Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.     

Determination of perchlorate at trace levels in drinking water by ion-pair extraction with electrospray ionization mass spectrometry

Perchlorate has been added to the U.S. Environmental Protection Agency's Drinking Water Contaminant Candidate List (CCL). The present work describes the analysis of perchlorate in water by liquid-liquid extraction followed by flow injection electrospray mass spectrometry (ESI/MS). Cationic surfactants, mostly alkyltrimethyl-ammonium salts, are used to ion-pair aqueous perchlorate, forming extractable ion pairs. The cationic surfactant associates with the perchlorate ion to form a complex detectable by ESI/MS. The selectivity of the extraction and the mass spectrometric detection increases confidence in the identification of perchlorate. The method detection limit for perchlorate based on 3.14 sigma n-1 of seven replicate injections was 100 ng L-1 (parts per trillion). Standard addition was used to quantitate perchlorate in a drinking water sample from a contaminated source, and the concentration determined agreed within experimental error with the concentration determined by ion chromatography.     

Combination of chiral capillary electrochromatography with electrospray ionization mass spectrometry: method development and assay of warfarin enantiomers in human plasma

The hyphenation of chiral capillary electrochromatography (CEC) with electrospray ionization mass spectrometry (ESI-MS) is very challenging but promising due to the fact that it combines sensitivity with high specificity and selectivity. In this work, CEC capillaries packed with (3R,4S)-Whelk-O1 chiral stationary phase were used for simultaneous enantioseparation of (+/-)-warfarin and its internal standard, (+/-)-coumachlor. Furthermore, both the chiral CEC separation and MS detection parameters were examined in detail. First, the influence of different column fabrication was investigated. Second, enantioseparation was optimized by varying CEC parameters, including acetonitrile concentration, buffer pH, and ionic strength. Under the optimum chiral CEC conditions, ESI-MS parameters such as sheath liquid pH and composition, sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizer pressure, and fragmentor voltage were investigated to achieve maximum MS signals of the separated enantiomers. Finally, using solid-phase extraction as sample preparation method, (+/-)-warfarin spiked in 100-microL human plasma samples were analyzed. The calibration curves showed good linearity for both (R)-warfarin (R = 0.9979) and (S)-warfarin (R = 0.9978) enantiomers. The experimental limit of detection was approximately 25 ng/mL for both enantiomers. Even though the data are still preliminary, we can state with confidence that chiral CEC-ESI-MS has the potential to establish itself as a very powerful technique for the determination of enantiomeric ratios in human body fluid.     

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.     

Development and characterization of an ionization technique for analysis of biological macromolecules: liquid matrix-assisted laser desorption electrospray ionization

We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.     

High mass measurement accuracy determination for proteomics using multivariate regression fitting: application to electrospray ionization time-of-flight mass spectrometry

Important factors that limit the mass measurement accuracy from a mass spectrometer are related to (1) the type of mass analyzer used and (2) the data processing/calibration methods used to obtain mass values from the raw data. Here, two data processing methods are presented that correct for systematic deviations when the mass of ions is measured using a time-of-flight (TOF) mass spectrometer. The first fitting method is one where m/z values are obtained from fitting peak distributions using double Gaussian functions. A second calibration method takes into account the slight nonlinear response of the TOF analyzer in addition to the drift in the calibration over time. Using multivariate regression, both of these two effects can be corrected for using a single calibration formula. Achievable performance was evaluated with a trypsin digestion of serum albumin and proteins from the organism D. radiodurans that was analyzed using gradient reversed-phase liquid chromatography combined with an electrospray ionization orthogonal TOF mass spectrometer. The root-mean-square deviation between the theoretical and experimental m/z values for serum albumin tryptic peptides was found to be 8 ppm using the double Gaussian-multivariate method compared to 29 ppm determined using linear calibration and normal peak centroiding. An advantage of the methods presented here is that no calibrant compounds need to be added to the mobile phase, thereby avoiding interference effects and signal suppression of analytes.     

超高效液相色谱-串联质谱技术检测番茄制品中矮壮素及缩节胺残留量的方法研究

本文采用超高效液相色谱-串联质谱(UPLC-MS/MS)技术,同时检测番茄制品中矮壮素及缩节胺残留的方法。结果表明,番茄制品经稀释定容、超声浸提、高速离心后,采用UPLC-ES(I+)MS/MS同时测定番茄制品中矮壮素及缩节胺的残留,在0.001～0.05mg/kg浓度范围内,平均加标回收率在80%-110%,方法检出限0.01mg/kg,可同时满足出口番茄酱制品中矮壮素及缩节胺检测工作的需要。     

Comparison of atmospheric pressure chemical ionization, electrospray ionization, and atmospheric pressure photoionization for the determination of cyclosporin A in rat plasma

Atmospheric pressure chemical ionization was compared with electrospray ionization and atmospheric pressure photoionization (APPI) as an interface of high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for the determination of cyclosporin A (CsA) in biological fluids in support of in vivo pharmacodynamic studies. These ion sources were investigated in terms of their suitability and sensitivity for the detection of CsA. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the photoionization efficiency of CsA in the positive ion mode under normal-phase HPLC conditions were explored. The ionization mechanism in the APPI environment with and without the use of the dopant was studied using two test compounds and a few solvent systems employed for normal-phase chromatography. The test compounds were observed to be ionized mainly by proton transfer with the self-protonated solvent molecules produced through photon irradiation. Furthermore, ion suppression due to sample matrix interference in the normal-phase HPLC-APPI-MS/MS system was monitored by the postcolumn infusion technique. The applicability of these proposed HPLC-API-MS/MS approaches for the determination of CsA at low nanogram per milliliter levels in rat plasma was examined. These proposed methods were then compared with respect to specificity, linearity, detection limit, and accuracy.     

Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: application to clinical enzymology

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.     

Desorption electrospray ionization mass spectrometry for high-throughput analysis of pharmaceutical samples in the ambient environment

Desorption electrospray ionization (DESI) allows mass spectrometry to be used for on-line high-throughput monitoring of pharmaceutical samples in the ambient environment, without prior sample preparation. Positive and negative ion DESI are used to characterize the active ingredients in pharmaceutical samples formulated as tablets, ointments, and liquids. Compounds of a wide variety of chemical types are detected in these complex matrices. The effects on analytical performance of operating parameters, including the electrospray high voltage, heated capillary temperature, solvent infusion rate, and solvent composition, are evaluated and optimized. In addition to experiments in which a simple solvent is sprayed onto the solid analyte samples, reactive desorption is performed by adding reagents to the solvent spray to generate particularly stable or characteristic ions with the analytes of interest. A variable-speed moving belt was built for high-throughput sampling and used to provide rapid qualitative and semiquantitative information on drug constituents in tablets. Sampling rates as high as 3 samples/s are achieved in the ambient environment. Relative standard deviations of the relative ion abundances for major components in the mass spectra are in the range of 2-8%. Impurities and components present at levels as low as approximately 0.1% are identified and carryover effects are minimized in high-throughput on-line analysis of pharmaceutical samples.     

Direct electrospray ionization mass spectrometry quantitative analysis of sebacic and terephthalic acids in biodegradable polymers

A direct, rapid, and easy electrospray ionization mass spectrometry (ESI-MS) method to determine concentrations of sebacic acid (SA) and terephthalic acid (TA) residues in biodegradable copolymers was developed. Copolyester samples were synthesized from 1,4-butanediol and sebacic and terephthalic acids by melt polymerization. Extraction of monomers was performed in methanol. Their concentrations were determined by direct infusion ESI-MS, without chromatographic separation, using 1,12-dodecanedioic acid (DDA) as an internal standard. Calibration curves were obtained by plotting the ratio of the areas of the peaks relative to monomers and DDA standard as a function of their concentration ratio. We validated the method by determining the concentration of TA residue using both the ESI-MS protocol and high-performance liquid chromatography (HPLC) analysis with UV detection. The linearity range and the detection limit of this assay were 0.1-5.0 and 0.01 ppm for SA and 0.1-6.0 and 0.03 ppm for TA. This assay represents a useful alternative to conventional methods currently employed for acid quantification, resulting advantageous for its speed and high sensitivity.     

