Nucleotide Separation with MALDI-Ion Mobility-TOF MS

Mar 19, 2004 - Amina S. Woods*. NIDA IRP, Baltimore, Maryland 21224. Michael Ugarov, Tom Egan, John Koomen, Kent J. Gillig, Katrin Fuhrer, Marc Gonin,...
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Anal. Chem. 2004, 76, 2187-2195

Lipid/Peptide/Nucleotide Separation with MALDI-Ion Mobility-TOF MS Amina S. Woods*

NIDA IRP, Baltimore, Maryland 21224 Michael Ugarov, Tom Egan, John Koomen, Kent J. Gillig, Katrin Fuhrer, Marc Gonin, and J. Albert Schultz

Ionwerks Inc., Houston, Texas 77030

Matrix-assisted laser desorption/ionization when combined with ion mobility-orthogonal time-of-flight mass spectrometry is a viable technique for fast separation and analysis of biomolecules in complex mixtures. Isobaric lipid, peptide, and oligonucleotide ions are preseparated before mass analysis by differences of up to 30% in mobility drift time. Ions of similar chemical type fall along well-defined “trend lines” (with deviations of ∼3%) when plotted in two-dimensional representations of ion mobility as a function of m/z. Discussion of fundamental and technical limitations of the technique point to its potential for being most useful when applied to systems such as bodily fluids and intact tissue, where an alternative chemical or chromatographic preseparation step prior to mass analysis is either impractical or undesirable. One of the current challenges of bioanalytical chemistry is the identification and quantification of exceedingly complex mixtures of biological macromolecules. Advances in mass analyzer technology and ion sources such as electrospray ionization and matrixassisted laser desorption/ionization (MALDI) have provided new opportunities for accurate molecular weight determinations of “pure” samples and subsequently aided high-throughput mass spectrometry approaches by identifying proteins in their native environment, i.e., complex mixtures,1 including tissue-imaging experiments,2 MALDI analysis of laser capture microdissection samples,3 and characterization of protein complexes and microorganisms.4 Because such samples are so complex, the mass spectrum often contains an unmanageable number of peaks. These interferences may of course be reduced by using 2D mass spectrometry techniques such as LC/MS, GC/MS, gel electrophoresis-MS, and MS/MS, which are becoming common among biology and biochemistry laboratories. Each of these separation* Corresponding author. Tel: 410-550-1507. Fax: 410-550-6859. awoods@ intra.nida.nih.gov. (1) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (2) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2002, 6, 676-681. (3) Palmer-Toy, D. E.; Sarracino, D. A.; Sgroi, D.; LeVangie, R.; Leopold, P. E. Clin. Chem. (Washington, D. C.) 2000, 46 (9), 1513-1516. (4) Champian, M. M.; Campbell, C. S.; Siegele, D. A.; Russell, D. H.; Hu, J. C. Mol. Microbiol 2003, 47, 383-396. 10.1021/ac035376k CCC: $27.50 Published on Web 03/19/2004

