Identification of pesticide transformation products in food by liquid chromatography/time-of-flight mass spectrometry via "fragmentation-degradation" relationships

The identification of transformation products of pesticides in foodstuffs is a crucial task difficult to tackle, due to the lack of standards and scarce information available. In this work, we describe a methodology for the identification and structural elucidation of pesticide transformation products in food. The proposed strategy is based on the use of liquid chromatography electrospray time-of-flight mass spectrometry (LC/TOFMS): accurate mass measurements of (molecule and fragment) ions of interest are used in order to establish relationships between fragmentation of the parent pesticides in the instrument (in-source CID fragmentation) and possible degradation products of these pesticides in food. Examples of this strategy showing the potential of LC/TOFMS to determine unknown pesticides in food are described in two different real samples, suggesting that pesticides often are transformed into degradation products in the same fashion that they are fragmented in the instrument. Using the proposed approach and without using standards a priori, based solely on accurate mass measurements of ions and "fragmentation-degradation" relationships, we have identified two parent pesticides (amitraz and malathion) along with six degradation products, m/z 253 (N,N'-bisdimethylphenylformamidine), 163 (N-2,4-dimethylphenyl-N-methyl formamidine), 150 (2,4-dimethylformamidine), and 122 (2,4-dimethylaniline) from amitraz, and m/z 317 and 303, due to ether hydrolysis of methyl and ethyl groups from malathion. Structures for these species were proposed, and the potential of the proposed approach was critically discussed.     

Study of the fragmentation mechanism of protonated 6-hydroxychlorzoxazone: application in simultaneous analysis of CYP2E1 activity with major human cytochrome P450s

The application of liquid chromatography tandem mass spectrometry for simultaneous analysis of major human cytochrome P450 activities via a single atmospheric pressure ionization (API) LC/MS/MS method has been hampered by the preferred detection of 6-hydroxychlorzoxazone (HCZ), the metabolite of the CYP2E1 probe, chlorzoxazone, under negative API. An initial simulation of the dissociation constants suggested the potential ionization of the enol form of HCZ at low pH, and the accurate mass measurements confirmed the presence of the protonated HCZ signal under (+) ESI at pH 3. However, the CID spectrum of the protonated HCZ resulted in a few intense, but uncommon, fragment ions that could be utilized for specific selected reaction monitoring (SRM) transitions. The deduced elemental compositions of these fragment ions indicated possible aromatic ring opening for the first two intense product ions at m/z 130 and 115, as well as chlorine radical loss for the third ion at m/z 151. Further precursor and product ion scan studies, along with the deuterium ion exchange in solution, revealed the involvement of three distinct pathways of fragmentation. The m/z 186-->130 transition, which was shown to be specific in human plasma and rat hepatic microsomes, was further combined with the SRM transition of reserpine (internal standard) and eight probe substrates for human cytochrome P450 isoforms. This led to the development of a full LC/MS/MS method capable of analyzing a total of nine human P450 activities within 3 min, including CYP2E1, using a single assay in the (+) ESI mode. The HCZ assay showed excellent linearity with a coefficient of determination (R2) greater than 0.98 at dynamic range of 0.05 (LOQ) to 40 microM. Preliminary data from the three-day validation of the HCZ assay indicated that the accuracy and precision for quality control samples was within +/- 15% of the spiked concentration at all levels.     

Intramolecular isobaric fragmentation: a curiosity of accurate mass analysis of sulfadimethoxine in pond water

Liquid chromatography with time-of-flight mass spectrometry (TOF-MS) and quadrupole-time-of-flight (Q-TOF) mass spectrometry/mass spectrometry (MS/MS) were used for the accurate mass analysis of sulfadimethoxine in pond water of a fish hatchery. Sulfadimethoxine is the most important sulfa antimicrobial used in aquaculture to treat bacterial disease in a wide variety of fish. Because correct identification is essential to environmental monitoring of antimicrobial pharmaceuticals, accurate mass analyses (TOF and Q-TOF-MS/MS) were compared to nominal mass measurement (quadrupole ion trap). It was known that all six members of the sulfa antimicrobial family gave a common 6-sulfanilamido ion at a nominal mass of m/z 156; thus, this ion was the focus of TOF confirmation (exact mass 156.0119 u) along with the protonated molecule (exact mass 311.0814 u). In the process of accurate mass confirmation of the 156 m/z fragment ion, a second isobaric ion (exact mass m/z 156.0773), was discovered at the same nominal mass, which was not differentiated by quadrupole ion trap. The structure was assigned as 2-4-dimethoxypyridine and is exactly the other protonated half of the sulfadimethoxine molecule. This discovery led to the subsequent use of Q-TOF-MS/MS and high-resolution identification of five other important ion fragments for the identification of sulfadimethoxine in pond water at environmental concentrations. The caveats of using low-resolution mass spectrometry without MS/MS for environmental monitoring are discussed in the light of high profile monitoring of sulfa antimicrobial pharmaceuticals in the aquatic environment.     

