Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.     

Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics

A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.     

Simultaneous determination of five tobacco-specific nitrosamines in mainstream cigarette smoke by isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry

Tobacco-specific nitrosamines (TSNAs) have been previously implicated as a source of carcinogenicity in tobacco and cigarette smoke. Accurate quantification of these chemicals is needed to help assess public health risk. We have developed and validated a specific and sensitive method to simultaneously measure five TSNAs in the particulate phase of mainstream tobacco smoke. Cigarette smoke particulate, produced using standardized machine smoking protocols, was collected on a Cambridge filter pad. The particulate matter was extracted using methylene chloride, back extracted into aqueous solution, further purified by solid-phase extraction, and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry using isotopically labeled analogues as internal standards. Limits of detection for this method ranged from 0.05 to 1.23 ng/mL using an injection volume of 20 microL. A linear calibration range spanning 2.5-2500 ng/mL was adequate to measure TSNA levels in cigarette smoke. The method achieved excellent reproducibility and accuracy. The identity of each TSNA was established by chromatographic retention time, analyte-specific fragmentation patterns, and relative peak area ratios of two product/precursor ion pairs. This new method provides higher sensitivity, specificity, and throughput than earlier methods for TSNA determination.     

Relative quantification of carboxylic acid metabolites by liquid chromatography-mass spectrometry using isotopic variants of cholamine

Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC-MS conditions, regardless of mobile-phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reversed-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for 10 observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements.     

Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures

The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.     

Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.     

Application of time-of-flight mass spectrometry with laser-based photoionization methods for time-resolved on-line analysis of mainstream cigarette smoke

The application of soft photoionization mass spectrometry methods (PIMS) for cigarette mainstream smoke analysis is demonstrated. Resonance-enhanced multiphoton ionization (REMPI) at 260 nm and vacuum ultraviolet light single-photon ionization (SPI) at 118 nm were used in combination with time-of-flight mass spectrometry (TOFMS). An optimized smoking machine with reduced memory effects of smoke components was constructed, which in combination with the REMPI/SPI-TOFMS instrument allows PIMS smoke analysis with a time resolution of up to 10 Hz. The complementary character of both PIMS methods is demonstrated. SPI allows the detection of various aliphatic and aromatic compounds in smoke up to approximately 120 m/z while REMPI is well suited for aromatic compounds. The capability of the instrument coupled to the novel sampling system for puff-by-puff resolved measurements is demonstrated. The feasibility of using the experimental system for intrapuff smoke measurements is also shown. Two main patterns of puff-by-puff behaviors are observed for different smoke constituents. The first group exhibits a constant increase in smoke constituent yield from the first to the last puff. The second group shows a high yield of the constituent in the first puff, with lower and constant or slowly increasing yields in the following puffs. A third group cannot be clearly classified and is a combination of both observed profiles.     

Characterization of triacetone triperoxide by ion mobility spectrometry and mass spectrometry following atmospheric pressure chemical ionization

The atmospheric pressure chemical ionization of triacetone triperoxide (TATP) with subsequent separation and detection by ion mobility spectrometry has been studied. Positive ionization with hydronium reactant ions produced only fragments of the TATP molecule, with m/z 91 ion being the most predominant species. Ionization with ammonium reactant ions produced a molecular adduct at m/z 240. The reduced mobility value of this ion was constant at 1.36 cm(2)V(-1)s(-1) across the temperature range from 60 to 140 °C. The stability of this ion was temperature dependent and did not exist at temperatures above 140 °C, where only fragment ions were observed. The introduction of ammonia vapors with TATP resulted in the formation of m/z 58 ion. As the concentration of ammonia increased, this smaller ion appeared to dominate the spectra and the TATP-ammonium adduct decreased in intensity. The ion at m/z 58 has been noted by several research groups upon using ammonia reagents in chemical ionization, but the identity was unknown. Evidence presented here supports the formation of protonated 2-propanimine. A proposed mechanism involves the addition of ammonia to the TATP-ammonium adduct followed by an elimination reaction. A similar mechanism involving the chemical ionization of acetone with excess ammonia also showed the formation of m/z 58 ion. TATP vapors from a solid sample were detected with a hand-held ion mobility spectrometer operated at room temperature. The TATP-ammonium molecular adduct was observed in the presence of ammonia and TATP vapors with this spectrometer.     

Hydrophilic interaction liquid chromatography coupled to electrospray mass spectrometry of small polar compounds in food analysis

Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile components in food. However, polar low-molecular-weight compounds such as hydrophilic amino acids, di- and tripeptides, and organic acids are often not sufficiently retained and represent a challenge for RPLC. Hydrophilic interaction liquid chromatography in combination with electrospray mass spectrometry (HILIC-ESI-MS) on a carbamoyl-derivatized stationary phase was successfully employed to separate free amino acids and small polar peptides in complex food matrixes such as wheat gluten hydrolysate and Parmesan cheese. Glutamyl dipeptides were separated in a sequence-specific order with peptides with N-terminal glutamic acid residues eluting prior to their reverse sequence analogues. ESI-MSn detection in the positive ionization mode provided the necessary information to unambiguously identify isobaric peptides due to their characteristic fragmentation patterns. The technique also proved useful to separate and identify glycoconjugates between amino acids and reducing sugars (Amadori compounds). The investigation of organic acids present in food used a mobile phase comprising ammonium acetate buffer at pH 7 and mass spectrometric detection in the negative ionization mode.     

