Monitoring the synthetic reaction of a polyamide/peptide conjugate using electrospray ionization mass spectrometry

Recently, the chemical structures of a series of monoimidazole/polyamine conjugates were studied in this laboratory using electrospray ionization mass spectrometry (ESI-MS) combined with tandem mass spectrometry (ESI-MS/MS). The method was found to be a powerful tool for the identification of this class of compounds. During the synthesis of targeted polyamide/peptide conjugates as derivatives or analogues of netropsin and distamycin, the method was applied to analyze and track the coupling reaction for the formation of the polyamide, which was difficult to achieve using thin layer chromatography (TLC). Characteristic fragmentation pathways for a nitro-monoimidazole conjugate, an amino-monoimidazole conjugate, and the final product (a nitro-diimidazole conjugate) were explored. The fragmentations of these conjugates were strongly affected by the presence of an amino group instead of a nitro group in the molecule, and led to the identification of the three compounds in the reacting solution or in the final reaction mixture. Consequently, the reaction could be monitored successfully and the synthetic route optimized.     

Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation

The alpha v beta 3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging alpha v beta 3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.     

Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.     

Electrospray tandem mass spectrometry of underivatised acetylated xylo-oligosaccharides

Acetylated neutral (Xyl(n)Ac(m)) and acidic xylo-oligosaccharides (Xyl(n)Ac(m)MeGlcA, and Xyl(n)Ac(m)MeGlcAHex) obtained by partial acid hydrolysis of Eucalyptus globulus wood glucuronoxylans and fractionated by preparative ligand exchange/size-exclusion chromatography were identified by electrospray ionisation mass spectrometry (ESI-MS). Low molecular weight acetylated xylo-oligosaccharides were studied by ESI-tandem mass spectrometry (MS/MS). All the acetylated xylo-oligosaccharides showed an abundant ion due to the neutral loss of 60 Da (CH(3)CO(2)H) in the MS/MS spectra. The presence of diacetylated xylo-oligosaccharides was confirmed by the ions formed by loss of two molecules of acetic acid. Furthermore, characteristic [Xyl(res)Ac(2)+Na](+) and [XylAc(2)+Na](+) ions, and ions due to loss of XylAc(2), indicate that both acetyl groups are located in the same Xyl residue. On the other hand, losses of Xyl(res)Ac and XylAc are also observed as well as [Xyl(res)Ac+Na](+) and [XylAc+Na](+) , indicating the location of both acetyl groups in different Xyl residues, in some cases even in adjacent xyloses. The MS/MS spectra of triacetylated xylo-oligosaccharides were complex due to the presence of different isobaric xylo-oligosaccharides containing the acetyl groups at different locations in the xylo-oligosaccharide backbone. In the MS/MS spectra of acidic xylo-oligosaccharides, the ion at m/z 387, [Xyl(res)AcMeGlcA+Na](+), indicates that the acetyl groups are preferentially linked to Xyl substituted with MeGlcA. However, acidic xylo-oligosaccharides with the acetyl and 4-O-methylglucuronic acid groups in different Xyl residues were also identified. In neutral and in acidic xylo-oligosaccharides several possible locations of the acetyl groups were identified, namely at terminal positions. In summary, ESI-MS/MS is shown to be a powerful tool for the characterisation of acetylated patterns in complex mixtures of oligosaccharides.     

(Bio)Molecular surface patterning by phototriggered oxime ligation

Making light work of ligation: A novel method utilizes light for oxime ligation chemistry. A quantitative, low-energy photodeprotection generates aldehyde, which subsequently reacts with aminooxy moieties. The spatial control allows patterning on surfaces with a fluoro marker and GRGSGR peptide, and can be imaged by time-of-flight secondary-ion mass spectrometry.     

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).     

Microcapillary liquid chromatography/tandem mass spectrometry using alkaline pH mobile phases and positive ion detection