© 2004 American Chemical Society

MS techniques has inherent limitations; for instance, special steps (removal of buffers, detergents, and ion-pairing reagents) are required to make LC and CE compatible with MS, gel-based separations are often slow and tedious, and sufficient reproducibility is difficult to achieve.5 Although ion mobility spectrometry has been used for analytical studies and trace level detection since the early 1980s, the analytical mass spectrometry community is just now beginning to recognize the enormous potential for coupling ion mobility and mass spectrometry.6 The coupling of ion mobility with mass spectrometry (IM-MS) has been recently used to solve biochemical problems ranging from separations coupled to mass spectrometry7,8 to detailed investigations of biomolecular conformations.9-10 IM-MS was used in the analysis of peptide mixtures,7,11-12 intact bacteria,13 peptide-peptide interactions,14 peptide-organic molecule interactions,15 and small molecules such as drugs of abuse. Both MALDI16,17 and electrospray ionization18,19 sources have been used. Ion mobility separates ions based on (5) Nilsson, C. L.; Davidson, P. Mass Spectrom. Rev. 2000, 19, 390-397. (6) Collins, D. C.; Lee, M. L. Anal. Bioanal. Chem. 2002, 372, 66-73. (7) Ruotolo, B. T.; Gillig, K. J.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Int. J. Mass Spectrom. 2002, 219, 253-267. (8) Ruotolo, B. T.; Verbeck, G. F., IV; Gillig, K. J.; Russell, D. H. J. Proteome Res. 2002, 1, 303-306. (9) Gidden, J.; Bushnell, J. E.; Bowers, M. T. J. Am. Chem. Soc. 2001, 123, 5610-5611. (10) Srebalus, C. A.; Li, J.; Marshall, W. S.; Clemmer, D. E. Anal. Chem. 1999, 71, 3198-3927. (11) Valentine, S. J.; Counterman, A. E.; Hoaglund, C. S.; Reilly, J. P.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 1213-1216. (12) Mindy, J. M.; Taraszka, J. A.; Regnier, F. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 950-958. (13) Schultz, J. A.; Ugarov, M.; Jackson, S. N.; Jae-Kuk, K.; Mishra, S.; Murray, K. K. ASMS meeting, Montreal, Canada, 2003. (14) Woods, A. S.; Koomen, J.; Ruotolo, B.; Gillig, K. J.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Egan, T.; Schultz, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 166-169. (15) Woods, A. S.; Fuhrer, K.; Gonin, M.; Egan, T.; Ugarov, M.; Gillig, K. J.; Schultz, J. A. J. Biomol. Technol. 2003, 14, 1-8. (16) Gillig, K. J.; Ruotolo, B. T.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Chem. 2000, 72, 3965-3971. (17) Von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 14831485. (18) Counterman, A. E.; Clemmer, D. E. J. Am. Chem. Soc. 1999, 121, 40314039. (19) Wu, C.; Siems, W. F.; Klasmeier, J.; Hill, H. H. Anal. Chem. 1998, 70, 49294938.

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their collision cross section (Ω).20 Structural information can be obtained by using IM-MS, since Ω is dependent on the gas-phase conformation of the ion. IM-MS can be used to distinguish structural isomers and different classes of molecules and is therefore a means of assigning mass spectral data to compound type while differentiating between chemical noise and analyte signal through differences in mass-mobility relationships.21,22 MALDI analysis of laser capture microdissection samples,3 biomolecular spatial analysis of tissue slices,2 and protein biomarker identification on intact bacteria are such possible applications.13 In anticipation of this potential, our paper is focused on MALDI with ion mobility-orthogonal time-of-filght (IM-OTOF) MS measurements of quantitative differences in mobilities of ions formed from test mixtures of typical examples of different types of biomolecules that would be encountered in complex mixture samples. With MALDI-IM-OTOF MS, we demonstrate substantial preseparation by ion mobility prior to mass analysis of ions generated from mixtures of small organics (drugs or matrix), peptides, lipids, and nucleotides. Implications of this work for MALDI probing of intact biological tissue surfaces, the status of instrumental prototype development, and the likelihood that MALDI-IM-OTOF MS can become as sensitive as conventional MALDI-TOF MS are all discussed. Data that illustrate the practical use of the technique for analysis of drugs in saliva is also presented. MATERIALS AND METHODS Peptides. Dynorphin fragments 1-7 [YGGLFRR] and 1-8 [YGGLFRRI], gastrin [LEEEEEAYGWMDF-NH2], and sulfated gastrin [pEGPWLEEEEEAY(SO3H)GWMDF-NH2] were purchased from Sigma (Saint Louis, MO), and P10 [SVLpYTAQPN] was purchased from Anaspec (San Jose, CA). All peptides were diluted to a concentration of 10 pmol/µL. Lipids. Lipid A monophosphoryl was purchased from Sigma; sphingomyelin and cerebroside sulfate were purchased from Avanti polar lipids (Alabaster, AL). All lipids were diluted to a concentration of 100 pmol/µL in chloroform/methanol/water, 8:4:3. Nucleotides. CATG, CATGA, and CATGAT were purchased from Amitof (Allston, MA) and diluted to 100 pmol/µL. Quaternary Amines. Acetylcholine was purchased from Aldrich Chemical Co. (Milwaukee, WI) and chlorisondamine iodide from Tocris (St. Louis, MO). Both were diluted in water to a concentration of 1 nmol/µL. Sample Solutions. Peptide and lipid mixtures; peptide, lipid, and chlorisondamine mixtures; peptide Matrix. 6-Aza-2-thiothymine (ATT) was purchased from Sigma. A saturated matrix solution in 50% ethanol was used. Sample Preparation. A 3-µL sample solution and a 3-µL matrix solution were codeposited on the MALDI-IM target. Traditional dried droplet MALDI sample preparation was used throughout this work. Instrumentation. MALDI-IM-TOFMS data were acquired with two prototype periodic focusing ion mobility instruments with (20) McDaniel, E. W.; Mason, E. A. The Mobility and Diffusion of Ions in Gases; Wiley: New York, 1973; pp 68-72. (21) Clemmer, D. E.; Jarrold, M. F. J. Mass Spectrom. 1997, 32, 577-592. (22) Koomen, J. M.; Ruotolo, B. T.; Gillig, K. J.; McLean, J. A.; Russell, D. H.; Kang, M.; Dunbar, K. R.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Bioanal. Chem. 2002, 373, 612-617.

