Anal. Chem. 1987, 59, 313-317
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Cesium Ion Desorption Ionization with Fourier Transform Mass Spectrometry I. Jonathan Amster, Joseph A. Loo, Jorge J. P. Furlong, and Fred W. McLafferty* Chemistry Department, Cornell University, Ithaca, New York 14853-1301
Secondary Ion mass spectrometry (SIMS) using a pulsed ceslum ion gun interfaced to a Fourier transform mass spectrometer has broad utiilty for analyzing nonvolatile organic moiecuks such as peptides and polymers. The resulting mass spectrum Is a function of experlmentai parameters, such as current denslty and acceleration energy of the primary Ion beam, sample preparation technique, sample substrate, and presence of codeposlted compounds. The best results are obtained for a monolayer or less of a sample codeposHed wlh the peptide glutathione onto a gold target, by using a low primary ion current (20 nA/cm*) of hlgh kinetic energy (11 keV). A detection limit of mol Is demonstrated for the peptide gramlcldin S. With thls compound, tandem mass spectrometry is shown to yield additional structural informatlon. Experimental evidence supports an ionization mechanlsm of gas-phase cationization of the desorbed neutral species.
Recent developments (1-14) in Fourier transform mass spectrometry (FTMS) (15) have made it a promising method for the analysis of high molecular weight compounds. For example, a resolution of 15OOOOhas been demonstrated at m/z 1180 (4). Detailed structural information from tandem mass spectrometry (MSJMS) using collisionally activated dissociation (CAD) has been obtained from peptide ions as large as m / z 1920 (13). For the mass range attainable by FTMS, inorganic cluster ions (14) at m/z 16241 and organic polymer ions a t m / z -7000 (3) have been measured. The FTMS multichannel detection of ions simultaneously over a wide mass range is especially advantageous for spectra of large molecules. Ionization methods are critical for such analyses (1);reported FTMS experiments include laser desorption (2-4) and fast atom bombardment (FAB) (4-6). In an earlier report on cesium ion desorption (secondary ion MS, SIMS) of vitamin B12with FTMS analysis (€9,the only ions observed appeared to be dissociation products of a cesium bound dimer, although molecular ion species have recently been reported for SIMS of P-cyclodextrin (m/z 1135) (9, IO). However, extensive capabilities have been found for such “static” SIMS using other mass spectrometers, especially by Benninghoven (1618). I t is the most sensitive of ionization methods for larger molecules, e.g., the detection of mol of bradykinin, MW 1060 (17). SIMS does not need a liquid matrix, helpful for the low pressure requirement of FTMS, and avoids the problems of matrix chemistry inherent to FAJ3 (19). Here the use of SIMS with FTMS is investigated further, also addressing the mechanism of ion formation in SIMS. EXPERIMENTAL SECTION The cesium SIMS experiments were performed on a prototype Nicolet FTMS 2000 with a 3-Tmagnet and single ion cell; fuIl details are described separately (20). A cesium ion gun is placed 60 cm from one trapping plate of the ion cell to direct ions along the axis that passes through the plate center, parallel with the magnetic field lines. A direct sample probe is brought within 1 0003-2700/87/0359-0313$01.50/0
cm of the opposite side of the cell. The cesium ion beam travels along this axis through 5-mm openings in the front and rear cell trapping plates, striking the sample mounted on the probe face, which is electrically grounded. The secondary ions produced pass back into the cell where they are trapped (1.3 V) and measured (75-ms scan, 64K data points, 2 zero fills, 500-kHz bandwidth, lOO-Hz/ps sweep rate, 1-ms quench, 250-ps quench delay, 1-ms each for ejection of Cs+ and (Cs2C1)+).The Antek (Palo Alto, CA) Cs+ ion source consists of a cesium aluminosilicate ceramic mounted on a resistively heated filament, floated at 11 kV for ion acceleration through a grounded screen. A 20-nA cesium ion beam is produced for 110 ps at the probe face by pulsing the potential of an intermediate grid (100 mesh stainless steel) by -100 V relative to the f i i e n t voltage. To improve the sensitivity for trapped organic ions, Cs+and Cs2C1+primary ions are ejected prior to ion detection. Collisionally activated dissociation was effected by rf acceleration of the chosen ions and admission of air through the gas inlet system raising the background pressure to lo-’ torr. Copper probe tips were machined from a solid bar of OFHC copper. Silver probes were produced by spot welding silver foil (0.5 mm) to the solid copper probe face. For gold probes, a 75-nm layer of gold is vacuum vapor-deposited onto the copper faces; these can be used -10 times before gold must be redeposited. All samples were obtained commercially and used without further purification. Two techniques were used to prepare solid-state samples on -1 cm2 of the probe face. In the first, electrospraying (21), 10 p L of a dilute solution is added to the hub of a 28-gauge hypodermic needle, floated at 3-5 kV,and held 1 cm above the probe face, with a 0.13-mm tungsten wire in the needle to control the flow. A scanning electron micrograph showed -0.25 pm diameter microcrystalsfrom electrosprayinga lo-’ M methanol solution of vitamin BIZ.Samples were also prepared M solutions (using a low surface by evaporating -10 p L of tension solvent) directly onto the probe face to give monolayer or submonolayer coverage.
RESULTS AND DISCUSSION Experimental variables investigated include sample preparation, sample adducts, sample substrates, and primary ion kinetic energy. Optimized experimental conditions were then used to ascertain the structural information and sensitivity attainable and to investigate ionization mechanisms. Sample Surface Charging. Surface charging accelerates the secondary ions leaving the surface, making their trapping in the ion cell more difficult. Benninghoven has found (18) that the ion yield for SIMS/TOF analysis increases linearly with surface coverage until a monolayer is formed, after which the ion yield is constant. Similar effects are found in SIMS/FTMS /20),except that ion yields decrease dramatically after several monolayers of an organic sample have been deposited, probably due to the formation of an electrically insulating layer. The degree of charging is dependent on the number and density of charged particles striking the surface. Best results were obtained by using short primary ion pulses (110 ps) of low current density (20 nA/cm2). Alkali Cationization. Our FTMS results (20) confirm previous SIMS investigations showing that the abundance of molecular ion species can be enhanced by codeposition of alkali halide or ammonium salts with the sample (22-24). The SIMS/FTMS spectrum of gramicidin S containing equimolar 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
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amounts of NaC1, KC1, LiC1, and CsI shows that sodium attachment is strongly favored, with (M + Na)+ nearly an order of magnitude more abundant than (M + Li)+ and (M K)+, which in turn are several times the abundance of (M + Cs)'. The spectra from separately added sodium, potassium, and cesium are quite similar, with approximately equal numbers of protonated and cationized fragment ions. However with added lithium, most of the fragment ions are lithium attached, not protonated, species; the propensity of the small lithium cation to substitute for a proton has been observed before (22). Organic Sample Adduct. In "Wf plasma desorption (PD) experiments it has been shown (25) that adding the tripeptide glutathione to peptide samples enhances the abundance and lifetime of the protonated molecular ion species. The addition of glutathione to gramicidin S and NaCl on a copper target increases [(M Na)+] from 20% to 100% (Figure 1). Glutathione could transfer the sodium ion to form (M + Na)+; in the PD study [(M + H)+] enhancement was postulated (25) to result from the transfer of a glutathione proton to the sample, perhaps via a redox reaction. Without added alkali ions, glutathione addition enhances (M + H)+formation. Similarly, SIMS/FTMS of the pentadecapeptide gramicidin D plus NaCl electrosprayed onto a copper target produces (M + Na)+, m / z 1904, only if the sample was codeposited with glutathione. With an equimolar amount of glutathione, none of the larger molecules studied here gave spectra containing additional ions which can be attributed to glutathione. Other organic adducts tested and found to enhance [ (M + H)+] and [(M + Na)+ without contributing additional peaks to the spectrum included hydroquinone and 1,4-naphthalenediol. Substrate Composition. Benninghoven (26) has shown that gold and platinum produce the highest yield of molecular ion species for SIMS using monolayer or submonolayer samples, with silver and copper giving intermediate and low yields, respectively. Similarly, we find that vitamin B12plus glutatione and sodium chloride deposited onto a copper target results in no molecular ion species and extensive fragmentation. However, silver and gold targets yield both (M + H)+ and (M + Na)+ (Figure 2), with the silver target showing a lower signal to noise ratio and more abundant fragment ions of m/z 350-900. The SIMS/FTMS spectra of gramicidin D on gold can also be compared with that on copper (Figure 3). For the range of samples tested, a gold surface was found to be best for SIMS/FTMS.
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Figure 3. SIMS/FTMS spectrum of gramldlcln D and giutathlone (1:l)
on gold (top)and copper (bottom)targets.
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Nitrocellulose has recently been shown to be a dramatically effective sample substrate for plasma desorption mass spectrometry (PDMS) experiments (27) in stabilization of the molecular ion species. This was tried with SIMS/FTMS, electrospraying nitrocellulose onto copper at coverages of 4 and 16 pg/cm2. Spectra of gramicidin S (Figure 4) adsorbed from solution show molecular ion species that are enhanced by this procedure, but still less abundant than those from glutathione addition. Nitrocellulose spectra of gramicidin D show no molecular ion species. Thus it would appear that glutathione addition yields more useful spectra under our SIMS conditions. Acceleration Voltage Variation. Increasing the acceleration energy of the primary ion beam has been shown to increase the yield of molecular ion species (16,223). We find similar effects for SIMS/FTMS spectra of gramicidin D plus glutathione for primary beam energies from 1.5 to 11 keV (Table I). At lower energies, fragment ions containing only two or three amino acids dominate the spectrum. Molecular ion species, undetectable at thse energies, become the most abundant species for Cs+ ion energies > -8 keV. There is an approximately linear relationship between the primary ion acceleration energy and the ratio of [(M + Na)+] to the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
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Flgure 5. SIMSIFTMS spectrum of gramicidin S and glutathione (1:2) on a gold target. Total samples deposited were as follows: 2 X lo-'' mol (middle), mol (bottom). mol (top), 5 X
Table I. Primary Ion Energy vs. Peak Intensities for Gramicidin D, MW 1881 Cs+ energy,
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