Electrospray ionization tandem mass spectrometry for structural elucidation of protonated brevetoxins in red tide algae

Brevetoxins, the toxic components of "red tide" algae, all share one of two robust polycyclic ether backbone structures, but they are distinguished by differing side-chain substituents. Electrospray ionization mass spectrometry analyses of brevetoxins have shown that the polyether structure invariably has a very high affinity for sodium cations that results in the production of abundant (M + Na)+ ions even when sodium cations are only present as impurities. Because the ionic charge tends to remain localized on the sodium atom and because at least two bonds must be broken in order to produce polycyclic backbone fragmentation, it is extremely difficult to obtain abundant product ions (other than Na+) from (M + Na)+ brevetoxin precursor ions in low-energy collision-induced dissociation (CID) MS/MS experiments. This report establishes that acid additives (oxalic acid, trifluoroacetic acid, and particularly hydrochloric acid) in aqueous methanol solutions can promote high yields of protonated brevetoxin molecules (MH+ ions) for Btx-1, -2, and -9 brevetoxins. Most importantly, unlike their (M + Na)+ counterparts, MH+ precursor ions offer readily detectable product ions in CID MS/MS experiments, even under low-energy collisions. This direct structural characterization approach has provided decomposition information from brevetoxins that was previously inaccessible, including the identification of diagnostic product ions for "type A" brevetoxins (m/z 611) and "type B" brevetoxins (m/z 779, 473, 179) and characteristic ions for Btx-1 (m/z 221, 139), Btx-2 (m/z 153), and Btx-9 (m/z 157, 85). Precursor ion scans and constant neutral loss scans are proposed to enable screening of individual type A or type B brevetoxins present in naturally occurring mixtures.     

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.     

Collisionally induced dissociation in the study of A-ring hydroxylated vitamin D type compounds.

Collisionally induced dissociation (CID) is often used to determine the structure of ions based on comparison with the CID spectra of known ions. The latter are generated from judiciously selected compounds taking into account basic principles of ion chemistry. We report here on the use of this approach toward determination of the site of A-ring hydroxylation of vitamin D. Although not intrinsically an aromatic compound, vitamin D gives rise in its mass spectrum to an aromatic methylstyryl cation at m/z 118. A-ring hydroxylated metabolites of vitamin D would thus incorporate the extra OH group on the ion at m/z 118, shifting it to m/z 134. The position of substitution of the extra OH group on a metabolite could then be ascertained by comparing the CID spectrum of its m/z 134 fragment to those of the four possible (hydroxymethyl)styryl cations generated from synthesized authentic compounds. Because of their propensity to polymerize, these cations were generated in situ via the McLafferty rearrangement of the corresponding (hydroxyphenyl)ethanols. For optimum differentiation of isomeric ions, preparation of permethylated derivatives of vitamin D was necessary. The validity of the hypothesis was verified using 1,25-dihydroxy-vitamin D3 as a test compound. This method provides a viable approach for the characterization of A-ring hydroxylated metabolites of vitamin D as well as for related aromatic compounds.     

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Development of a proton-transfer reaction-linear ion trap mass spectrometer for quantitative determination of volatile organic compounds

Currently, proton-transfer reaction mass spectrometry (PTR-MS) allows for quantitative determination of volatile organic compounds in real time at concentrations in the low ppt range, but cannot differentiate isomers or isobaric molecules, using the conventional quadrupole mass filter. Here we pursue the application of linear quadrupole ion trap (LIT) mass spectrometry in combination with proton-transfer reaction chemical ionization to provide the advantages of specificity from MS/MS. A commercial PTR-MS platform composed of a quadrupole mass filter with the addition of end cap electrodes enabled the mass filter to operate as a linear ion trap. The rf drive electronics were adapted to enable the application of dipolar excitation to opposing rods, for collision-induced dissociation (CID) of trapped ions. This adaptation enabled ion isolation, ion activation, and mass analysis. The utility of the PTR-LIT was demonstrated by distinguishing between the isomeric isoprene oxidation pair, methyl vinyl ketone (MVK) and methacrolein (MACR). The CID voltage was adjusted to maximize the m/ z 41 to 43 fragment ratio of MACR while still maintaining adequate sensitivity. Linear calibration curves for MVK and MACR fragments at m/ z 41 and 43 were obtained with limits of detection of approximately 100 ppt, which should enable ambient measurements. Finally, the PTR-LIT method was compared to an established GC/MS method by quantifying MVK and MACR production during a smog chamber isoprene-NO x irradiation experiment.     

Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.     

Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

Ultrahigh Mass Accuracy in Isotope-Selective Collision-Induced Dissociation Using Correlated Sweep Excitation and Sustained Off-Resonance Irradiation: A Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Case Study on the [M + 2H](2+) Ion of Bradykinin

Using electrospray ionization with a 9.4 T Fourier transform mass spectrometer, fragment ion spectra were acquired for a single isotopomer of doubly protonated bradykinin (molecular mass, 1059.6 Da). Correlated sweep excitation methods were applied to mass-select the single isotopomer (m/z = 530.8). Sustained off-resonance irradiation was used to activate and fragment the ions. The accuracy (in terms of m/z) in detection of the fragment ions was on average 1.2 ppm, making the assignments unambiguous. The methods employed would be generally applicable to ions in the mass range of approximately 50 Da to 50 kDa.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Application of atmospheric pressure ionization time-of-flight mass spectrometry coupled with liquid chromatography for the characterization of in vitro drug metabolites

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 microL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic "A" ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.     

Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges

A kinetic model, based on the "mobile proton" model of peptide fragmentation, has been reported previously for quantitative prediction of low-energy collision-induced dissociation (CID) spectra of singly or doubly charged peptides. For peptides with three or more charges, however, the simulation process is complex and time-consuming. This paper describes a simplified model for quantitative prediction of CID spectra of peptide ions with three or more charges. Improvements on other aspects of the model were also made to accommodate large peptides. The performance of the simplified model was evaluated by generating predictions for many known highly charged peptides that were not included in the training data set. It was shown that the model is able to predict peptide CID spectra with reasonable accuracy in fragment ion intensities for highly charged peptide ions up to 5000 u in mass.     

Phosphate group-driven fragmentation of multiply charged phosphopeptide anions. Improved recognition of peptides phosphorylated at serine, threonine, or tyrosine by negative ion electrospray tandem mass spectrometry

The nanoelectrospray product ion spectra of multiply charged phosphopeptide anions reveal the occurrence of phosphate-specific high-mass fragment ions of the type [M - nH - 79](n-1)-. These so far unrecognized fragments, which are observed for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides, are the counterparts of the established inorganic phosphopeptide marker ion found at m/z 79 = [PO3]-. The high-mass marker ions are formed with high efficiency at moderate collision offset values and are particularly useful for sensitive recognition of pSer-, pThr-, and pTyr-peptides due to the low background level in MS/MS spectra at m/z values above those of the precursor ions. By virtue of this feature, the detection of the new phosphorylation-specific fragment ions appears to be more sensitive than the detection of the low-mass phosphate marker ion at m/z 79, where a higher interference by nonspecific background signals is generally observed. The number of phosphate groups within a phosphopeptide can also be estimated on the basis of the [M - nH - 79](n-1)- ions, since these exhibit an effective, sequential neutral loss of H3PO4 of the residing phosphate groups. A mechanistic explanation for the formation of the [M - nH - 79](n-1)- ions from multiply charged phosphopeptides is given. The high-mass marker ions are proposed to originate from phosphopeptide anions, which carry two negative charges located at the phosphate group. A new search tool denominated "variable m/z gain analysis", which utilizes these newly recognized high-mass fragments for spotting of phosphopeptides in a negative ion parent scan, is proposed. The findings strengthen the value of negative ion ESI-MS/MS for analysis of protein phosphorylation.     

