Structural analysis of styrene oxide/haemoglobin adducts by mass spectrometry: identification of suitable biomarkers for human exposure evaluation

The structural characterisation of adducts formed by the in vitro reaction of haemoglobin (Hb) with styrene oxide (SO), the most reactive metabolite of the industrial reagent styrene, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human Hb chains. The reactive sites of human Hb towards SO were identified through characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation with tandem mass spectrometry (MALDI-MS/MS). A procedure was set up based on this characterisation, allowing Hb modification to be assessed by monitoring SO/Hb adducts using HPLC with selected ion recording (SIR) mass spectrometry. By this methodology it was also possible to compare advantages and disadvantages of presently available strategies for the measurement of Hb adducts with SO. The results obtained could most plausibly lead to the optimisation of molecular dosimetry of SO adducts, and the analytical procedure described herein could be applied to the biological monitoring of styrene exposure in the workplace.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

Atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sulfonic acid derivatized tryptic peptides.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH(3)SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MS(n). These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins.     

Structural analysis of drug-DNA adducts by tandem mass spectrometry

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.     

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.     

Oligosaccharide sequences in Quillaja saponins by electrospray ionization ion trap multiple-stage mass spectrometry

Ten different samples with 13 previously identified saponin structures from Quillaja saponaria Molina were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive and negative ion modes. Both positive and negative ion mode MS(1)-MS(4) spectra were analyzed, showing that structural information on the two oligosaccharide parts in the saponin can be obtained from positive ion mode spectra whereas negative ion mode spectra mainly gave information on one of the oligosaccharide parts. Analysis of MS(1)-MS(4) spectra identified useful key fragment ions important for the structural elucidation of Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from MS(1)-MS(3) spectra was constructed for the identification of structural elements in the saponin. Peak intensity ratios in MS(3) spectra were found to be correlated with structural features of the investigated saponins and are therefore of value for the identification of terminal monosaccharide residues.     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

Structural analysis of 2,6-[bis(alkyloxy)methyl]-phenyltin derivatives using electrospray ionization mass spectrometry

Electrospray ionization (ESI) mass spectrometry was applied to the structural analysis of 23 2,6-[bis(alkyloxy)methyl]phenyltin(IV) derivatives. The mass spectra were measured in both polarity modes and multistage tandem mass spectrometric (MS(n)) measurements were performed on the ion trap analyser for positively charged tin-containing ions. The sum of complementary ions observed in the positive-ion mode (i.e. [M-R(3)](+) ion) and in the negative-ion mode (i.e. [R(3)](-) ion) permits molecular mass determination in spite of the fact that the molecular adducts were often missing even in the first-order mass spectra. The subsequent fragmentation of [M-R(3)](+) ions studied by MS(n) and the correlation of observed fragment ions with the expected structures of synthesized organotin(IV) compounds allowed us to understand the fragmentation behaviour and the mechanism of the ion formation for studied compounds. The typical neutral losses are alkenes, alcohols and aldehydes. The fragmentation pattern of one selected compound was supported by MS(n) measurements of an isotopically labelled analogue to confirm unusual ion-molecule reactions of some fragment ions with water in the ion trap.     

The influence of structural features on facile McLafferty-type, even-electron rearrangements in tandem mass spectra of carboxylate anions

In contrast to the well-described McLafferty rearrangement in odd-electron cations, relatively little has been reported on comparable rearrangements in even-electron ions, especially negative ions. This work reports a systematic study using tandem mass spectrometry (MS/MS) fragment ion spectra of carboxylate anions having a suitably acidic proton in the gamma position. The rearrangement process was studied in both ion trap and triple quadrupole mass spectrometers; characteristic enolate anions and stable neutral products were formed at low collision energies. The process has diagnostic and analytical potential in, for example, the analysis of peptides having C-terminal serine residues and of 3-hydroxy- or 3-aminocarboxylic acids in complex mixtures.     

Atmospheric pressure chemical ionisation reversed-phase liquid chromatography/ion trap mass spectrometry of intact bacteriohopanepolyols

Atmospheric pressure chemical ionisation liquid chromatography/multi-stage ion trap mass spectrometry (APCI-LC/MS(n)) has been applied to the study of intact bacteriohopanepolyols. Spectral characterisation of bacteriohopanepolyols of known structure present in bacterial extracts (Zymomonas mobilis and a fermenter containing methanotrophs including Methylococcus capsulatus) has revealed greater structural detail than previous liquid chromatography/mass spectrometry (LC/MS) methods and identified characteristic fragmentations indicative of numerous biohopanoid structures. Analysis of a Recent sedimentary extract from Lake Druzhby (Antarctica) has demonstrated the power of this technique to detect biohopanoids in complex samples including at least partial characterisation of previously unknown composite structures.     

