Inlet ionization: a new highly sensitive approach for liquid chromatography/mass spectrometry of small and large molecules

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.     

Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good agreement of the measured value of (116 ± 3) s(-1) with that measured by the stopped-flow optical method (105 ± 2) s(-1) validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.     

Determination of pKa values of individual histidine residues in proteins using mass spectrometry

We developed a mass spectrometric method to determine the p K a values of individual histidine residues in proteins. The method is based on the fact that the imidazole C 2-proton undergoes pH-dependent hydrogen-deuterium exchange reaction, of which the rate constant ( k phi) reflects the p K a for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D 2O solvent at various pH values, (2) protein digestion by proteolytic enzyme(s), during which all the rapidly exchanging deuterons such as those in amide and hydroxyl groups are back-exchanged for protons, and (3) measurement of the mass spectrum of each histidine-containing peptide by LC/ESI-MS. The k phi of the H-D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The p K a value is then determined from a plot of k phi versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the p K a values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The p K a values of three of four histidine residues (His12, -105, and -119) in RNase A were successfully determined by this method and were in good agreement with those determined by (1)H NMR and hydrogen-tritium exchange methods. The method uses subnanomole quantities of protein, allowing measurement at a much lower concentration than that of 1 mM required for the conventional NMR approach that is currently almost exclusively the method of choice.     

Optimization and application of ICPMS with dynamic reaction cell for precise determination of 44Ca/40Ca isotope ratios

An inductively coupled plasma mass spectrometer with dynamic reaction cell (ICP-DRC-MS) was optimized for determining (44)Ca/(40)Ca isotope ratios in aqueous solutions with respect to (i) repeatability, (ii) robustness, and (iii) stability. Ammonia as reaction gas allowed both the removal of (40)Ar+ interference on (40)Ca+ and collisional damping of ion density fluctuations of an ion beam extracted from an ICP. The effect of laboratory conditions as well as ICP-DRC-MS parameters such a nebulizer gas flow rate, rf power, lens potential, dwell time, or DRC parameters on precision and mass bias was studied. Precision (calculated using the "unbiased" or "n - 1" method) of a single isotope ratio measurement of a 60 ng g(-1) calcium solution (analysis time of 6 min) is routinely achievable in the range of 0.03-0.05%, which corresponded to the standard error of the mean value (n = 6) of 0.012-0.020%. These experimentally observed RSDs were close to theoretical precision values given by counting statistics. Accuracy of measured isotope ratios was assessed by comparative measurements of the same samples by ICP-DRC-MS and thermal ionization mass spectrometry (TIMS) by using isotope dilution with a (43)Ca-(48)Ca double spike. The analysis time in both cases was 1 h per analysis (10 blocks, each 6 min). The delta(44)Ca values measured by TIMS and ICP-DRC-MS with double-spike calibration in two samples (Ca ICP standard solution and digested NIST 1486 bone meal) coincided within the obtained precision. Although the applied isotope dilution with (43)Ca-(48)Ca double-spike compensates for time-dependent deviations of mass bias and allows achieving accurate results, this approach makes it necessary to measure an additional isotope pair, reducing the overall analysis time per isotope or increasing the total analysis time. Further development of external calibration by using a bracketing method would allow a wider use of ICP-DRC-MS for routine calcium isotopic measurements, but it still requires particular software or hardware improvements aimed at reliable control of environmental effects, which might influence signal stability in ICP-DRC-MS and serve as potential uncertainty sources in isotope ratio measurements.     

Portable laser ablation sampling device for elemental fingerprinting of objects outside the laboratory with laser ablation inductively coupled plasma mass spectrometry

Laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) is a powerful method for elemental fingerprinting of solid samples in a quasi-nondestructive manner. In order to extend the field of application to objects outside the laboratory, a portable laser ablation sampling device was assembled using a diode pumped solid state laser and fiber-optics. The ablated materials were sampled on membrane filters and subsequently quantified by means of LA-ICPMS. The analytical performance of this approach was investigated for glass and gold reference materials. Accuracies of better than 20% were reached for most elements and typical limits of detection were found to be in the range of 0.01-1 μg/g. In summary, this approach combines spatially resolved sampling with the detection power of ICPMS and enables elemental fingerprinting of objects which cannot be transferred to the laboratory, e.g., archeological artifacts in museums.     

Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra

High-resolution and high-accuracy Fourier transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5-3.0-fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS spectra which are then combined into a single concatenated file and submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations.     