Extending the dynamic range of microcapillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) peptide sequencing methods is essential for extracting new discoveries from proteomic studies. The complexity of global protein digests and in vivo processed peptide repertoires (as isolated from immunologically important HLA complexes) have led to the development of novel separation methods to increase the number of peptides identified by a single analysis. Separation of complex mixtures by multidimensional high-performance liquid chromatography (HPLC) decreases the number of isolated peptides contained in each fraction and increases the likelihood of detecting low abundant peptides in a background of dominant signals. In this study, we have evaluated the use of two dimensions of reversed-phase chromatography for resolving and sequencing naturally processed HLA-A2 presented peptide repertoires. The first dimension of separation was reversed-phase chromatography using the strong ion pairing reagent trifluoroacetic acid (TFA) to ensure the highest efficiency of peptide fractionation. The second dimension of reversed-phase chromatography was online with an electrospray ionization (ESI) ion trap mass spectrometer. Mobile phases used for the second dimension of chromatography were modified with volatile reagents including a contemporary acetate-modified acidic solvent, which was compared with mobile phases prepared with ammonium hydroxide at an alkaline pH. As expected, we demonstrate improved separation of the HLA-A2 presented fractions using the alkaline pH conditions. However, less obvious was the improved peptide signal-to-noise detected for peptide signals by positive ion ESI ion trap mass spectrometric detection, which was attributed to a reduced chemical background when using the alkaline pH mobile phases that allowed the ion trap to fill with the peptide ions until the automatic gain control detected a full trap. The term 'wrong-way-round ionization' has been used to describe intense [M+H](+) ions generated during ESI under strongly basic solutions. Ultimately, a larger number of the HLA-A2 peptide repertoire was sequenced by coupling TFA-modified reversed-phase fractionation with alkaline-modified microcapillary LC/MS/MS analysis of each fraction. In the present report, we compare the two second-dimension approaches and demonstrate the quality of data that was acquired using alkaline pH reversed-phase conditions.     

Tandem mass spectrometry in the clinical analysis of variant hemoglobins

A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.     

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry

A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein.     

Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis

Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.     

Nanoscale separations combined with tandem mass spectrometry.

High-efficiency separations of peptide mixtures, tryptic digest and other biological compounds have been achieved using nanoscale packed capillaries and capillary zone electrophoresis (CZE). The coaxial continuous-flow fast atom bombardment design is an excellent interface for coupling these separation techniques with mass spectrometry (MS). In addition, this interface is very useful for the acquisition of MS-MS data from compounds separated by nanoscale packed capillary liquid chromatography and CZE. Structurally informative daughter-ion spectra can be obtained at the low picomole to femtomole level.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.     

Directly coupled liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry for the identification of drug metabolites in urine: application to diclofenac using chlorine and sulfur detection

We report the application of high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICPMS) and orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) for the identification of phase I and II urinary metabolites of diclofenac. The metabolites were separated by reversed-phase HPLC monitored with a UV diode array detector (UV-DAD) after which 90% of the eluent was directed to an ICPMS source, with the remainder going to an oa-TOF mass spectrometer. Compounds containing (35)Cl, (37)Cl and (32)S were detected specifically using ICPMS and identified by oa-TOFMS. The metabolites detected and identified in this way included glucuronic acid and sulfate conjugates, mono- and dihydroxylated and free diclofenac. In addition a previously unreported in vivo metabolite, an N-acetylcysteinyl conjugate of diclofenac, was also characterised. This is the first application of the combination of HPLC/UV-DAD/ICPMS/oa-TOFMS for the investigation of the metabolic fate of chlorinated xenobiotics by direct biofluid analysis.     

Analysis of a commercial preparation of erythromycin estolates by tandem mass spectrometry and high performance liquid chromatography/electrospray ionization tandem mass spectrometry using an ion trap mass spectrometer

Because of the lack of a UV chromophore and their much smaller abundances in comparison with the major component, the minor components in erythromycin estolate preparations are difficult to analyze by high performance liquid chromatography ultraviolet (HPLC-UV). Tentative assignment of the major and minor components can be achieved with the combination of full scan and ZoomScan using an ion trap mass spectrometer. Tandem mass spectrometry (MS/MS) provided an effective method to quickly identify most components without chromatographic separation, and all the related compounds, except the isobaric pair ECE and PdMeEA, could be identified in this way. The best result was obtained by using liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring mode. The major compound, the estolate of erythromycin A (EAE), and seven other minor components, could be separated and identified, with semiquantitative estimates of relative concentrations.     

Investigation of the tris(trimethoxyphenyl)phosphonium acetyl charged derivatives of peptides by electrospray ionization mass spectrometry and tandem mass spectrometry

Charged derivatives of peptides are useful in obtaining simpler collision-activated dissociation (CAD) mass spectra. An N-terminal charge-derivatizing reagent capable of reacting with picomole levels of peptide has been recently reported (Huang et al. Anal. Chem. 1997, 69, 137-144) in the contexts of analyses by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Electrospray ionization (ESI) mass spectrometric investigation of these tris(trimethoxyphenylphosphonium) acetyl derivatives are described in this article, including studies by in-source fragmentation (ISF) and tandem mass spectrometry (MS/MS). Results from ISF are compared with those from MS/MS. Similarities and differences between ESI-ISF, MALDI-post-source decay (PSD), and FAB-CAD data are presented. Differences in fragmentation of these charged derivatives in the triple quadrupole and ion trap mass spectrometers also are discussed. Application of this derivatizing procedure to tryptic digests and subsequent analysis by liquid chromatography-mass spectrometry is also shown.     

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble metabolites

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.     

Synthesis of branched oxime-linked peptide mimetics of the MUC1 containing a universal T-helper epitope

Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive deprotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.