A mobile mass spectrometer for comprehensive on-line analysis of trace and bulk components of complex gas mixtures: parallel application of the laser-based ionization methods VUV single-photon ionization, resonant multiphoton ionization, and laser-induced electron impact ionization

A newly developed compact and mobile time-of-flight mass spectrometer (TOFMS) for on-line analysis and monitoring of complex gas mixtures is presented. The instrument is designed for a (quasi-)simultaneous application of three ionization techniques that exhibit different ionization selectivities. The highly selective resonance-enhanced multiphoton ionization (REMPI) technique, using 266-nm UV laser pulses, is applied for selective and fragmentationless ionization of aromatic compounds at trace levels (parts-per-billion volume range). Mass spectra obtained using this technique show the chemical signature solely of monocyclic (benzene, phenols, etc.) and polycyclic (naphthalene, phenathrene, indol, etc.) aromatic species. Furthermore, the less selective but still fragmentationless single photon ionization (SPI) technique with 118-nm VUV laser pulses allows the ionization of compounds with an ionization potential below 10.5 eV. Mass spectra obtained using this technique show the profile of most organic compounds (aliphatic and aromatic species, like nonane, acetaldehyde, or pyrrol) and some inorganic compounds (e.g., ammonia, nitrogen monoxide). Finally, the nonselective ionization technique laser-induced electron-impact ionization (LEI) is applied. However, the sensitivity of the LEI technique is adjusted to be fairly low. Thus, the LEI signal in the mass spectra gives information on the inorganic bulk constituents of the sample (i.e., compounds such as water, oxygen, nitrogen, and carbon dioxide). Because the three ionization methods (REMPI, SPI, LEI) exhibit largely different ionization selectivities, the isolated application of each method alone solely provides specific mass spectrometric information about the sample composition. Special techniques have been developed and applied which allow the quasi-parallel use of all three ionization techniques for on-line monitoring purposes. Thus, a comprehensive characterization of complex samples is feasible jointly using the characteristic advantages of the three ionization techniques. Laboratory applications show results on rapid overview characterization of mineral oil-based fuels and coffee headspace. The first reported field applications include timely resolved on-line monitoring results on automobile exhausts and of waste incineration flue gas.     

Simultaneous quantitative analysis of anionic, cationic, and nonionic surfactants in water by electrospray ionization mass spectrometry with flow injection analysis

A rapid method is described for the quantitative analysis of anionic, cationic, and nonionic surfactants in water samples by flow injection analysis coupled to electrospray ionization mass spectrometry (FIA/ESI-MS). All surfactants were isolated by liquid-liquid extraction and quantified using labeled triethoxylated nonylphenol ([13C6]-NP3EO) and sodium dibutylnaphthalenesulfonate as internal standards. FIA/ESI-MS was performed by alternating both positive and negative ionization modes, which allows simultaneous analysis of most common surfactants in a short time. Quality parameters of the method, such as linear range, repeatability, reproducibility, and limits of detection were studied. This method was applied to the analysis of wastewater treatment plant effluents from Catalonia (NE Spain).     

Direct analysis of fatty acid vapors in breath by electrospray ionization and atmospheric pressure ionization-mass spectrometry

Real time analysis of human breath is achieved in an atmospheric pressure ionization mass spectrometer (API-MS) by negatively charging exhaled vapors via contact with an electrospray cloud. The spectrum observed is dominated by a wide range of deprotonated fatty acids, including saturated chains up to C14. Above C14, the background from cutaneous sources becomes dominant. We also tentatively identify a series of unsaturated fatty acids (C7-C10), ketomonocarboxylic acids (C6-C10), and a family of aldehydes. The ionization probability of large fatty acids increases drastically when the humidity changes from 20% to 95%. Accordingly, distinguishing lung vapors (humid) from those in the background (dry) requires special precautions. Estimated fatty acid vapor concentrations in breath based on our measurements ( approximately 100 ppt) are in fair agreement with values expected from blood concentrations in the range for which data are available (C3-C6).     