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Electron-transfer reagent anion formation via electrospray ionization and collision-induced dissociation

A strategy is described and demonstrated for the formation of reagent anions via electrospray ionization (ESI) for electron-transfer dissociation (ETD). To circumvent difficulties associated with formation of high mass-to-charge ratio (m/z) reagent anions, it is desirable to form ETD reagents via means other than those that require reagent molecule vaporization. ESI is a candidate method, but anions that are generally generated efficiently by ESI tend to react with multiply protonated polypeptides via proton transfer. The strategy described herein involves the use of a precursor reagent molecule that ionizes efficiently via electrospray ionization and that can subsequently be converted to an ETD reagent via gas-phase dissociation. The approach is demonstrated with arenecarboxylic acids that yield strong signals associated with the deprotonated molecule and that subsequently undergo collision-induced dissociation (CID) by loss of CO(2). In the present work, triply protonated KGAILKGAILR served as a test substrate for the CID product ions to give rise to ETD. Several precursor molecules were shown to be capable of generating ETD reagents via ESI followed by CID. These included 9-anthracenecarboxylic acid, 2-fluoro-5-iodobenzoic acid, and 2-(fluoranthene-8-carbonyl)benzoic acid. The latter molecule has the most attractive set of characteristics as a precursor for a relatively high m/z ratio ETD reagent.     

Nanostructured diamond-like carbon on digital versatile disc as a matrix-free target for laser desorption/ionization mass spectrometry

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.     

Online comprehensive two-dimensional characterization of puff-by-puff resolved cigarette smoke by hyphenation of fast gas chromatography to single-photon ionization time-of-flight mass spectrometry: quantification of hazardous volatile organic compounds

This work presents the direct coupling of a custom-made smoking machine (SM) to fast gas chromatography combined with single-photon ionization mass spectrometry (GC × SPI-MS) utilizing a six-port, two-position valve for online puff-resolved comprehensive two-dimensional investigation of cigarette smoke. The innovative electron-beam pumped rare gas excimer light source (EBEL) filled with argon provided vacuum ultraviolet (VUV) photons of 9.8 ± 0.4 eV (126 ± 9 nm) for SPI. Puff-by-puff quantification of 14 hazardous volatile organic smoke constituents from the 2R4F Kentucky research cigarette was enabled for two smoking regimes, i.e., ISO and Canadian Intense, after determination of photoionization cross sections. The investigated analytes comprised NO, acetaldehyde, butadiene, acrolein, propanal, acetone, isoprene, furan, crotonaldehyde, isobutanal, butanal, 2-butanone, benzene, and toluene. The determined amounts of these compounds in cigarette smoke agreed excellently with the literature values. Furthermore, the two well-known patterns of puff-by-puff behaviors for these different smoke constituents were obtained for both whole smoke and gas-phase measurements.     

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.     

顶空-气相色谱法测定卷烟主流烟气粒相物中吡啶

为研究气相色谱测定卷烟主流烟气粒相物中吡啶含量,采用碳酸钠水溶液作基质校正剂,通过顶空进样、DB-WAXETR色谱柱分离、火焰离子化检测器检测。结果表明:吡啶在0.20~16.20μg/mL质量浓度范围内线性拟合度为0.9998,加标回收率在98.4%~104.7%之间,定量限为0.08μg/支,RSD小于5%,该方法可以快速、准确地测定卷烟主流烟气粒相物中吡啶。     

Ionizable isotopic labeling reagent for relative quantification of amine metabolites by mass spectrometry

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.     

Analysis of cigarette smoke condensates by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry I acidic fraction

Cigarette smoke condensate is a complex chemical matrix, and analysis of its components is very difficult because of the limitation of the peak capacity and sensitivity of conventional chromatography and the extensive and laborious sample preparation that is frequently required. In this study, the acidic fraction of mainstream cigarette smoke condensate has been investigated by using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS). Different column systems were tested and compared under proper GCxGC/TOFMS conditions. Auto data processing by TOFMS software combined with manual identification was used to assign the components. Over 1000 compounds, with S/N > or = 100, including 139 organic acids and over 150 phenols were tentatively identified by the developed method.     

Averagine-scaling analysis and fragment ion mass defect labeling in peptide mass spectrometry

A method termed as the averagine-scaling analysis (ASA) is proposed for predictive design and selection of chemical reagents for modifying peptides, as well as for facile mass spectral analysis of peptide fragment ions with increased mass defects. The ASA method scales mass spectral data using the mass of the hypothetical averagine residue as reference. The scaling analysis is used in conjunction with a strategy of fragment ion mass defect labeling (FIMDL) for effectively using the broad, unoccupied mass zones in the low m/ z region of mass spectra. The FIMDL approach involves the solution modification of peptide termini with chemical reagents of large mass defects and the gas-phase generation of peptide terminal fragment ions that carry the FIMDL groups. The scaling analysis reveals that iodine has the highest FIMDL efficiency among halogens. Iodine-containing reagents, 4-iodophenylisocyanate and 4-iodophenylisothiocyanate, are used to label primary amines on peptides to demonstrate the scaling analysis. The ASA method successfully distinguishes peptide fragment ions with and without an FIMDL group and specifically and efficiently reduces the data complexity of peptide tandem mass spectra. The combination of ASA with FIMDL extends the instrument suitability for the mass defect analysis from mass spectrometers of ultrahigh mass resolution and accuracy to those of medium ones. This combination is expected to have a profound impact on peptide tandem mass spectrometry.