Scrubbing ions with molecules: kinetic studies of chemical noise reduction in mass spectrometry using ion-molecule reactions with dimethyl disulfide

The kinetics and product distributions of the reactions of dimethyl disulfide (DMDS) have been investigated with a group of chemical background ions commonly observed in atmospheric pressure ionization (API) mass spectrometry (MS) in order to assess the value of this molecule in filtering (or "scrubbing") these ions by changing their mass/charge (m/z) ratio. The measurements were taken with a novel electrospray ionization/selected ion flow tube/QqQ tandem mass spectrometer. The background ions studied include those with m/z 42 (protonated acetonitrile, ACN), 83 (protonated ACN dimer), 99 (protonated phosphoric acid), 117 (water cluster of m/z 99), 131 (methanol cluster of m/z 99), 149 (protonated phthalic anhydride, formed from the phthalates), and 327 (protonated triphenyl phosphate). In addition, reactions of DMDS have been studied with two model analytes--protonated caffeine and doubly protonated bradykinin--in order to assess the selectivity of DMDS reactivity. All the measurements were taken at 295 +/- 2 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. DMDS was observed to react efficiently with m/z 42 (ACNH+), 149 (from phthalates), and 99 (protonated phosphoric acid), with k/kc=0.91, 0.47, and 0.38, respectively. Only proton transfer was observed with ACNH+, followed by the secondary reaction of [DMDSH]+ with DMDS to yield [CH3S-S(CH3)-SCH3]+. Ligation of DMDS was the dominant primary channel observed for the reaction of the m/z 149 background ion; however, some proton transfer also was observed. Both of these primary product ions react further with DMDS to yield [CH3S-S(CH3)-SCH3]+, the structure of which we have determined computationally using DFT calculations. Only the sequential ligation with two DMDS molecules was observed for the reaction of the m/z 99 ion. Reactions of DMDS with m/z 117 [H3PO4 + H + H2O]+ and m/z 131 [H3PO4 + H + MeOH]+ were observed to proceed with k/kc=0.71 and 0.058, respectively. Ligand substitution of DMDS for H2O predominated ( approximately 94%) over DMDS ligation ( approximately 6%) in the reaction with m/z 117, while only DMDS ligation was observed for the reaction of m/z 131 with DMDS. In contrast, the reactions of DMDS with ions of m/z 83 (protonated dimer of ACN) and 327 (protonated triphenyl phosphate) were extremely inefficient, with k/kc=0.0042 and 0.0079, respectively. The higher reactivity of DMDS toward ACNH+ (m/z 42) compared to (ACN)2H+ (m/z 83) is attributed to the lower proton affinity of the unsolvated ACN. The reactivity of DMDS toward the two model analyte ions studied-protonated caffeine and doubly protonated bradykinin-was negligible, with k/kc=0.0073 and 0.010, for the respective reactions. These results suggest that, under appropriate reagent pressure conditions, DMDS can be an appropriate reagent for chemically filtering out many common API-MS background ions, without significantly affecting the observed intensity of analyte peaks.     

Simple identification of a cross-linked hemoglobin by tandem mass spectrometry in human serum

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).     

Primary structure determination of peptides and enzymatically digested proteins using capillary liquid chromatography/mass spectrometry and rapid linked-scan techniques

Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the low-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that it is possible to generate interpretable collision-induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating in the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ ions were then selected for subsequent LC/MS/MS analysis (typically 1/5 of the remaining protein digest). Elution times for each chromatographic peak were generally greater than 30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the signal/noise ratio at these low-picomole levels. Unit resolution was observed for product ions below m/z 1000. Rapid linked scanning by LC/frit-FABMS/MS provided mass assignments for product ions within 0.2-0.3 amu of theoretical values. Side-chain fragment ions (wn and dn) were also observed, which allowed for the differentiation of isobaric amino acids (e.g., leucine and isoleucine). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated beta-casein.     

Prediction of low-energy collision-induced dissociation spectra of peptides

A kinetic model, based on the "mobile proton" model of peptide fragmentation, was developed to quantitatively simulate the low-energy collision-induced dissociation (CID) spectra of peptides dissociated in a quadrupole ion trap mass spectrometer. The model includes most fragmentation pathways described in the literature, plus some additional pathways based on the author's observations. The model was trained by optimizing parameters within the model for predictions of CID spectra of known peptides. A best set of parameters was optimized to obtain best match between the simulated spectra and the experimental spectra in a training data set. The performance of the mathematical model and the associated optimized parameter set used in the CID spectra simulation was evaluated by generating predictions for a large number of known peptides, which were not included in the training data set. It was shown that the model is able to predict peptide CID spectra with reasonable accuracy in fragment ion intensities for both singly and doubly charged peptide parent ions up to 2000 u in mass. The optimized parameter set was evaluated to gain insight into the collision-induced peptide fragmentation process.