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Two very different analytical instruments are featured in this perspective paper on mass spectrometer design and development. The first instrument, based upon the curved-field reflectron developed in the Johns Hopkins Middle Atlantic Mass Spectrometry Laboratory, is a tandem time-of-flight mass spectrometer whose performance and practicality are illustrated by applications to a series of research projects addressing the acetylation, deacetylation and ADP-ribosylation of histone proteins. The chemical derivatization of lysine-rich, hyperacetylated histones as their deuteroacetylated analogs enables one to obtain an accurate quantitative assessment of the extent of acetylation at each site. Chemical acetylation of histone mixtures is also used to determine the lysine targets of sirtuins, an important class of histone deacetylases (HDACs), by replacing the deacetylated residues with biotin. Histone deacetylation by sirtuins requires the co-factor NAD+, as does the attachment of ADP-ribose. The second instrument, a low voltage and low power ion trap mass spectrometer known as the Mars Organic Mass Analyzer (MOMA), is a prototype for an instrument expected to be launched in 2018. Like the tandem mass spectrometer, it is also expected to have applicability to environmental and biological analyses and, ultimately, to clinical care.
Instrument development in the Cotter laboratory at the Johns Hopkins University School of Medicine began in the late 1970s amidst a flurry of interest in the so-called rapid-heating methods as alternatives to field desorption1 for the mass spectral analysis of non-volatile compounds. These techniques included the direct electron ionization (EI) method of McLafferty and Baldwin in 1973,2 flash volatilization by Doyle Daves in 1977,3 in-beam EI of Ohashi4 and direct chemical ionization (CI) by Hansen and Munson in 19785 and the direct exposure CI method from Cotter and Fenselau.6,7 All of these studies were motivated by the suggestion of Buehler and Friedman8 that a crossover in the Arrhenius plots for the decomposition vs desorption of non-volatiles was such that rapid heating to a very high temperature would render the desorption process more favorable. Using a not-so-rapid heating approach, Cotter and Yergey9,10 showed that quaternary ammonium salts, generally considered intractable for mass spectral analysis, could be ionized directly by heating (that is: without an electron beam) and that the co-desorption of marginally volatile neutral sugars in a matrix of alkali salt would produce a stable beam of gas-phase adduct ions.
An entirely logical progression for these techniques was, of course, the use of fast pulsed lasers for ion desorption, first reported by Kistemaker and the group at FOM in Amsterdam in 197811 and followed by reports from Heinen,12 Cooks et al.13 and Hercules et al.14 who utilized a laser microprobe time-off-light mass spectrometer (LAMMA 1000, Leybold Hereaus). Our own work in this area began with a double focusing sector instrument in which the ions were laser desorbed directly from within a CI source. Relying on the longer source residence times for ions formed in the high pressure source and fast scanning of the ion acceleration voltage, it was possible to record mass spectra within a millisecond time frame.15 However, it was obvious that the full advantage from these fast pulsed lasers could be best realized using a time-of-flight (TOF) mass spectrometer, and in 1979–80 we constructed an instrument based upon a modified CVC Products (Rochester, NY, USA) model 2000 and a pulsed carbon dioxide laser at 10.6 microns.16 This instrument was uniquely different to other laser time-of-flight configurations, as it was the first to incorporate a delay time between the laser desorption of ions and their extraction into the mass analyzer. An additional delay, the so-called time-lag focusing, developed earlier by Wiley and McLaren,17 enabled focusing of either directly desorbed ions or desorbed neutrals ionized by electron impact.18 This instrument demonstrated the possibility for sequencing peptides up to around 1200 Da, yielding fragment ions which, according to a subsequently developed nomenclature,19 would be known as b-ions and cationized y-ions.
