Electrospray ionization with quadrupole mass analysis

Advances in mass spectrometry have made it a tool of exceptional power for analysis of large biomolecules. Electrospray ionization, MALDI, and other soft ionization techniques for nonvolatile compounds and macromolecules make possible analyses of proteins, nucleic acids, and other biologically relevant compounds with molecular weights up to and in excess of 100,000 daltons. Electrospray ionization with quadrupole mass analysis is now routine for biomolecule analysis as is analysis using MALDI-TOF instruments. Extremely high resolution can be achieved using Fourier transform-ion cyclotron resonance (FT ICR, or FTMS). We shall discuss ESI and MALDI applications of mass spectrometry to protein sequencing and analysis in Sections 24.5E, 24.13B, and 24.14. 

Different mass analysers can be combined with the electrospray ionization source to effect analysis. These include magnetic sector analysers, quadrupole filter (Q), quadrupole ion trap (QIT), time of flight (TOF), and more recently the Fourrier transform ion cyclotron resonance (FTICR) mass analysers. Tandem mass spectrometry can also be effected by combining one or more mass analysers in tandem, as in a triple quadrupole or a QTOF. The first analyzer is usually used as a mass filter to select parent ions that can be fragmented and analyzed by subsequent analysers. 

Preferably, electrospray ionization (ESI) is used in combination with quadrupole mass filters [16,17], whereas MALDI is commonly used in combination with the time-of-flight (TOF) analyzer [18], The relatively simple construction of these two types of analyzers and the resulting price advantage has led to their replacing the traditional magnetic sector instruments as the workhorses of mass spectrometric analysis. A more recent development is the ion-cyclotron-resonance (ICR) analyzer [19] which can be used for both ES-and MALDI-ionization. 

Many metabolites are polar, ionic, and nonvolatile. Thus comprehensive metabolic analysis is not well suited for gas chromatographic techniques. Metabolite mixtures in complex biological samples such as lymph and blood, however, can be rapidly characterized by electrospray ionization with ion mobility coupled to mass spectrometry. As with GC-IMS, coupling ion mobility spectrometers with mass spectrometers produces two dimensions of information. There are many types of ion mobility-mass spectrometers (IM-MS) and most of these will be discussed in other chapters of this book. The focus of this chapter, however, is on atmospheric pressure IMS with quadrupole or time-of-flight mass spectrometry. Other types of IM-MS have also been recently been reviewed. Advantages of coupling atmospheric pressure IMS with a mass spectrometer include ... 

Hsu, RR, Turk, J., Rhoades, E.R., Russell, D.G., Shi, Y.X. and Groisman, E.A., Structural characterization of cardiohpin by tandem quadrupole and mulhple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. J. Am. Soc. Mass Spectrom., 16, 491-504 (2005). Hsu, RR and Turk, J., Characterization of cardiohpin from Escherichia coli by electrospray ionization with multiple stage quadrupole ion-trap mass spectrometric analysis of [M — 2H -I Na](—) ions. J. Am. Soc, Mass Spectrom., 17, 420-429 (2006). 

Reverse-phase columns with a gradient elution in combination with UV-Vis spectrophotometers using photodiode-array (PDA) (Fig. 1.6) and spectrofiuorimeters are common devices employed in this technique. In a lesser extent, MS, tandem mass spectrometry (MS-MS), and nano liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry (nanoLC-nanoESI-Q-qTOF-MS-MS) has been used as detection system. This instrumentation has been mainly used in the analysis of dyes and proteinaceous media, and in some extent, in the analysis of drying oils and terpenoid varnishes [47,48],... 

ToF mass spectrometers as dynamic instruments gained popularity with the introduction of matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) as effective pulsed ion sources for the soft ionization of large biomolecules (up to 10s dalton) due to their high ion transmission.38 ToF mass spectrometers, quadrupole analyzers and/or magnetic sector fields can be combined in tandem mass spectrometers (MS/MS) for the analysis of organic compounds. 

Figure F2.4.1 Liquid chromatography/mass spectrometry (LC/MS) analysis of isomeric carotenes in a hexane extract from 0.5 ml human serum. Positive ion electrospray ionization MS was used on a quadrupole mass spectrometer with selected ion monitoring to record the molecular ions of lycopene, p-carotene, and a-carotene at m/z (mass-to-charge ratio) 536. A C30 HPLC column was used for separation with a gradient from methanol to methyl-ferf-butyl ether. The a -trans isomer of lycopene was detected at a retention time of 38.1 min and various c/ s isomers of lycopene eluted between 27 and 39 min. The all-frans isomers of a-carotene and P-carotene were detected at 17.3 and 19.3 min, respectively.

Figure A.3A.3 LC/MS analysis of a dietary supplement consisting of extract of Trifolium pratense (red clover). Reversed-phase C18 HPLC and negative ion electrospray ionization mass spectrometry were used with a quadrupole mass spectrometer analyzer (Agilent also see Table A.3A.1). The map illustrates the abundance of information provided by this hyphenated technique with HPLC mass chromatograms in one dimension and mass spectra in another dimension.

Even the lawn format still does not represent the final limit of miniaturization. The one well/one catalyst or one bead/one catalyst strategy, where catalyst identity is spatially coded, can be replaced by in situ synthesis combined with mass spectrometry [48]. The advantage of this strategy is the use of a mass spectrometer for the synthesis, reaction and analysis. The described electrospray ionization procedure helps to avoid the cleavage of chemical bonds, which would falsify the results. The synthesis step does not have to deliver clean and isolated products. Instead, after synthesis, the reactants are first separated by a quadrupole. In a second step, they are further reacted in an octapole and the reaction products are finally isolated in a second quadrupole and analyzed. Figure 3.15 describes the screening process in detail [49],... 

Mass spectrometry Positive ion electrospray ionization mass spectrometry (ESl-MS) analysis was performed on a PE API 2000 triple quadrupole mass spectrometer (Sciex, Toronto, Canada). Spray voltage was set to 4.8 kV, and 30 V orifice voltage was applied. Samples were dissolved in a methanol-water (1 1, v/v) mixture containing 0.1% acetic acid, and 5 pL of sample was injected with a flow rate of 100 pL/min. The instmment was used in a Qj scan mode in the range of m/z 400-1700, with a step size of 0.3 amu and a dwell time of 0.5 ms. Other chimeric peptides in this study were purified and characterized in the same or a very similar way. 

All mass spectra were obtained on a VG Quattro triple-quadrupole mass spectrometer (Micromass Inc., Altrincham, U.K.). Peptides were ionized with electrospray ionization under the following conditions mobile phase, methanol/water (50/50 v/v) needle voltage, 2.8 kV high voltage lens (counter electrode), 0.05 kV and skimmer potential, -12 V. The flow rate of the mobile phase for the spectra of the whole library was 100 pl/min rather than 10 pl/min, which was used for the samples after binding. All samples were dissolved in methanol. Either 5 or 10 pi of sample was injected. Two injections were performed for each sample. Data were acquired in Multichannel Analysis... 

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