides from salts and proteins using RP-HPLC with a formic acid/acetonitrile/water mobile phase system and on-line electrospray mass spectrometry analysis in the positive ion mode. Under the solution conditions employed, both neutral and acidic (sialylated) oligosaccharides are protonated and therefore detected. In contrast to MALDI-TOF MS, no loss of sialic acid is observed when operating in the positive ion mode. Furthermore, the capability of this method to provide quantitative estimates of the relative abundance of each oligosaccharide mass has been demonstrated using fetuin as a model compound.

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene–divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of α-lactalbumin and β-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD≤5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7–10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of ≤0.3% powder mass and a limit of quantitation of ≤1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.

Detection of artefacts and peak identification in reversed-phase HPLC of metallothioneins by electrospray mass spectrometry

The use of ion-spray mass spectrometry rendered it possible to characterize the signals obtained during studies of the polymorphism of metallothionein (MT) by reversed-phase (RP) HPLC in terms of the molecular mass. Artefact signals due to incomplete metallation, exchange of metals with the impurities of the column stationary phase and cross-contamination of the preparations purified by size-exclusion and anion-exchange chromatography may be present. On the other hand, some signals in RP HPLC with UV detection considered to belong to a single species were found to be composed of several complexes eluting precisely at the same time. On-line electrospray mass spectrometry was used to systematize the knowledge of the MT isoforms and subisoforms by attributing to each of the eluting peaks the molecular mass of the form involved and can be used to compare the results obtained for the different groups.

Characterization of metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection

Post-column acidification of the chromatographic effluent was developed to eliminate artefacts in investigations of the polymorphism of metallothionein (MT) by microbore reversed-phase HPLC with detection by pneumatically assisted electrospray mass spectrometry. Metallated species (Cd, Zn and mixed Cd–Zn complexes) were decomposed on-line to produce apo metallothioneins of which the molecular masses were determined by MS. Besides the simplification of the mass spectra taken at the apexes of the chromatographic peaks, the method resulted in a 10-fold improvement of the detection limit of metallothionein and allowed a more comprehensive and less ambiguous detection and identification of the iso- and subisoforms. The method was applied to the characterization of rabbit liver metallothioneins: rabbit liver MT (purified by size-exclusion chromatography only) and MT-1 and MT-2 isoform fractions purified additionally by anion-exchange chromatography.

Silver-phase high-performance liquid chromatography–electrospray mass spectrometry of triacylglycerols

Silver-phase chromatography hyphenated with on-line electrospray mass spectrometry can be applied to characterize triacylglycerols present in vegetable oils with respect to their degree of unsaturation, the position of the most unsaturated fatty acid and the carbon number (CN). The CN information obtained with silver-phase HPLC–ESP–mass spectrometry is complementary to the unsaturation information obtained by silver-phase HPLC-flame ionization detection. Both information is essential to monitor or study modified vegetable oils on the presence of non-natural triacylglycerols. The quantitative results obtained with the method are in agreement with the results obtained in the silver-phase HPLC-flame ionization detection and with theoretical values calculated from the fatty acid distribution of the oil. Silver-phase HPLC–ESP-mass spectrometry gives direct information on fatty acid position and triacylglycerol CN, for each of the triacylglycerols in the sample. This in contrast with non-aqueous reversed-phase HPLC hyphenated with on-line atmospheric pressure chemical ionization-mass spectrometry, which requires a more extensive data processing. The results obtained with silver-phase HPLC–ESP-mass spectrometry can be presented in a three-dimensional overview (relative amount, CN, fatty acid position) serving as a fingerprint for the oil.

Structural analysis of photo-oxidized (ethylenediamine)bis(2,2′-bipyridine)ruthenium(II) complexes by using on-line electrospray mass spectrometry of labeled compounds

Photo-oxidation of Ru(bpy)2(en)2+, where bpy = 2,2′-bipyridine, en = ethylenediamine, was studied in isotopic labeling experiments by using on-line electrospray mass spectrometry (ESMS). The complex was known to undergo photochemical dehydrogenation of a fourelectron oxidation, giving the α,α′-diimine complexes in a stepwise manner via a two-electron-oxidized intermediate that represents loss of two hydrogen atoms from the en ligand. On-line mass analysis after photoirradiation (λ > 420 nm) of Ru(bpy)2(ed)2+ (ed = ethylene-d4diamine) showed that the ligand of the intermediate with loss of two hydrogen atoms was not an enamine but had an imine structure. Also, a ligand-oxygenated complex that has mass 14 amu higher than the Ru(bpy)2(en)2+ complex was observed in the ES mass spectra. The ligand of this complex was proposed to have a nitroso structure as a primary product in 18O2 experiments. The oxygenated complex was not generated in a stepwise manner via the imine intermediate, but directly by loss of two amino hydrogen atoms and addition of an oxygen atom. The source of the oxygen atom would be from oxygen dissolved in solution rather than from water in solution. Another oxygenated complex Ru(bpy)2(NO 2 #x2212; )+ was produced by irradiation and the structure was identified in 18O2 experiments.

