Exact Mass FAB and EI/FAB MS with Peptides Using a Direct Insertion FAB Probe

Abstract

We have successfully installed and tested the first direct insertion probe (DIP) fast atom bombardment (FAB) gun on a Kratos MS-80 mass spectrometer. the installation requires only minor modifications on the standard EI/CI source which do not interfere with normal EI or CI operation. A small 2 inch extender must be added to the solid inlet to accomodate the extra length of the probe tip; this can be replaced in a few minutes by the standard seal to accept the regular solid probe. We have operated the source under simultaneous EI/FAB conditions with excellent results. The use of sorbitol syrup helps to keep source contamination and background spectrum to a minimum making it possible to operate at sufficiently high resolution in order to make exact mass operation routine. The performance evaluation has been carried out with synthetic peptides and the results are presented here.     

Improved detection limits for fast atom bombardment mass spectrometry: A study of time-dependent desorption using a model pulsed bombardment ionization method

Certain sample preparations for fast atom bombardment (FAB) yield an intense but brief pulse of sample ions at the onset of ionization. A model system is used to study this phenomenon. This system utilizes a conventional source of a constant flux of fast atoms and a probe that permits mechanical movement of the sample stage. This is used to simulate the effect of pulsing the atom beam. Experiments with model samples and selected ion monitoring detection show that, following bombardment initiation, sample ions are preferentially desorbed with respect to ions from the FAB matrix. Exhibition of preferential sample desorption depends upon the analyte behaving as a surfactant in the selected matrix. When used in conjunction with an array detector that permits data collection in a time-resolved manner, this system allows collection of useful mass spectra with significantly enhanced sensitivity compared to normal bombardment. When applied to the undecapeptide eledoisin (sequence pyro-EPSKDAFIGLM-NH₂, MW 1187.6 Da) this novel methodology allows an improvement in detection limit of at least three to four orders of magnitude over that observed when using conventional continuous FAB and a point detector.     

Characterization of an improved thermal ionization cavity source for mass spectrometry

A new thermal ionization source for use with a quadrupole mass spectrometer has been designed and characterized. The new source provides significant advantages over the previously reported prototype source and traditional filament-type thermal ionization sources. The operational interface between the source and the quadrupole mass spectrometer has been redesigned. A vacuum interlock, a translational stage, and an adjustable insertion probe are added to improve the source performance. With these modifications, the source is easier to operate while maximizing sample throughput. In this work, the performance of the newly developed source is examined. The ionization efficiencies are measured with a quadrupole mass spectrometer. The efficiency values obtained with this system are comparable to those obtained from a large scale isotope separator. The relationships among the ionization potential, vapor pressure, and measured ionization efficiency results are discussed. The crucible lifetime has been quantitatively estimated by measuring the crucible sputtering rate. Diagnostic studies of the new source show that the crucible position is a crucial parameter for sensitivity and performance. Stability tests demonstrate that the source can be run several weeks at a fixed emission current without significant degradation.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

Direct analysis of drugs by continuous-flow fast-atom bombardment and tandem mass spectrometry

The techniques of continuous-flow fast-atom bombardment (CF-FAB) and tandem mass spectrometry (MS/MS) are combined and applied to the analysis of small molecular mass drugs (mol.wt less than 500 Da). The approach involves the interfacing of a CF-FAB inlet with a triple-stage quadrupole mass spectrometer, enabling the acquisition of collision-activated decomposition mass spectra of the drugs after FAB ionization. The relationship between a stable sample surface on the CF-FAB probe tip and the quality of the mass spectrum is discussed, as are practical methods for obtaining and maintaining surface stability. CF-FAB MS/MS spectra for several drugs are presented, including penicillin G, phentolamine, cocaine and benzoylecgonine. Minimum detection limits range from 50-500 pg injected, depending on the compound. The reproducibility of the integrated areas of peaks from repetitive injections is approximately five per cent. Data are also presented for the direct CF-FAB MS/MS analysis of cocaine and benzoylecgonine in spiked urine samples.     

A fully automated mass spectrometer for the analysis of organic solids

Automation of a mass spectrometer—computer system makes it possible to process up to 30 samples without attention after sample loading. An automatic sample changer introduces the samples successively into the ion source by means of a direct inlet probe. A process control unit determines the operation sequence. Computer programs are available for the hardware support, system supervision and evaluation of the spectrometer signals. The most essential precondition for automation — automatic evaporation of the sample material by electronic control of the total ion current — is confirmed to be satisfactory. The system operates routinely overnight in an industrial laboratory, so that day work can be devoted to difficult analytical problems. The cost of routine analyses is halved.     

Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.     

Comparison of collisionally activated dissociation mass spectra for the identification of cyclopeptides and cyclodepsipeptides

Collisionally activated dissociation (CAD) spectra of protonated molecules of cyclopeptides and cyclodepsipeptides obtained with two different mass spectrometry systems were compared. Fragmentations were obtained either from collisions induced in the ion source of an electrospray mass spectrometer fitted with a single quadrupole by increasing the extracting cone voltage or from collisions with an inert gas in a free-field area of a fast-atom bombardment (FAB) mass spectrometer. Similar fragmentation pathways were produced with the two configurations even though actual tandem mass spectrometry experiments with magnetic and electric sectors provided more information than cone-induced dissociations. However, only the latter mode allowed us to perform mass spectrometric analyses coupled to liquid chromatography (LC/ESI-MS) at low cost on commercially widespread instruments. Copyright © 1999 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption using a fast-atom bombardment ion source and a magnetic mass spectrometer.

A conventional fast-atom bombardment (FAB) ion source was used to achieve matrix-assisted laser desorption (MALD) in a high-mass, double-focusing, magnetic mass spectrometer. The pulsed ion signals generated by irradiation of a mixture of sample and matrix (2,5-dihydroxybenzoic acid) with either a XeF excimer laser (353 nm) or a nitrogen laser (337 nm) were recorded with a focal-plane detector. A resolution (full-width at half maximum) of 4500 was achieved at m/z 1347.7 (the peptide substance P), 2500 for CsI cluster ions at m/z 10,005.7, and 1250 for the isotope cluster of the small protein cytochrome c (horse) [M+H]+ = m/z 12,360 (average). Sensitivity is demonstrated with 11 fmol of substance P. A survey scan is taken to locate the m/z of the sample molecular ion. The segment that contains the sample can then be integrated for a longer time to produce a better signal-to-noise ratio. In addition to higher sensitivity and lower matrix interference, the advantage of MALD over FAB is the former's lower susceptibility to the presence of salts, and competition between hydrophobic and hydrophilic components of a mixture. This feature is demonstrated by the complete MALD spectrum of a crude partial tryptic digest of sperm-whale apomyoglobin, containing 24 peptides, representing the entire sequence of this protein.     

Analysis of an egyptian mummy resin by mass spectrometry

Fast atom bombardment combined with mass spectrometry (FAB/MS), high resolution FAB/MS, FAB tandem mass spectrometry (MS/MS), and gas chromatography/mass spectrometry (GC/MS) were used to determine the composition of the resinous material recovered from the wrappings of an Egyptian mummy believed to be from the Roman period (100–350 A.D.). FAB/MS and MS/MS studies identified several oxidation products of abietic acid as the principal resin components, indicating that one or more species of coniferous trees were used by the Egyptians as a source of the resin. GC/MS studies also identified several n-alkanes with carbon numbers from C₁₉ to C₃₃ in the sample. The relative amounts of these n-alkanes, along with characteristic trace metals, indicate that bitumen, an asphalt native to the region, was added to the resin. The presence of this ancient source of carbon in this sample explains the inconsistent date assigned to the mummy by carbon-14 analysis.     

双电离源(辉光放电/激光溅射电离)四极杆质谱仪的研制

本研究将辉光电离源与激光溅射电离源巧妙地结合在同一台仪器中,使固体样品在离子源腔体中既能辉光电离,也能激光电离;并且使用同一质量分析器,两种离子源的结果可以相互比对,进而得到更为准确的分析结果.此仪器主要由真空系统、离子源、离子传输系统、四极杆质量分析器及检测与数据采集系统等组成.实验中分别用两种离子源测试了标准样品SRM 1262b,并获得了半定量结果.结果表明,仪器具有定性能力强,分析速度快,检测灵敏度高等优点,对固体样品元素分析的检出限可达μg/g量级.实验表明,激光溅射电离质谱的性能优于辉光放电质谱.     