RT-PSM, a real-time program for peptide-spectrum matching with statistical significance

The analysis of complex biological peptide mixtures by tandem mass spectrometry (MS/MS) produces a huge body of collision-induced dissociation (CID) MS/MS spectra. Several methods have been developed for identifying peptide-spectrum matches (PSMs) by assigning MS/MS spectra to peptides in a database. However, most of these methods either do not give the statistical significance of PSMs (e.g., SEQUEST) or employ time-consuming computational methods to estimate the statistical significance (e.g., PeptideProphet). In this paper, we describe a new algorithm, RT-PSM, which can be used to identify PSMs and estimate their accuracy statistically in real time. RT-PSM first computes PSM scores between an MS/MS spectrum and a set of candidate peptides whose masses are within a preset tolerance of the MS/MS precursor ion mass. Then the computed PSM scores of all candidate peptides are employed to fit the expectation value distribution of the scores into a second-degree polynomial function in PSM score. The statistical significance of the best PSM is estimated by extrapolating the fitting polynomial function to the best PSM score. RT-PSM was tested on two pairs of MS/MS spectrum datasets and protein databases to investigate its performance. The MS/MS spectra were acquired using an ion trap mass spectrometer equipped with a nano-electrospray ionization source. The results show that RT-PSM has good sensitivity and specificity. Using a 55,577-entry protein database and running on a standard Pentium-4, 2.8-GHz CPU personal computer, RT-PSM can process peptide spectra on a sequential, one-by-one basis in 0.047 s on average, compared to more than 7 s per spectrum on average for Sequest and X!Tandem, in their current batch-mode processing implementations. RT-PSM is clearly shown to be fast enough for real-time PSM assignment of MS/MS spectra generated every 3 s or so by a 3D ion trap or by a QqTOF instrument.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.     

A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry

Liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) has been employed to identify carotenoid esters present in raw organic extracts of pigmented freshwater microalgae and to gain structural information on these compounds. In particular, acyl carotenoid derivatives of Haematococcus pluvialis and Euglena sanguinea have been characterised by tandem mass spectrometry (MS/MS) in a quadrupole ion trap. ESI-MS/MS allows recognition of the presence of carotenoid esters in complicated mixtures without any initial chromatographic work-up and without the need to use UV-Vis photo-diode array (PDA) detectors. Product ion scans of the [M + Na]+ ion lead to known neutral losses of the C7H8 and C8H10 residues from the conjugated polyene moiety of the carotenoid unit, that permit the unambiguous identification of the carotenoid itself. These structurally relevant ions are not observed in positive or negative ion APCI (atmospheric pressure chemical ionisation) mass spectra. Moreover, the several product ions observed in positive and/or negative ion ESI-MS/MS not only are a diagnostic signature of the main structural features of the acyl chains such as length, position and unsaturation, but also display the nominal mass of the parent xanthophyll. Our methodology has been validated (i) by using esters of astaxanthin obtained from off-line purification of the H. pluvialis extracts and structurally elucidated through proton nuclear magnetic resonance (1H-NMR) spectroscopy and (ii) by product analysis of esters by alkaline hydrolysis. The characterisation of the unknown carotenoid esters of E. sanguinea is a demonstration of the capabilities of this methodology.     

Mass spectrometric fragmentation of cyclic peptides belonging to the polymyxin and colistin antibiotics studied by ion trap and quadrupole/orthogonal-acceleration time-of-flight technology

Electrospray ionization linked to quadrupole/orthogonal-acceleration time-of-flight (Q/oaTOF) and ion trap equipment was used to study the fragmentation behavior of the linear side-chain cyclized peptides of the polymyxin B and E antibiotics. This study exemplifies both the benefits and the drawbacks of mass spectrometric techniques for the determination of the sequence of such complex linear side-chain cyclized peptides. Q/oaTOF accurate mass measurements did not help sufficiently to assign the product ions observed in the product ion spectra. An ion trap mass spectrometer providing MS(n) capability was used to eliminate ambiguities encountered with a single MS/MS approach. The complex fragmentation behavior of these compounds of well-established structure is described which could be useful for structural characterization of unknown substances related to polymyxin B and E in the future.     

Alkylation of complementary ribonucleotides by 1,2‐dodecyl‐epoxide in a micellar environment: a liquid chromatography—electrospray ionization—sequential mass spectrometry investigation

Alkylation of a pair of complementary ribonucleotides, adenosine monophosphate (AMP) and uridine monophosphate (UMP), was accomplished by 1,2‐dodecyl‐epoxide (DE) in a oil‐in‐water microemulsion based on the cationic surfactant Cetyl‐trimethyl‐ammonium‐bromide, providing a suitable catalytic interface for the reagents. Several, often isomeric, alkylation products, bearing one or two hydroxy–dodecyl moieties on their structures, were identified in the reaction mixtures by high‐performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry. In particular, mass spectrometry (MS)/MS spectra, implemented by extracted ion chromatograms obtained for peculiar MS/MS product ions, indicated alkylation to occur on uracil and on uracil/phosphate OH groups in singly and doubly alkylated UMP, respectively. Adenine NH₂ group and phosphate or ribose OH groups were found to be involved as such (single alkylation) or in combination, in the case of alkylated derivatives of AMP. The reaction of both endocyclic N and C?O groups (tautomerized to C? OH groups) of uracil and the predominance of nucleophilic attack to the more accessible carbon of the DE epoxydic bridge (the only exception being the reaction by the NH₂ group of adenine) were inferred from MS³ spectra with the help of extracted ion chromatograms for specific fragment ions, after their structural characterization. Interestingly, alkylation on one of the uracil C?O groups and, partially, on the adenine NH₂ group, both potentially involved in AMP/UMP base pairing in the micellar environment, were found to be hindered when both ribonucleotides were present in the reaction mixtures. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.     

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.     

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.