An optimized Boc synthesis of indolicidin

Indolicidin, an antimicrobial peptide from bovine neutrophils containing five tryptophan out of a total of 13 residues, has the highest molar proportion of tryptophan of any known peptide sequence and is thus considered a difficult synthetic target. Conventional Boc chemistry can be applied to the synthesis of indolicidin with an appropriate choice of scavenger mixtures, reaction times and temperatures at the crucial acidolytic cleavage and deprotection step. In particular, treatment with HF/p-cresol/p-thiocresol (90:7:3) for 40 min at −8°C results in a crude product containing ca. 90% indolicidin, from which the target compound can be isolated in satisfactory yields and purity after reverse-phase purification. The main byproducts arising during the synthesis and cleavage steps have been identified by HPLC with on-line electrospray mass spectrometric detection.

On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites.

Metabolism of 2'-deoxyphosphorothioate oligonucleotides ISIS 11061 and ISIS 11637 was examined with capillary gel electrophoresis (CGE) and on-line HPLC electrospray mass spectrometry (HPLC/ES-MS). Oligonucleotides were isolated from plasma, liver, and kidneys of rats injected with ISIS 11061 and ISIS 11637. Metabolites found in plasma were consistent with 3'-exonuclease activity. Metabolites isolated from liver and kidney were consistent with 3'- and/or 5'-exonuclease activity. HPLC/ES-MS analysis of ISIS 11061 isolated from kidney indicated extensive degradation from the 3' terminus, but metabolites consistent with 5' degradation and combinations of 3' and 5' truncations also were observed. ISIS 11061 isolated from liver showed less extensive degradation. The 5' truncated metabolites represented the predominant species in contrast to the kidney sample. Metabolites with masses consistent with combinations of 3' and 5' truncations were also observed in liver. The metabolic profiles generated by CGE analysis of these samples agreed qualitatively with mass spectrometric results. HPLC/ES-MS enabled the simultaneous determination of degradation products that are the same length but differ in composition. CGE could discriminate species that differed by one nucleotide in length. HPLC/ES-MS was shown to be a useful tool to study the complex metabolism of antisense oligonucleotides in vivo.

On-line electrospray mass analysis of photoallylation reactions of dicyanobenzenes by allylic silanes via photoinduced electron transfer

Photoinduced electron transfer reaction of 1,4-dicyanobenzene with allyltrimethylsilane in acetonitrile was analyzed by on-line electrospray mass spectrometry. A reductive allylation product of 1,4-dicyanobenzene was detected for the first time as a key intermediate in the photoinduced electron transfer reaction. The electrospray technique, combined with a flowing photoreaction cell, was shown to be a useful tool for detecting and identifying unstable intermediates or primary products in organic photochemical reactions.

Separation and characterization of the metal-thiolate-cluster domains of recombinant sea urchin metallothionein.

Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.

Isolation and identification of glycated aminophospholipids from red cells and plasma of diabetic blood

Glycosylation is a major pathway for posttranslational modification of tissue protein and begins with non-enzymatic addition of carbohydrate to the primary amino groups. Excessive glycation of tissue protein has been implicated in the pathogenesis of diabetes and ageing. While glycation of aminophospholipids has also been postulated, glycated aminophospholipids have not been isolated. Using normal phase HPLC with on-line electrospray mass spectrometry we found glycated ethanolamine phospholipids to make up 10–16% of the total phosphatidylethanolamine (PE) of the red blood cells and plasma of the diabetic subjects. The corresponding values for glycated PE of control subjects were 1–2%.

Identification of stress proteins in lysates of human cell lines separated by two-dimensional electrophoresis and electroblotted simultaneously onto two different membranes.

A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two-dimensional electrophoresis (2-DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N-terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on-line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2-DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).

Photolysis of (Diamine)bis(2,2'-bipyridine)ruthenium(II) Complexes Using On-Line Electrospray Mass Spectrometry.

Photolysis of Ru(bpy)(2)(en)(2+) and Ru(bpy)(2)(tn)(2+), where bpy = 2,2'-bipyridine, en = ethylenediamine, and tn = 1,3-propylenediamine, was studied in acetonitrile using on-line electrospray mass spectrometry (ES-MS). These complexes are known to undergo a four-electron oxidation photochemically, giving the alpha,alpha'-diimine complexes. The monoimine complexes involved in this stepwise process were detectable after photoirradiation (lambda >420 nm). Also, new ligand-oxidized complexes Ru(bpy)(2)(en+14)(2+) and Ru(bpy)(2)(tn+14)(2+) were observed together with photosubstitution products such as Ru(bpy)(2)(AN)(2)(2+) and Ru(bpy)(2)(AN)(2)X(+) (AN = acetonitrile). The notation (en+14) and (tn+14) represents loss of two hydrogen atoms and addition of an oxygen atom to the en and tn ligands. Photosubstitution intermediates with the monodentate diamine, Ru(bpy)(2)(tn)(AN)(2+) and Ru(bpy)(2)(tn)(AN)X(+), were detected in the ES mass spectrum of the tn complex but not in that of the en complex. Other photosubstituted intermediates with the monodentate (en+14) and (tn+14) ligands were detected by on-line mass analysis. The electrospray technique combined with use of a flow-through photoreaction cell was shown to be a useful tool for studying photolysis of inorganic metal complexes.