Negative Corona Discharge Ion Source Under Ambient Conditions with Mini Line-cylinder Electrodes

A novel ambient negative corona discharge ion source with mini line-cylinder electrodes is designed. The diameters of inner and outer electrode are 0.16 and 4 mm respectively. With a special assembly method, a perfect coaxiality of the two electrodes is obtained. An injection system utilizing a temperature control technique, achieves a constant and stable concentration of the sample, which is critical to the experiment. The formulas of the corona onset voltage of line-cylinder electrodes are also introduced. The experiment results show that negative substances such as formic acid and acetic acid can be ionized under ambient conditions. When combined with micro electrical mechanical system fabricationprocess, the volume of the ion source can be reduced dramatically, but there is an undesirable surface discharge. To solve the surface discharge problem, an improved structure was designed and tested. The simplicity of the interface of the ion source makes it suitable for mass spectrometer, micro mass spectrometer, ion mobility spectrometer, and high-field asymmetric waveform ion mobility spectrometer applications.     

Super-Atmospheric Pressure Electrospray Ion Source: Applied to Aqueous Solution

This is a follow-up paper of our previous report on an ion source, which was operated at an operating pressure higher than the atmospheric pressure. Besides having more working gas for desolvation, the reduction of mean free path of electrons in a higher pressure environment increases the threshold voltage for gaseous breakdown, thus enabling a stable electrospray for the sample solution with high surface tension without the occurrence of electric discharge. In our previous work, the ion source was not coupled directly to the mass spectrometer and significant amount of ions were lost before entering the vacuum of the mass spectrometer. In this paper, we report the new design of our second prototype in which, by using a modified ion transport capillary, the pressurized ESI ion source was coupled directly to the first pumping stage of the mass spectrometer without additional modification on the vacuum pumping system. Demonstrations of the new ion source on the sensitive detection of native proteins from aqueous solution in both positive and negative ion modes are presented.     

Evaluation of a surface-sampling probe electrospray mass spectrometry system for the analysis of surface-deposited and affinity-captured proteins

A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size < or =200 microm) was approximately 1.0 fmol (approximately 100 fmol/mm2) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm2 depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.     

Miniaturized time-of-flight mass spectrometer for peptide and oligonucleotide analysis

A matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer was developed which uses a novel reflectron composed of a grounded cylinder and an adjustable endcap electrode to provide high-order kinetic energy focusing for a miniaturized mass analyzer. The nearly quadratic potential form of the reflecting field focuses ions desorbed from a source of very small dimensions formed by placing the sample probe within the centered hole of the coaxial dual channel plate detector. At the same time, the depth of the reflectron can be adjusted to accommodate a short drift length between the source/detector and the reflectron. For larger drift lengths, in particular to allow the addition of an XY stage for the analysis of sample arrays, endcap reflectron focusing can be combined with time-delayed ion extraction to achieve good mass resolution. The instrument has been used for the analysis of peptides digested with trypsin or carboxypeptidase, and also small DNA oligomers.     

Modeling the effect of the cathode geometry in a DC glow discharge ion source for mass spectrometry

A self-consistent, two-dimensional hybrid fluid-particle model is used to study the effect of cathode geometry on the plasma produced in an argon glow discharge for conditions typically of the commercially available glow discharge mass spectrometer system (VG9000 spectrometer and Megacell source). For a given power supply voltage and gas pressure, we show that the spatial distribution of the plasma in the discharge volume is strongly dependent on the cathode geometry. The plasma created in a discharge with a pin cathode tends to form a ring around the cathode, while the plasma in a discharge with a larger diameter, disk cathode is centered on-axis between the cathode face and the anode. The ion current arriving at the entry plane of the mass spectrometer thus depends strongly on the cathode geometry. This suggests that analytical performance can be enhanced by optimization of the cathode (sample) geometry.     

基于敞开式直接电离质谱技术的尿液中毒品检测北大核心CSCD

尿液作为一种易于获取的体内毒品检材,在吸毒人员快速筛查中被广泛应用。针对传统快速筛查技术存在假阳性率高、定量能力不足以及实验室质谱技术在快速检测中存在前处理复杂、检测耗时长、使用环境苛刻等问题,该文提出了一种基于敞开式直接电离质谱技术的生物样本快速检测方法。该研究采用探针式电喷雾离子源与便携式质谱仪联用快速检测平台,优化了喷雾电压和质谱入口毛细管温度,开发了高效快速的前处理技术。基于该平台和前处理技术,5种常规毒品(甲基苯丙胺、氯胺酮、可卡因、O^(6)-单乙酰吗啡和3,4-亚甲双氧甲基苯丙胺)的尿液加标溶液的检出限为0.5~30 ng/mL,且其中4种毒品定量检测的线性相关系数大于0.99。除此之外,5种常规毒品在3个不同水平下的加标回收率为56.1%~103.7%,多次检测结果的相对标准偏差为9.0%~27.8%,说明联用检测平台与前处理方法结合可以达到良好的准确度。为了进一步检验该联用仪器的实战能力,测试了某社区戒毒康复中心40份阳性和110份阴性实际尿液样本,总体检测的准确率接近99%,且通过一次进样在20 s内可同时检测多种毒品。该研究成果有利于推动快速检测技术的发展,促进敞开式直接电离质谱仪技术的推广应用,提升一线执法服务水平。     

A New Matrix Assisted Ionization Method for the Analysis of Volatile and Nonvolatile Compounds by Atmospheric Probe Mass Spectrometry

Matrix assisted ionization of nonvolatile compounds is shown not to be limited to vacuum conditions and does not require a laser. Simply placing a solution of analyte dissolved with a suitable matrix such as 3-nitrobenzonitrile (3-NBN) or 2,5-dihydroxyacetophenone on a melting point tube and gently heating the dried sample near the ion entrance aperture of a mass spectrometer using a flow of gas produces abundant ions of peptides, small proteins, drugs, and polar lipids. Fundamental studies point to matrix-mediated ionization occurring prior to the entrance aperture of the mass spectrometer. The method is analytically useful, producing peptide mass fingerprints of bovine serum albumin tryptic digest consuming sub-picomoles of sample. Application of 100 fmol of angiotensin I in 3-NBN matrix produces the doubly and triply protonated molecular ions as the most abundant peaks in the mass spectrum. No carryover is observed for samples containing up to 100 pmol of this peptide. A commercial atmospheric samples analysis probe provides a simple method for sample introduction to an atmospheric pressure ion source for analysis of volatile and nonvolatile compounds without using the corona discharge but using sample preparation similar to matrix-assisted laser desorption/ionization.

Global optimization of the infrared matrix-assisted laser desorption electrospray ionization (IR MALDESI) source for mass spectrometry using statistical design of experiments

Design of experiments (DOE) is a systematic and cost-effective approach to system optimization by which the effects of multiple parameters and parameter interactions on a given response can be measured in few experiments. Herein, we describe the use of statistical DOE to improve a few of the analytical figures of merit of the infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source for mass spectrometry. In a typical experiment, bovine cytochrome c was ionized via electrospray, and equine cytochrome c was desorbed and ionized by IR-MALDESI such that the ratio of equine:bovine was used as a measure of the ionization efficiency of IR-MALDESI. This response was used to rank the importance of seven source parameters including flow rate, laser fluence, laser repetition rate, ESI emitter to mass spectrometer inlet distance, sample stage height, sample plate voltage, and the sample to mass spectrometer inlet distance. A screening fractional factorial DOE was conducted to designate which of the seven parameters induced the greatest amount of change in the response. These important parameters (flow rate, stage height, sample to mass spectrometer inlet distance, and laser fluence) were then studied at higher resolution using a full factorial DOE to obtain the globally optimized combination of parameter settings. The optimum combination of settings was then compared with our previously determined settings to quantify the degree of improvement in detection limit. The limit of detection for the optimized conditions was approximately 10 attomoles compared with 100 femtomoles for the previous settings, which corresponds to a four orders of magnitude improvement in the detection limit of equine cytochrome c.     

Performance of an ion trap mass spectrometer modified to accept a direct insertion membrane probe in analysis of low level pollutants in water

Modifications to a Finnigan ITS40 ion trap mass spectrometer are described which allow its use with a direct insertion probe. Details are given of the fabrication of a membrane probe for such an instrument. The membrane probe, which includes facilities for heating the fluid, employs a tubular membrane which is located just outside the electrode structure of the ion trap. Direct analysis of organic compounds in aqueous solution is demonstrated using a silicone membrane, with compounds such as benzene, chlorobenzene and dichloroethene being studied below the 1 ppb level. The effects of operating parameters including probe temperature, ion trap temperature, solution flow rate, mass spectrometer scan speed, and instrument tune procedures are explored in detail. Optimum performance characteristics are identified and trace level detection of eight organic compounds in the parts per trillion range is demonstrated. In seven of the eight cases studied, detection limits are below the EPA practical limit of quantitation levels. It is shown that the most sensitive mode of operation is when steady state passage of the analyte across the membrane is achieved, however, the time required for this is long in the case of some samples, and a dynamic flow injection analysis procedure is then favored. Use of the modified inlet system for solid sample introduction via a standard solids probe is also demonstrated.