Use of bioactive glass slides for matrix-assisted laser desorption/ionization analysis: application to microorganisms

Glass slides are widely used in high-throughput analysis and are available commercially with surfaces activated, etched, and channeled. Thin glass microscope slides are shown here to be suitable sample supports for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As a demonstration, lectins immobilized on glass slides with activated surfaces are used to concentrate and purify agglutinated Bacillis spores. It is expected that such slides will provide a rapid, inexpensive way to evaluate and implement new strategies involving MALDI MS readout.     

Protocol for an electrospray ionization tandem mass spectral product ion library: development and application for identification of 240 pesticides in foods

Modern determination techniques for pesticides must yield identification quickly with high confidence for timely enforcement of tolerances. A protocol for the collection of liquid chromatography (LC) electrospray ionization (ESI)-quadruple linear ion trap (Q-LIT) mass spectrometry (MS) library spectra was developed. Following the protocol, an enhanced product ion (EPI) library of 240 pesticides was developed by use of spectra collected from two laboratories. A LC-Q-LIT-MS workflow using scheduled multiple reaction monitoring (sMRM) survey scan, information-dependent acquisition (IDA) triggered collection of EPI spectra, and library search was developed and tested to identify the 240 target pesticides in one single LC-Q-LIT MS analysis. By use of LC retention time, one sMRM survey scan transition, and a library search, 75-87% of the 240 pesticides were identified in a single LC/MS analysis at fortified concentrations of 10 ng/g in 18 different foods. A conventional approach with LC-MS/MS using two MRM transitions produced the same identifications and comparable quantitative results with the same incurred foods as the LC-Q-LIT using EPI library search, finding 1.2-49 ng/g of either carbaryl, carbendazim, fenbuconazole, propiconazole, or pyridaben in peaches; carbendazim, imazalil, terbutryn, and thiabendazole in oranges; terbutryn in salmon; and azoxystrobin in ginseng. Incurred broccoli, cabbage, and kale were screened with the same EPI library using three LC-Q-LIT and a LC-quadruple time-of-flight (Q-TOF) instruments. The library search identified azoxystrobin, cyprodinil, fludioxinil, imidacloprid, metalaxyl, spinosyn A, D, and J, amd spirotetramat with each instrument. The approach has a broad application in LC-MS/MS type targeted screening in food analysis.     

Quantitative analysis of amoxycillin and its major metabolites in animal tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A sensitive and specific method for the quantitative determination of amoxycillin and its major metabolites (amoxycilloic acid, amoxycillinpiperazine-2',5'-dione) in animal tissue samples using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. A liquid extraction using an aqueous 0.01 M potassium dihydrogenphosphate solution as the extraction solvent was performed for a preliminary sample cleanup. The extracts were further purified by a solid-phase extraction using an octadecyl (C18) column. Ampicillin was used as the internal standard. Chromatographic separation of the analytes of interest was achieved on a reversed-phase Hypersil column (100 x 3 mm i.d., dp, 5 microm), using a mixture of 9.6 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. To obtain as high sensitivity and selectivity as possible, the mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated for the analysis of amoxycillin and its investigated metabolites in various porcine tissues, kidney, liver, muscle, and fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues, and good linearity was achieved over the concentration range tested (25-500 ng/g, r > or = 0.9974, and goodness of fit < or = 9.6). A limit of quantification of 25 ng/g was obtained for amoxycillin and its metabolites in all tissues, which corresponds to half the maximum residue limit for amoxycillin. Limits of detection ranged from 2.3 to 12.0 ng/g for amoxycillin and from 1.1 to 15.1 ng/g and 0.2 to 2.4 ng/g for amoxycilloic acid and amoxycillinpiperazine-2',5'-dione, respectively. The results for the within-day precision and the trueness fell within the ranges specified. The method has been successfully used for the quantitative determination of amoxycillin and its major metabolites in tissue samples from pigs medicated via the drinking water, proving the usefulness of the developed method for application in the field of residue analysis.