The introduction of the matrix-assisted laser desorption/ionization (MALDI) techniques20,21 and this unique instrument provided the first opportunity for utilizing time-delayed extraction with this new ionization method.22 At the same time, the possibilities for peptide sequencing by MALDI time-of-flight mass spectrometry led to our interest in developing a higher performance tandem (MS/MS) TOF instrument. Key to such an instrument is the design of a suitable reflectron, as the reflectron mass analyzer is what distinguishes precursor ions from product ions formed after the source. Specifically, the flight time of an ion of mass, m, in a single-stage reflectron instrument is given by:
where L1 and L2 are the field-free flight distances before and after the ion traverses the reflecting region and d is the penetration depth into the reflecting region. If, however, a precursor ion, m1, dissociates to form a product ion, m2, that is: m1 → m2 + neutral, then the reflectron causes these ions to have different arrival times:
but such reflectrons focus the product ions more poorly as m2 becomes much less than m1. This occurs because the kinetic energy of the product ion:
lies outside the energy bandwidth of most reflectrons. While, in theory, the quadratic reflectron has an infinite energy bandwidth,23 the non-axial fields inside these reflectrons result in reduced ion transmission. In addition, as they focus from a point at the entrance, these reflectrons do not accommodate a linear drift region from which to carry out the dissociation.
Thus, we developed the so-called curved-field reflectron (CFR), whose axial potential is a small slice of the arc of a circle.24 Differing only slightly from the linear (or constant field) profiles of a single-stage reflectron, this reflectron retains the high ion transmission of other reflectrons while focusing a wider range of product ions. The CFR was first implemented on a tandem time-of-flight MS with two reflectron analyzers,25 a Z-geometry in which the first single-stage reflectron was used to focus precursor ions, while the second incorporated the curved field. Schematically, the configuration is shown in Figure 1(a). The collision chamber was, in fact, an open region, with the high pressure collision gas created by a pulsed valve with a duration of about 100 ms. With this unique configuration, nearly 100% beam attenuation could be achieved without differential pumping. Figure 2(a) shows the MS/MS spectra for C60 taken at low, medium and high attenuation.26 The collision energy was 5 keV (in the laboratory frame).
The two-reflectron design has a number of limitations. The first is that precursor ions that undergo metastable decomposition in the first analyzer, after passing through the first reflectron, do not arrive at the ion gate at the correct time, reducing their contributions to the product ion mass spectrum. The second is that even a well focused ion beam entering the reflectron exits with a planar (or ribbon) profile that reflects the range of corrected energies and, thus, leads to a focusing mismatch going into the second mass analyzer. Commercial tandem TOF instruments, those available from Applied Biosystems (Billerica, MA, USA) and Bruker Daltonics (Bremen, Germany), utilize a geometry in which the first mass analyzer is a linear time-of-flight, focusing ions to an ion gate using correlated velocity/space focusing by delayed ion extraction.27 The second mass analyzer is a reflectron type, with the energy bandwidth addressed either by decelerating the ions before collision followed by pulsed reacceleration28 or using a lift cell.29 The CFR provided us with an opportunity to utilize this basic geometry in a much simpler configuration. Implemented first as a modification to a Kratos (Manchester, UK) AXIMA CFR mass spectrometer, the instrument incorporated a collision tube in the field-free region between the ion source and a mass selection gate [Figure 1(b)]. Because ions are not decelerated prior to the collision chamber, metastable fragments retain the same velocity as their precursors and contribute to the final product ion spectra along with those formed by collision induced dissociation (CID). This improves sensitivity, and reduces the number of laser shots required to build a product ion mass spectrum with good signal/noise. This design also means that collision energies correspond to the full 20 keV (laboratory frame) derived from the initial ion acceleration from the source. Figure 2(b) shows the improvement in the product ion mass spectra for C60 following 20 keV collisions.30
Because precursors and their associated product ions retain the same velocity in an instrument with a curved-field reflectron, the collision region can, in theory, be located at any point in the drift space between the source and reflec-tron and before or after the ion selection gate which sits at the focal point for each mass analyzer. However, the design implemented in the Shimadzu Biotech (Manchester, UK) TOF2 instrument places the collision chamber close to the reflectron with some important consequences [Figure 1(c)]. There are, in fact, very slight differences between the final ion velocities of fragments formed by metastable decomposition and those formed by collisions, as the latter result in conversion of a portion of that collision kinetic energy into internal excitation.31 The deleterious effects of these Derrick shifts32 upon flight times and peak shapes are minimized by locating the collision chamber as far into the flight path as possible. Second, CID does result in some low angle scattering, so that locating the collision chamber just ahead of the reflectron increases acceptance of the ions. Thus, this configuration provides improvements in both sensitivity (ion transmission) and mass resolution.
While on-line liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) methods, using electrospray ionization (ESI),33 currently dominate much of current proteomics research, these are useful primarily in biomarker discovery and quantitation methods.34,35 Many protein (and protein modification) problems benefit from the direct mass spectral analyses that can be provided by MALDI time-of-flight mass spectrometry. Our own laboratory, as part of an NIH multi-investigator effort known as the Technical Center for Networks and Pathways of Lysine Modifications (TCNP), has had an interest in characterizing the post-translational modifications to histones that control gene expression, silencing etc., the so-called histone code. Histones H2a, H2b, H3 and H4, are the major protein components of chromatin and they can be acetylated, mon-, di- or trimethylated, or ubiquitylated on lysine residues, methylated on arginines and phosphorylated on serines. The amino acid sequences of histones, and their acetylating enzymes, incorporate many lysine residues which are generally hyper-acetylated, so that an important part of our analytical strategy involves acetylation or deutero-acetylation of lysine residues prior to tryptic digestion and bottom up, de novo sequence analysis by MALDI MS.
The acetylation of lysine residues to form ε-N-acetyllysine constitutes the major modification to histones. Acetylation is carried out through the action of histone acetyltrans-ferases (HATs) in the presence of acetyl-CoA, as shown in the acetylation cycle in Figure 3. Removal of acetyl groups from lysine utilizes any of a number of histone deacetylases (HDACs) with many of these requiring NAD+ as a cofactor. These include the sirtuins: hst1 to hst4 in yeast, Sir1 to Sir7 in mouse and SIR1 to SIR7 in humans. In addition to their acetylase activity, the sirtuins Sir4/SIR4 and Sir5/SIR5 in mitochondria are also responsible for ADP-ribosylation, as also shown in Figure 3.
The protein p300, also known as CBP or cAMP response element binding protein, contains a histone acetyltrans-ferase domain that is, itself, highly lysine-acetylated. Figure 4 shows the MALDI TOF MS/MS spectrum of a tryptic peptide from the HAT region, obtained using the Shimadzu TOF2, which includes two acetylated lysine residues (K1542 and K1546, using the numbering from the intact protein). Because trypsin generally fails to cleave acetylated lysines, it is possible to view these within their sequence context and to achieve very high sequence coverage. Using MALDI MS/MS analysis we were able to examine the HAT regions for a series of synthetic mutants that established that the acetylation of three specific lysine residues, K1549, K1558 and K1560 in the so-called activation loop, were required for HAT activity.36,37
A method first suggested by Christine Smith et al.38 was used for quantifying the acetylation of histones as specific sites. Purified histones were deuteroacetylated prior to tryptic cleavage, in a solution of deuterated acetic acid and deuterated acetic anhydride. Tryptic digestion then yields a set of peptides formed by cleavage only at arginine residues, producing a series of isotopically distinguishable isoforms that can be directly quantitated (Reference 39 and supplementary material). Figure 5 shows the mass spectrum of the tryptic fragment GKGGKGLGKGGAKR of histone H4 from HeLa cells with four potential acetylation sites at K5, K9, K12 and K16.40 Note that the lowest mass 1438.91 would correspond to the fully acetylated peptide (which is not seen), while peptides not fully acetylated will incorporate deuterated acetyl groups that increase the mass in increments of 3 Da.
The relative abundances, corrected for 13C contributions to the isotopic distribution, are shown in Table 1. Also shown are similar results for yeast cells reported earlier [39 suppl]. MS/MS analysis of the latter showed that the major singly-acetylated species was acetylated at K16, while the doubly-acetylated species was primarily K8 + K16 acetylation.
Referring again to the acetylation/deacetylation pathways shown in Figure 3, we developed, in collaboration with Cynthia Wolberger et al.,41 a method for locating lysine residues that are substrates for specific deacetylases. In this case, mixtures of calf thymus H1, H2A, H2B, H3 and H4 were hyperacetylated. These were then reacted either with the sirtuin Hst2 (control) or Hs2 plus cofactor NAD+. They were then biotinylated at all of the deacetylated lysines, digested with trypsin and analyzed by MALDI mass spectrometry. Acetylation at lysine residues increases the mass of each tryptic peptide by 42 Da per acetyl group. For each lysine that is deacetylated by Hst1, that 42 mass units is substituted with a mass of 226 Da from biotin, or a net shift in mass of 184 Da if one compares the acetylated and biotinylated species. A number of Sirtuins were utilized in this study, and in no case were biotinylated lysines observed without the presence of cofactor. Thus, as shown in Figure 6, the mass spectrum from the mixture of histone and Hst2 without cofactor serves as a control. The mass spectrum of the peptide mixture from Hst2 and the cofactor NAD+ reveals five peaks that correspond to biotinylation at one or more lysine residues. These are identified in Table 2.
Note that in this example, the peak at 1377.3 appearing in both mass spectra corresponds to the acetylated form of the H3 peptide EIAQDFKTDLR. Thus, for this lysine deacetylation by Hst2 is incomplete. Using this method, the sirtuins Hst1, Hst2, Sir2, TmSir2 and TbSir2 all showed deacetylase activity in the presence of NAD+, while Hst4, Hst2 dm and Hst2 dm traut showed no deacetylase activity.
As noted earlier, many of the sirtuins carry ADP-ribosylation activity in the presence of NAD+. ADP-ribosylation can, in fact, take place at arginine residues. In an ongoing study with the Wolberger group,42 we have been able to show that this reaction is not always a side product of deacetylation. Figure 7 shows the reaction of Sirtuin TpSIR2 from Trypanosome bruceii using as a substrate a synthetic peptide variant of H1.1 with multiple arginine residues. The reaction is clearly enzymatic, as ADP-ribosylation does not occur when only NAD+ is present.
The development of a tandem TOF mass spectrometer based upon the curved-field reflectron has been both an instrumental success and a critical tool for our own biological research. It is one among many instruments that have been developed in the Cotter laboratory, which has now embarked on an exciting new instrumental adventure. The Mars Organic Mass Analyzer project (MOMA) is a cooperative effort of NASA and the European Space Agency (ESA) whose overall aim is to search for specific molecular signs of life from core samples obtained 2 m below the Martian surface. The mass spectrometer is an ion trap, intended to accommodate both the direct laser desorption/ionization of solid samples at Mars atmospheric pressure and electron ionization of the effluent from a gas chromatograph. The US team responsible for the MS design is headed by PI Luann Becker from the Physics and Astronomy department and Deputy PI Robert Cotter from the School of Medicine at Johns Hopkins. Management of the project and assembly of the instrument will be carried out by the Space Science group at the Johns Hopkins Applied Physics Laboratory. Other groups involved in the overall project include the Max Planck Institute for Solar Research, the University of Leiden, Université Nice, Laboratoire Inter-Universitaire des Systèmes Atmosphériques, Universität Bremen and Laser Zentrum Hannover. The current launch date is 2018.
Direct UV laser desorption was selected as an ionization technique that would suitably ionize non-volatile organics with masses up to 2000 Da without introducing organic solvents and risking contamination. The ion trap was then selected over other possible mass analyzers because it provided the opportunity for laser desorption in situ, outside the vacuum region, while not requiring the ultra-high vacuums of orthogonal TOF instruments. In addition, it is possible for MS/MS analyses on a reasonably sized instrument. While miniaturization is an important factor for instruments designed for space, ion traps are, themselves, relatively compact; but the commercial instruments running the 1.1 MHz trapping RF frequency at an amplitude of 7.5 kV0–p consume considerable power. An ion trap instrument, aboard the Rosetta spacecraft expected to rendezvous with the comet Churyumov–Gerasimenko in 2014,43 uses a frequency, Ω, of 600 kHz with an amplitude Vmax of only 300 volts and a radius, r0, of 0.8 cm to achieve a mass range of 150 Da according to the equation:
where qeject is normally 0.908 in the mass-selective instability mode.44 The MOMA instrument, also intended for operation at a maximum voltage of 300 V, has a frequency of 735 kHz and a trap radius of 0.67 cm resulting in a mass range (in the mass selective instability mode) of 173 Daltons. Different from the Rosetta instrument, however, the MOMA ion trap also includes a supplemental frequency44 applied to the endcaps to increase the mass range. At the maximum amplitude of 300 V, the supplemental frequency required to record ions at m/z 2000 using the resonance ejection mode would be 30.4 kHz. In fact, the instrument utilizes a third scan mode to record the mass range. With the fundamental RF voltage kept constant, the supplemental frequency is scanned. This enables operation below the maximum voltage Vmax when used to record mass spectra from the gas chromatograph.
Ions formed in the Mars atmosphere (5 Torr to 10 Torr, consisting primarily of carbon dioxide) will enter the mass spectrometer through an orifice or capillary. They will then be collimated and injected through one of the endcap electrodes using a quadrupole or hexapole ion guide [Figure 8(a)]. To accommodate the gas chromatograph, electron ionization will be done internally (inside the trap) using an electron beam source mounted in the ring electrode as is also shown in Figure 8(a). Figure 8(b) shows an electron impact ionization mass spectrum of the calibrant PFTBA obtained on a test prototype (using an external EI source) operating at an RF frequency of 760 kHz, an amplitude of 237 V and a supplemental frequency scan from 350 kHz to 15 kHz. Similar mass spectra have also been obtained using carbon dioxide (rather than helium) as the bath gas, anticipating the use of the Mars atmosphere for this purpose.
The instrument is certainly a work in progress as the next generation prototype is constructed to include both the electron beam and laser desorption ionization modes. However, the current instrument illustrates the feasibility and practicality of designing a low voltage ion trap with high mass range that can be expected to provide a portable analytical instrument for environmental of point-of-care measurements.
The authors acknowledge support of this work from several sources, in particular: a contract N01HV28180 Proteomics of Adaptation to Ischemia/Hypoxia in Heart, Lung and Blood (Jennifer VanEyk, PI) from the NHLBI for support of the tandem TOF development, a grant U54 RR020839 Networks and Pathways of Lysine Modification (Jef Boeke, PI) from the National Institutes of Health for the histone studies, and a contract NNX08AO82G Mars Organic Mass Analyzer (MOMA) Instrument (Luann Becker, PI) from the National Aeronautics and Space Administration. The authors also note the contributions of several colleagues and collaborators, including Timothy Cornish (JHU/APL), Serguei Ilchenko (Case Western Reserve), Vladimir Doroshenko (Mass Technologies/SESI), Philip Cole (Pharmacology), Wendell Griffith (University of Toledo), Dwella Nelson (Johns Hopkins), Rocio Montes de Oca and Katherine L. Wilson (Cell Biology, JHU), Poonam Bheda, Kamau Fahie, Po Hu, Yingkai Zhang and Cynthia Wolberger (HHMI, JHU).
*Perspective article based upon a Keynote address by RJC at the 18th International Mass Spectrometry Meeting in Bremen (August 16, 2009) and a paper presented at the American Chemical Society Analytical Division Award Symposium (August 16, 2009).