Chapter 6
Fourier Transform Mass Spectrometry of Large (m/z >5,000) Biomolecules 1
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Curtiss D. Hanson , Mauro E. Castro , David H . Russell , Donald F. Hunt , and Jeffrey Shabanowitz Downloaded by EAST CAROLINA UNIV on January 3, 2018 | http://pubs.acs.org Publication Date: November 23, 1987 | doi: 10.1021/bk-1987-0359.ch006
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Department of Chemistry, Texas A&M University, College Station, TX 77843 Department of Chemistry, University of Virginia, Charlottesville, VA 22901 2
Recent experimental results demonstrate the high mass (>10,000 amu) capabilities of Fourier transform mass spectrometry, however the data reveal non-theoretical limits in the resolution at high mass. These advances can be attributed to the development of methods for coupling high pressure ion sources to the ultra-high vacuum FT-ICR analyser. Specifically, external ion sources permit the utilization of liquid matrix secondary ionization mass spectrometry for the desorption of large involatile, thermally labile biomolecules with ion detection by FT-ICR. Limitations in the mass resolution arise from the inability to effectively trap the ions and produce a coherent packet of ions for detection. The lack of spatial and phase coherence of the injected ions leads to the loss of the frequency domain signal. According to our model, a principle factor contributing to the lack of spatial and phase coherence is field inhomogeneities coupled with the kinetic energies of the ions along the Z-axis.
The development of an ion excitation scheme for ion cyclotron resonance (ICR) compatible with Fourier transform data analysis methods has greatly increased the analytical utility of the method. Although the potential utility of Fourier transform mass spectrometry (FTMS) for the analysis of large biomolecules was recognized early, the development of suitable ionization methods and experimental hardware for biomolecule F T M S proved to be a rather difficult task. One consideration in designing a system for analysis of biomolecules is the high vacuum requirements of ion detection by F T - I C R methods. The requirements for maintaining high vacuum (10~ torr or less) for ion trapping and high resolution mass measurements has led to the adaption of ionization methods for F T M S such as laser desorption and C s ion SIMS. Although these ionization methods are quite useful for the analysis of some biomolecules, the success with molecules larger than 2,500 daltons has been limited. 1,2
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0097-6156/87/0359-0100S06.00/0 © 1987 American Chemical Society
Buchanan; Fourier Transform Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
β. HANSON ET AL.
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FTMS of Large Biomolecules
The introduction of the tandem quadrupole-Fourier transform mass spectrometer , the dual cell analyzer, and the external ion source opens new possibilities for analyzing biomolecules by F T M S . The first real success with biomolecules was sample ionization by liquid matrix SIMS and introduction of the sample ions to the ion cell by the tandem quadrupole ion injection system. Although the transmission efficiency (fraction of ions trapped in ion cell relative to the total secondary ion yield) of the device has not been fully characterized, especially for large molecules, it is clear that the sample detection levels are comparable to the most sensitive magnetic sector instruments and time-of-flight mass analyzers. An additional advantage of the tandem quadrupole-FTMS system ( Q - F T M S ) has been recently demonstrated by the photodissociation of large peptides. 7,8
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Downloaded by EAST CAROLINA UNIV on January 3, 2018 | http://pubs.acs.org Publication Date: November 23, 1987 | doi: 10.1021/bk-1987-0359.ch006
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The work performed on the Q - F T M S reveals a fundamental problem with the method, viz., the inability to preform high resolution mass measurement at high mass. Although impressive mass resolution data has been reported for ions of 1500-2000 daltons, the limited duration of the frequency domain signal for ions above m/z 2500 is insufficient for high resolution measurements. Similar limitations apply to solid-state SIMS experiments performed in a Nicolet FTMS-1000 system equipped with a single section ion c e l l . In an attempt to understand this problem, we will consider the physics of trapping ions in the ion cell and the effect of the initial velocity of the ions and field inhomogeneities on the ion trapping. 11
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Detection of High Mass Ions using F T M S A major thrust of recent work on F T M S of large biomolecules has dealt with questions concerning the lifetime of molecular ions formed by highenergy particle bombardment. Chait and Field have reported that a large fraction of the molecular ions of chlorophyll A formed by C f fission fragment ionization decomposes with lifetimes of less than a few microseconds. A later study on the [M+H] ion of insulin showed that extensive dissociation occurs on both the nanosecond and microsecond time scale. These results raise questions concerning the utility of F T M S for the analysis of large biomolecules. For example, to acquire a low resolution F T mass spectrum from m/z 100 to m/z 10,000 requires data acquisition times of 300-500 ms. On the basis of Chait and Field's study it is reasonable to suspect that molecular ions of large molecules may not survive the long times required for F T detection. The problem is even more severe when considering the requirements for acquiring high mass resolution data at high mass, e.g.. conditions that require data acquisition times of several seconds to even tens of seconds. 2 5 2
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Earlier studies on the detection of C s ( C s I ) cluster ions were directed at addressing the general problem discussed above. Although there is ample sensitivity to detect high mass (>m/z 10,000) C s ( C s I ) cluster ions in the low and high mass resolution modes, there are questions concerning how well the behavior of such cluster ions models the behavior of large organic molecules. The analysis of biomolecules by FTMS methods at Texas A & M University has been limited to C s desorption ionization from a solid-state matrix in order to maintain the high vacuum requirements. Due to the low ion yields for [M+H] type ions by keV energy particle bombardment from the solid-state, the number of successful analyses with molecules larger n
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Buchanan; Fourier Transform Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
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FOURIER TRANSFORM MASS SPECTROMETRY 12
than 2000-2500 amu are limited. Owing to the differential pumping arrangement of the ion source and the analyzer, sample ionization on the tandem quadrupole-FTMS instrument at the University of Virginia can be performed by desorption ionization from a liquid matrix, i.e., keV energy Cs ion particle bombardment of a liquid matrix. Early studies with this instrument demonstrate both the sensitivity and mass resolution of the method. Recent modifications to the Q - F T M S instrument have greatly increased the signal-to-noise ratio and resolution for high mass samples. The modifications involved milling out the ion source block to increase the gas flow in the vicinity of the sample probe. Aberth has discussed the importance of gas pressure on the performance of particle bombardment ion sources. The second modification involved pulsing of the trapping plate voltage of the ion cell from 1 to 10 volts during the ionization step. These two relatively simple modifications have increased the sensitivity of the instrument by roughly two orders of magnitude and extended the working mass range for organic samples beyond mass 10,000. +
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Downloaded by EAST CAROLINA UNIV on January 3, 2018 | http://pubs.acs.org Publication Date: November 23, 1987 | doi: 10.1021/bk-1987-0359.ch006
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The data for neurotensin (a tridecapeptide) shown in Figure 1 illustrate the available sensitivity and mass resolution. This spectrum was obtained from a 100 pmol sample dissolved in thioglycerol/glycerol matrix and by using a primary C s ion beam irradiation time of 4 ms. The secondary ions formed on particle bombardment were injected into the ion cell by the rfonly quadrupole rods operated with a low-mass cut-off of ca. m/z 400. As noted above, the trapping voltage was set to zero during ionization and then pulsed positive (to 3 volts for this sample) following the ionization step. The purpose of pulsing the trapping voltage positive following ionization is to restrict the motion of the ions in Z-direction (vide infra). In the broadband (low mass resolution) mode the only ion detected was the [M+H] ion, and in the narrowband mode a mass resolution of ca. 90,000 (resolution specified at F W H M ) was obtained. Note also that the signal-tonoise ratio in the broadband mode is > 100:1 for a sample size of 100 pmol, and in the narrowband (high resolution) mode the signal-to-noise ratio is greater than 500:1. Owing to the pulsed ionization used for F T M S , the total ionization time required to produce this spectrum was approximately 80 ms. That is, the C s ion beam was turned on for 4 ms to produce each mass spectrum and 20 individual ionization events were signal-averaged. +
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Tandem quadrupole-FTMS data for two 2000-6000 mass range peptides are shown in Figure 2. Melittin (26 residues) and glucagon (29 residues) were analyzed in earlier work, but even with long sample ionization times (5 s) the signal-to-noise in the region of the [M+H] was low (