Determination of arsenobetaine in fish tissue by species specific isotope dilution LC-LTQ-Orbitrap-MS and standard addition LC-ICPMS

An accurate and precise method for the determination of arsenobetaine (AsB, (CH(3))(3)(+)AsCH(2)COO(-)) in fish samples using exact matching species specific isotope dilution (ID) liquid chromatography LTQ-Orbitrap mass spectrometry (LC-LTQ-Orbitrap-MS) and standard addition LC inductively coupled plasma mass spectrometry (LC-ICPMS) is described. Samples were extracted by sonication for 30 min with high purity deionized water. An in-house synthesized (13)C enriched AsB spike was used for species specific ID analysis whereas natural abundance AsB, synthesized and characterized by quantitative (1)H NMR (nuclear magnetic resonance spectroscopy), was used for reverse ID and standard addition LC-ICPMS. With the LTQ-Orbitrap-MS instrument in scan mode (m/z 170-190) and resolution set at 7500, the intensities of [M + H](+) ions at m/z of 179.0053 and 180.0087 were used to calculate the 179.0053/180.0087 ion ratio for quantification of AsB in fish tissues. To circumvent potential difficulty in mass bias correction, an exact matching approach was applied. A quantitatively prepared mixture of the natural abundance AsB standard and the enriched spike to give a ratio near one was used for mass bias correction. Concentrations of 9.65 ± 0.24 and 11.39 ± 0.39 mg kg(-1) (expanded uncertainty, k = 2) for AsB in two fish samples of fish1 and fish2, respectively, were obtained by ID LC-LTQ-Orbitrap-MS. These results are in good agreement with those obtained by standard addition LC-ICPMS, 9.56 ± 0.32 and 11.26 ± 0.44 mg kg(-1) (expanded uncertainty, k = 2), respectively. Fish CRM DORM-2 was used for method validation and measured results of 37.9 ± 1.8 and 38.7 ± 0.66 mg kg(-1) (expanded uncertainty, k = 2) for AsB obtained by standard addition LC-ICPMS and ID LC-LTQ-Orbitrap-MS, respectively, are in good agreement with the certified value of 39.0 ± 2.6 mg kg(-1) (expanded uncertainty, k = 2). Detection limits of 0.011 and 0.033 mg kg(-1) for AsB with LC-ICPMS and ID LC-LTQ-Orbitrap-MS, respectively, were obtained demonstrating that the technique is well suited to the determination AsB in fish samples. To the best of our knowledge, this is first application of species specific isotope dilution for the accurate and precise determination of AsB in biological tissues.     

Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.     

Improved LC-MS method for the determination of fatty acids in red blood cells by LC-orbitrap MS

We report a new method for fast and sensitive analyses of biologically relevant fatty acids (FAs) in red blood cells (RBC) by liquid chromatography mass spectrometry (LC-MS). A new chemical derivatization approach was developed forming picolylamides from FAs in a quantitative reaction. Fourteen derivatized FA standards, including saturated and unsaturated FAs from C14 to C22, were efficiently separated within 15 min. In addition, the use of a recently introduced benchtop orbitrap mass spectrometer under positive electrospray ionization (ESI) full scan mode showed a 2-10-fold improvement in sensitivity compared with a conventional tandem MS method, with a limit of detection in the low femtomole range for saturated and unsaturated FAs. The developed method was applied to determine FA concentrations in RBC with intra- and interday coefficients of variation below 10%.     

Alpha-particle energy loss measurement of microgram depositions of biomolecules

A commercially available alpha-particle spectrometer and 210Po alpha-particle source were used to determine the mass of microgram quantities of biomolecules. Samples were deposited in microliter volumes on thin silicon nitride windows and dried. The energy loss of the alpha-particles after traversing the sample was converted to a mass using tabulated alpha-particle stopping powers. The measurement was absolute, independent of biomolecule species, and no standards were needed for quantitation. The method has a dynamic range of 0.1-100 microg for deposits of diameter 1-2 mm. The precision varies from approximately 20% at 100 ng to a few percent at 5-100 microg. The silicon nitride windows allow multimodal analysis of the same quantified sample, including PIXE probing of elemental abundances, molecular identification by mass spectrometry, and isotopic quantitation of interactions. The method was used with accelerator mass spectrometry to quantify specific activities of microgram quantities of 14C-labeled proteins.     

Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) associated with capillary electrophoresis (CE) has been used for structural characterization of mannooligosaccharide caps from Mycobacterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs). The mannooligosaccharide caps were released by mild acid hydrolysis, labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS) prior to being separated by CE, collected, and analyzed by MALDI-TOF-MS and post-source decay experiments. This approach was optimized using standard APTS-labeled oligosaccharides. With the selected (9:1) mixture of 2,5-dihydroxybenzoic acid (DHB) and 5-methoxysalicylic acid (MSA) as matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTS-maltotriose was successfully detected down to 50 fmol using linear-mode negative MALDI-TOF-MS. Moreover, using extraction delay time, only 100 and 500 fmol of this standard were required, respectively, to obtain accurate reflectron mass measurements and sequence determination through post-source decay experiments. Applied to only 5 microg (294 pmol) of M. tuberculosis ManLAMs, this analytical approach allowed successful mass characterization of the mannooligosaccharide cap structures from the deprotonated molecular ions [M - H]- and the y-type ion fragments obtained in post-source decay experiments. This powerful analytical approach opens new insights into both the characterization of oligosaccharides and the capping motifs displayed by ManLAMs purified from mycobacteria isolated from tubercular patients without in vitro culturing.     

An ICP-OES method with 0.2% expanded uncertainties for the characterization of LiA1O2

An improved inductively coupled plasma-optical emission spectrometry (ICP-OES) method has been applied to the determination of Li and A1 mass fractions and the Li/A1 amount-of-substance ratio in representative samples of LiA1O2. This ICP-OES method has uncertainty on the order of 0.2%,(2,3) comparable to the best analytical methods. This method is based on several strategies, which are detailed in this work. The mean measured mass fractions of Li and A1 in eight samples were 0.10151 +/- 0.00016 (+/-0.16%) and 0.41068 +/- 0.00056 (+/-0.14%), and the mean Li/A1 amount-of-substance ratio was 0.9793 +/- 0.0017 (+/-0.17%). The uncertainty is dominated by sample handling and heterogeneity-about a factor of 2 larger than the ICP-OES instrumental uncertainties, which were 0.04% for A1 and 0.07% for Li.     

Complete quantitative online analysis of methanol electrooxidation products via electron impact and electrospray ionization mass spectrometry

We report on a novel approach for complete quantitative online product analysis in electrocatalytic reactions, combining electron impact ionization mass spectrometry (EI-MS) and electrospray ionization mass spectrometry (ESI-MS) for simultaneous detection of both volatile and nonvolatile reaction products. The potential of this method is demonstrated using continuous methanol oxidation in a flow cell. The overall reaction rate was followed via the Faradaic current; CO(2) formation was monitored mass spectrometrically via a membrane inlet system, and formaldehyde and formic acid were detected by ESI-MS after a derivatization-extraction-separation procedure introduced recently (Zhao, W.; Jusys, Z.; Behm, R. J. Anal. Chem.2010, 82, 2472-2479) providing quantitative data on the product distribution. In a more general sense, this approach is applicable for a wide range of reactions at the solid-liquid interface or in liquid phase.     

Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.     

Pulsed acceleration charge detection mass spectrometry: application to weighing electrosprayed droplets

We describe a new approach to measuring the masses of individual macroions. The method employs a pulsed acceleration tube located between two sensitive image charge detectors. The charge and velocity of the macroion are recorded with the first image charge detector. The ion is pulse accelerated through a known voltage drop, and then the charge and velocity are remeasured using the second image charge detector. The mass of the ion is deduced from its charge and its initial and final velocities. The approach has been used to measure masses in the 10(10)-10(14) Da range with z = 10(3)-10(6) and m/z = 10(6)-10(9). It should be extendable to masses of <10(6) Da. We have used the method to determine the size and charge of water droplets transmitted through a capillary interface and an aperture interface. The droplets detected from the aperture interface are approximately 1 order of magnitude smaller in mass than those detected from the capillary interface. The droplets from both interfaces have relatively low charges, particularly with the capillary interface where they are only charged to a small fraction of the Rayleigh limit. These results suggest that the aerodynamic breakup of the droplets plays a significant role in the mechanism of electrospray ionization.     

Dynamic range and mass accuracy of wide-scan direct infusion nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry-based metabolomics increased by the spectral stitching method

Direct infusion nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry (DI nESI FT-ICR MS) offers high mass accuracy and resolution for analyzing complex metabolite mixtures. High dynamic range across a wide mass range, however, can only be achieved at the expense of mass accuracy, since the large numbers of ions entering the ICR detector induce adverse space-charge effects. Here we report an optimized strategy for wide-scan DI nESI FT-ICR MS that increases dynamic range but maintains high mass accuracy. It comprises the collection of multiple adjacent selected ion monitoring (SIM) windows that are stitched together using novel algorithms. The final SIM-stitching method, derived from several optimization experiments, comprises 21 adjoining SIM windows each of width m/z 30 (from m/z 70 to 500; adjacent windows overlap by m/z 10) with an automated gain control (AGC) target of 1 x 10(5) charges. SIM-stitching and wide-scan range (WSR; Thermo Electron) were compared using a defined standard to assess mass accuracy and a liver extract to assess peak count and dynamic range. SIM-stitching decreased the maximum mass error by 1.3- and 4.3-fold, and increased the peak count by 5.3- and 1.8-fold, versus WSR (AGC targets of 1 x 10(5) and 5 x 10(5), respectively). SIM-stitching achieved an rms mass error of 0.18 ppm and detected over 3000 peaks in liver extract. This novel approach increases metabolome coverage, has very high mass accuracy, and at 5.5 min/sample is conducive for high-throughput metabolomics.     