Detection of Reaction Intermediates: Photosubstitution of (Polypyridine)ruthenium(II) Complexes Using Online Electrospray Mass Spectrometry

Photosubstitution of Ru(bpy) 2 BX 2 (bpy = 2,2'-bipyridine ; B = 3,3'-dimethyl-2,2'-bipyridine (dmbpy) or 2-(aminomethyl)pyridine (ampy) ; and X = ClO 4 - or PF 6 - ) has been studied in both acetonitrile and pyridine by using on-line electrospray mass spectrometry. For the complexes with B = dmbpy and ampy, intermediates with a monodentate (one-legged) B ligand, Ru(bpy) 2 BS 2 and Ru(bpy) 2 BSX + (S = solvent molecule), were detected for the first time in the electrospray mass spectra, together with the photosubstitution products Ru(bpy) 2 S 2 2+ and Ru(bpy) 2 S 2 X + . For the complex with B = ampy, minor photoproducts that resulted from dissociation of bpy, Ru(bpy)BS 2 2+ and Ru(bpy)BS 2 X + , were detected. The solvent-released ions Ru(bpy) 2 S 2+ , Ru(bpy) 2 2+ , and Ru(bpy) 2 SX + , which were secondary products from collision-induced dissociation of the photosubstitution reaction, were also seen. The effects of solvent and intensity of irradiation on the photoreactions were also studied. It was shown for the first time that the reaction intermediates with B = dmbpy can be detected after photoirradiation of electrosprayed charged droplets at the spray needle of the electrospray mass spectrometer. The electrospray technique, combined with use of a flow-through photoreaction cell or with direct photoirradiation of electrosprayed charged droplets, was demonstrated to be a powerful tool for the study of photosubstitution and for the detection of reaction intermediates

Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry

Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C 9 aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C 5 and mono-C 9 aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses

Electrospray Liquid Chromatography/Mass Spectrometry of Ginsenosides

An electrospray liquid chromatography/mass spectrometry (LC/MS) method has been developed for the analysis of ginseng saponins (ginsenosides) contained in extracts of the root of Panax ginseng (Korean ginseng) and Panax quinquefolius (American ginseng). The LC/MS method consists of separation of ginsenosides using an (aminopropyl)silica HPLC column, followed by detection using a photodiode array UV absorbance detector and then on-line electrospray mass spectrometry. Ginsenosides eluted from the HPLC column in order of increasing molecular weight and were detected as [M + Na] + or [M + 138] + adducts. Occurrence of [M + 138] + adducts was most prominent when new HPLC columns were used, and none were observed when solutions of ginsenoside standards were infused into the mass spectrometer without use of HPLC. The cation weighing 138 was identified as (3-ammoniumpropyl)trihydroxysilane, + NH 4 (CH 2 ) 3 Si(OH) 3 , which was either a byproduct of stationary phase synthesis or a stationary phase degradation product. LC/MS chromatograms of extracts of Korean ginseng and American ginseng demonstrated substantial differences between the relative amounts of each ginsenoside. Based on molecular weight and coelution with standards, ginsenosides R g1 , R e , R d , R c , R b2 , and R b1 (in order of elution from the HPLC column) were identified in both ginseng extracts. Four other ginsenosides were detected by mass spectrometry for which no standards were available, and their molecular weights were 801 (possibly corresponding to ginsenoside R f ), 817, 947, and 963 (possibly 20-gluco-R f ). The ginsenosides weighing 817 and 963 were detected only in the Korean ginseng extract.

Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization-time of flight mass spectrometry to the characterization of single-chain plasminogen activator

The analysis of recombinant Desmodus salivary plasminogen activator (DSPAα1), a heterogeneous glycoprotein, is demonstrated through the use of high-performance liquid chromatography (HPLC), high-performance capillary electrophoresis (HPCE), liquid chromatography-electrospray mass spectrometry (LC-ES-MS), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The protein is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein, and fractions from the proteolytic digest. A method for “on-column” collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI-TOF-MS is shown.

An agarose-based gel-concentration system for microsequence and mass spectrometric characterization of proteins previously purified in polyacrylamide gels starting at low picomole levels.

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting.

An Agarose-Based Gel-Concentration System for Microsequence and Mass Spectrometric Characterization of Proteins Previously Purified in Polyacrylamide Gels Starting at Low Picomole Levels

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 μl. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spec-trometry, allowing their identification by peptide mass fingerprinting.

Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis —electrospray mass spectrometry

A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, α-chymotrypsinogen, myoglobin, carbonic anhydrase II and α-lactalbumin) in acetic acid—sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid–sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500 000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris · HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls