Coupling capillary electrophoresis and high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of complex lipopolysaccharides

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technology for atmospheric pressure, room temperature separation of gas-phase ions. The FAIMS system acts as an ion filter that can continuously transmit one type of ion, independent of mass-to-charge ratio (m/z). Capillary electrophoresis-electrospray mass spectrometry (CE-MS) has been extensively used for the analysis of complex bacterial lipopolysaccharides (LPS). The coupling of FAIMS to CE-MS provides a sensitive technique for the characterization of these complex glycolipids, permitting the separation of trace-level LPS oligosaccharide glycoforms for subsequent structural characterization using tandem mass spectrometry. This was demonstrated for LPS from nontypeable Haemophilus influenzae strain 375 following O-deacylation with anhydrous hydrazine. This strain of H. influenzae can express a triheptosyl-containing glycoform to which four hexose residues are linked forming the outer-core region of the molecule. This has been referred to as the Hex4 glycoform. Glycoforms have been identified which differ in the number of phosphoethanolamine substituents in the inner-core. With the use of CE-FAIMS, isomeric Hex4 glycoforms containing two PEtn groups were separated and characterized by MS/MS. FAIMS provided a significant reduction in mass spectral noise, leading to improved detection limits ( approximately 70 amol of the major glycoform). The extracted mass spectrum showed that the apparent noise was virtually eliminated. In addition to the reduction of chemical background, the ion current was increased by as much as 7.5 times as a result of the atmospheric pressure ion-focusing effect provided by the FAIMS system. The linearity of response of the CE-FAIMS-MS system was also studied. The calibration curve is linear for approximately 3 orders of magnitude, over a range of 40 pg/microL to 10 ng/microL.     

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.     

Analysis of protein tyrosine phosphorylation by nanoelectrospray ionization high-resolution tandem mass spectrometry and tyrosine-targeted product ion scanning

A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.     

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.     

Mobility labeling for parallel CID of ion mixtures

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.     

Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.     

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).     

Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.     

Large scale pesticide multiresidue methods in food combining liquid chromatography--time-of-flight mass spectrometry and tandem mass spectrometry

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) are powerful and complementary techniques that can independently cover the majority of the challenges related with pesticide residue food control. The sequential combination of both systems benefits from their complementary advantages and assists to increase the performance and to simplify routine large scale pesticide multiresidue methods. The proposed approach consists of three stages: (1) automated pesticide screening by LC-TOFMS; (2) identification by LC-TOFMS accurate mass measurements; and (3) confirmation and quantitation by LC-MS/MS. We have developed a fast comprehensive (identification/confirmation + quantitation) automated screening method for 100 target pesticides in crops. In the first stage, a set of data including m/z accurate mass windows (within 20 mDa width) and retention time is obtained (using a standard solution containing all the targeted pesticides) in order to build the automated screening procedure, which is created automatically by assigning retention time and the m/z mass window for each target pesticide. Samples are then analyzed, and the method enables the screening and preliminary identification of the species first by retention time and m/z mass window, followed by subsequent identification (only if positive results) by LC-TOFMS accurate mass measurements. After that, final confirmation of the positive findings using two MRM transitions and accurate quantitation is performed by LC-MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) mass spectrometer. In addition, the use of this QqLIT instrument also offers additional advantageous scanning modes (enhanced product ion and MS3 modes) for confirmatory purposes in compounds with poor fragmentation. Examples of applications to real samples show the potential of the proposed approach, including the detection of nonselected "a priori" compounds as a typical case of retrospective evaluation of banned or misused substances.     

Enhanced screening of glutathione-trapped reactive metabolites by in-source collision-induced dissociation and extraction of product ion using UHPLC-high resolution mass spectrometry

A selective and sensitive approach, called extraction of product ion (XoPI) method, was developed for the detection of l-glutathione (GSH)-trapped reactive metabolites employing an Orbitrap high resolution mass spectrometer. Fragmentation of GSH conjugates in the negative ion mode leads to a product ion, deprotonated γ-glutamyl-dehydroalanyl-glycine (m/z 272.0888). As a means of utilizing this property, negative ion high resolution MS data were collected from in vitro incubations by monitoring ions from m/z 269.5 to 274.5 under in-source collision-induced dissociation. Extraction of product ions at m/z 272.0888 ± 5 ppm from this data resulted in a chromatogram exhibiting deprotonated γ-glutamyl-dehydroalanyl-glycine as the major peaks with no or very few interferences. Therefore, peaks in this extracted product ion chromatogram potentially came from GSH-trapped reactive metabolites. The GSH conjugate parent ions were then confirmed in the corresponding full scan MS data, and their structures were identified from their MS(2) fragmentation patterns. The effectiveness of the approach was assessed with four model compounds, amodiaquine, clozapine, diclofenac, and fipexide, all well-known to form GSH-trapped reactive metabolites, following incubation in human liver microsomes supplemented with β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) and GSH. The results from XoPI method were compared to two other commonly employed liquid chromatography-mass spectrometry (LC-MS) methods: precursor ion scan method and mass defect filter method. Overall, the XoPI method was more selective and sensitive in detecting the GSH conjugates. Many GSH conjugates previously not reported were detected and characterized in this study.     

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

Investigation of the unusual behavior of metolachlor under chemical ionization in a hybrid 3D ion trap mass spectrometer

This article describes the strange behavior of the widely used herbicide metolachlor under chemical ionization conditions in a hybrid source ion trap mass spectrometer in gas chromatography/mass spectrometry (GC/MS) coupling. With the use of ammonia as the reagent gas, metolachlor provides a chlorinated ion at m/z 295/297, almost as abundant as the protonated molecule at m/z 284/286, which cannot be isolated to perform tandem mass spectrometry (MS(n)) experiments. Curiously, this ion at m/z = M + 12 is not observed for the herbicides acetochlor and alachlor, which present very similar chemical structures. The chemical structure of the m/z 295/297 ions and the explanation of the observed phenomenon based on the metastable behavior of these ions were elucidated on the basis of experiments including isotopic labeling and modifications of the operating conditions of the ion trap mass spectrometer. This work allows one to give new recommendations for an optimized use of hybrid source ion trap mass spectrometers.     

Lipid/peptide/nucleotide separation with MALDI-ion mobility-TOF MS

Matrix-assisted laser desorption/ionization when combined with ion mobility-orthogonal time-of-flight mass spectrometry is a viable technique for fast separation and analysis of biomolecules in complex mixtures. Isobaric lipid, peptide, and oligonucleotide ions are preseparated before mass analysis by differences of up to 30% in mobility drift time. Ions of similar chemical type fall along well-defined "trend lines" (with deviations of approximately 3%) when plotted in two-dimensional representations of ion mobility as a function of m/z. Discussion of fundamental and technical limitations of the technique point to its potential for being most useful when applied to systems such as bodily fluids and intact tissue, where an alternative chemical or chromatographic preseparation step prior to mass analysis is either impractical or undesirable.     

Gas chromatography/electron ionization-mass spectrometry-selected ion monitoring screening method for a thorough investigation of polyhalogenated compounds in passive sampler extracts with quadrupole systems

Nontarget analysis and identification of unknown polyhalogenated compounds is important in acquiring a thorough picture of the present pollution status as well as for identifying emerging environmental problems. Such analyses usually require the application of electron ionization mass spectrometry because the resulting mass spectra frequently allow for compound identification. When quadrupoles are used as mass separators, the full scan technique often suffers from low sensitivity along with nonspecificity for polyhalogenated trace compounds which often result in interference by matrix compounds. We have developed a novel nontarget gas chromatography/electron ionization-mass spectrometry-selected ion monitoring (GC/EI-MS-SIM) method that overcomes these sensitivity and selectivity issues. Our method is based on the fact that the molecular ions and isotope patterns of polyhalogenated compounds involve the most relevant primary information with regard to the structure of polyhalogenated compounds. Additionally, the retention times of polyhalogenated compounds generally increase with increasing molecular weight. The retention time range of polyhalogenated compounds was divided in three partly overlapping segments of 112 u (segment A: m/z 300-412; segment B: m/z 350-462; segment C: m/z 450-562) that were screened in eight GC runs consisting of 15 consecutive SIM ions. This method was tested with a passive water sampler extract known to contain over 30 polyhalogenated compounds according to the sensitive analysis by GC/electron capture negative ion (ECNI)-MS. While none of these polyhalogenated compounds could be detected by GC/EI-MS in full scan mode, our nontarget GC/EI-MS-SIM method allowed for the detection of 38 polyhalogenated compounds. Only seven could be identified by means of reference standards while more than 15 of the unknowns could be traced back to at least the class of compounds based on the mass spectrometric data from the nontarget SIM runs. All compounds identified originated from halogenated natural products. The nontarget GC/EI-MS-SIM method combines the high sensitivity obtainable with quadrupole systems for trace analysis with the structural information essential for the identification of unknown pollutants.     

Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode. A majority of the monosaccharide structural isomers exhibited different mobility drift times in either system, depending on differences in their anomeric and stereochemical configurations. In general, drift time patterns (relative drift times of isomers) matched between the two instruments. Higher resolving power was observed using the atmospheric pressure drift tube. Collision cross section values of monosaccharide structural isomers were directly calculated from the atmospheric pressure ion mobility experiments, and a collision cross section calibration curve was made for the traveling wave ion mobility instrument. Overall, it was demonstrated that ion mobility-mass spectrometry using either drift tube or traveling wave ion mobility is a valuable technique for resolving subtle variations in stereochemistry among the sodium adducts of monosaccharide methyl glycosides.     

Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification

Proteolytic peptide mass mapping as measured by mass spectrometry provides a major approach for the identification of proteins. A protein is usually identified by the best match between the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. Without ultrahigh instrumental accuracy, it is possible to increase the specificity of the assignments of particular proteolytic peptides by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence. Here we report this novel method of generating residue-specific mass-tagged proteolytic peptides for accurate and efficient protein identification. Selected amino acids are labeled with 13C/15N/2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tags can then be readily distinguished from other peptides in mass spectra. This method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency, and accuracy for protein identifications.     

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.     

Laser desorption and matrix-assisted laser desorption/ionization mass spectrometry of 29-kDa Au:SR cluster compounds

Positive and negative ions generated by laser-based ionization methods from three gold:thiolate cluster compounds are mass analyzed by time-of-flight mass spectrometry. The three compounds have similar inorganic core masses ( approximately 29 kDa, approximately 145 Au atoms) but different n-alkanethiolate ligands associated with each cluster compound (Au:SR, R = butane, hexane, dodecane). Irradiation of neat films (laser desorption/ionization) and films generated by dilution of the cluster compounds in an organic acid matrix (matrix-assisted laser desorption/ionization) with a nitrogen laser (337 nm) produced distinct ion abundances that are relevant to different structural aspects of the cluster compound. Laser desorption/ionization of neat Au:SR compound films produces ions consistent with the inorganic core mass (i.e., devoid of original hydrocarbon content). Matrix-assisted laser desorption/ionization produces either ions with m/z values consistent with the core mass of the cluster compounds or ions with m/z values consistent with the approximate molecular weight of the cluster compounds, depending on ionization conditions. The ion abundances, and ionization conditions under which they are detected, provide insight into desorption/ionization processes for these unique cluster compounds as well as other analytes typically studied by matrix-assisted laser desorption/ionization.     

Combination of LC/TOF-MS and LC/Ion trap MS/MS for the identification of diphenhydramine in sediment samples

Diphenhydramine (Benadryl) is a popular over-the-counter antihistaminic medication used for the treatment of allergies. After consumption, excretion, and subsequent discharge from wastewater treatment plants, it is possible that diphenhydramine will be found in environmental sediments due to its hydrophobicity (log P = 3.27). This work describes a methodology for the first unequivocal determination of diphenhydramine bound to environmental sediments. The drug is removed from the sediments by accelerated solvent extraction and then analyzed by liquid chromatography with a time-of-flight mass spectrometer and an ion trap mass spectrometer. This combination of techniques provided unequivocal identification and confirmation of diphenhydramine in two sediment samples. The accurate mass measurements of the protonated molecules were m/z 256.1703 and 256.1696 compared to the calculated mass of m/z 256.1701, resulting in errors of 0.8 and 2.3 ppm. This mass accuracy was sufficient to verify the elemental composition of diphenhydramine in each sample. Furthermore, accurate mass measurements of the primary fragment ion were obtained. This work is the first application of time-of-flight mass spectrometry for the identification of diphenhydramine and shows the accumulation of an over-the-counter medication in aquatic sediments at five different locations.