Imaging MS methodology for more chemical information in less data acquisition time utilizing a hybrid linear ion trap-orbitrap mass spectrometer

A novel mass spectrometric imaging method is developed to reduce the data acquisition time and provide rich chemical information using a hybrid linear ion trap-orbitrap mass spectrometer. In this method, the linear ion trap and orbitrap are used in tandem to reduce the acquisition time by incorporating multiple linear ion trap scans during an orbitrap scan utilizing a spiral raster step plate movement. The data acquisition time was decreased by 43-49% in the current experiment compared to that of orbitrap-only scans; however, 75% or more time could be saved for higher mass resolution and with a higher repetition rate laser. Using this approach, a high spatial resolution of 10 μm was maintained at ion trap imaging, while orbitrap spectra were acquired at a lower spatial resolution, 20-40 μm, all with far less data acquisition time. Furthermore, various MS imaging methods were developed by interspersing MS/MS and MS(n) ion trap scans during orbitrap scans to provide more analytical information on the sample. This method was applied to differentiate and localize structural isomers of several flavonol glycosides from an Arabidopsis flower petal in which MS/MS, MS(n), ion trap, and orbitrap images were all acquired in a single data acquisition.     

人血清白蛋白纯品含量的同位素稀释质谱方法研究

同时建立了人血清白蛋白(:HSA)纯品含量质量平衡测定方法和同位素稀释质谱测定方法。质量平衡法首先采用高效液相色谱法测定了人血清白蛋白的纯度,然后分别采用卡尔费休法和灼烧残渣法测定其水分和灰分,计算得到HSA的含量为0.861 g/g。在同位素稀释质谱法中,HSA经酸水解后,采用液相色谱-同位素稀释质谱联用法测定了水解液中的Pro、Val和Phe3种氨基酸,进一步计算得出HSA纯品的含量。在最优条件下,HSA含量测定结果为0.853 g/g,相对标准偏差为0.8%,扩展不确定度为0.015 g/g(k=2),检出限和定量限分别为1.37×10~(-5)g/g和4.55×10~(-5)g/g。与质量平衡法测定结果相比,同位素稀释质谱方法测定结果只有0.9%的相对偏差。     

碳纤维丝束起毛量测试方法

为定量测试碳纤维丝束的断丝起毛程度,建立了碳纤维丝束起毛量测试方法,并搭建了测试装置。采用所建立的方法对2种国产T800级碳纤维进行起毛量测试,研究了丝束展宽、张力、毛丝吸附材料等测试条件对测试结果的影响。采用优化的测试条件,测试了几种进口、国产T700级、T800级碳纤维丝束的起毛量。实验表明,所建方法操作简单,适用性较广。对于12K的T800级碳纤维,当纤维张力为1~2N,海绵孔隙大小在0.1~0.6mm,且受到的载荷大小为1~4N时,测试结果比较理想。对于起毛量较高的碳纤维,需保证一定的纤维展宽使丝束内部的毛丝暴露,从而保证测试结果的准确性。     

Improving protein identification sensitivity by combining MS and MS/MS information for shotgun proteomics using LTQ-Orbitrap high mass accuracy data

We investigated and compared three approaches for shotgun protein identification by combining MS and MS/MS information using LTQ-Orbitrap high mass accuracy data. In the first approach, we employed a unique mass identifier method where MS peaks matched to peptides predicted from proteins identified from an MS/MS database search are first subtracted before using the MS peaks as unique mass identifiers for protein identification. In the second method, we used an accurate mass and time tag method by building a potential mass and retention time database from previous MudPIT analyses. For the third method, we used a peptide mass fingerprinting-like approach in combination with a randomized database for protein identification. We show that we can improve protein identification sensitivity for low-abundance proteins by combining MS and MS/MS information. Furthermore, "one-hit wonders" from MS/MS database searching can be further substantiated by MS information and the approach improves the identification of low-abundance proteins. The advantages and disadvantages for the three approaches are then discussed.