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different drift cell designs.23 The previous instrument parameters are described elsewhere. A mobility resolution of 40 and a mass resolution of 400 using an orthogonal linear time-of-flight mass spectrometer (o-TOFMS) for detection is routinely achieved.14 One drift cell is 30 cm long with 1.5-cm-inner diameter electrodes operating at ∼1500 V with 1-10 Torr helium present. The second design, under development, is 12 cm in length and operates at higher field strength (∼60 V/cm) with a mobility resolution equal to that of the longer cell. Due to a greater drift velocity (proportional to the cell voltage) over a shorter flight tube, drift times in the shorter cell are a factor of 3.4 times faster than the longer cell. A schematic diagram of the upgraded instrument is shown in Figure 1. A higher resolution o-TOFMS incorporating a home-built reflectron has a theoretical resolution of 3000 and achieves an experimental resolution of 2400 for m/z 1000. Another important feature is the addition of a multisample wheel on which up to 32 MALDI dried droplet spots can be deposited. This allows quick intercomparison between different samples while constant experimental conditions are maintained. The reproducibility of the ion mobility measurements from the same sample preparation can vary by up to 5% when the sample is removed from the analysis chamber, reinserted, and reanalyzed. This makes accurate intercomparisons of the mobility measurements difficult and is the reason for using the multitarget wheel. The instrumental stability seems to be determined in great part by gas purity and by any drift in the gas pressure in the ion mobility cell. The sample target is at ground potential to ease sample introduction, which requires the application of an attractive high-voltage bias to the exit end of the ion mobility cell to drag the ions through the He gas. Furthermore, the entire mass spectrometer must be floated on top of this bias voltage. These biases can be switched so that either negative or positive ions can be sequentially analyzed from a particular MALDI preparation. The experiments are performed typically using 2 Torr of He drift gas and an electric field strength of ∼40 V/cm to maximize the mobility resolution (defined as the arrival time divided by peak width at half-height) The ion arrival times ranged from hundreds of microseconds for the short cell to 1.5 milliseconds for the longer cell. The sequence for the MALDI-IM-OTOF MS experiment is as follows. A nitrogen laser (LSI, Cambridge, MA) is used to perform MALDI at threshold laser fluence. In contrast to highvacuum MALDI, the ablation plume is collisionally cooled within microseconds by interaction with the He drift gas, allowing ions to remain intact during long drift times. Indirect evidence for this comes from intercomparison of MALDI spectra from the same peptide digest samples obtained from a traditional high-vacuum MALDI MS and from a MALDI-IM-OTOF MS. Significantly higher peptide coverages were obtained from the MALDI-IM-OTOF MS data.7 The ideal He pressure for operation of the periodic focusing mobility cell is controlled as precisely as possible within an optimal range of between 1 and 10 Torr. A 2 Torr pressure was used throughout this work and represents a compromise between obtaining the highest voltage without gas breakdown and the highest mobility cell pressure consistent with using small (250 L/s) turbomolecular pumps to maintain adequate pressure in the (23) Gillig, K. J.; Russell, D. H. Periodic field focusing ion mobility spectrometer, U.S. Patent Application 20010032930, 2001. Fuhrer, K.; Gillig, K. J.; Gonin, M.; Russell, D. H.; Schultz, J. A. Mobility spectrometer, U.S. Patent Application 20010032929, 2001.

Figure 1. Instrument diagram.

mass spectrometer. The MALDI ions drift to the end of the mobility cell under the force of a high-voltage field applied between the sample plate and successive electrode rings within the mobility spectrometer. The ions then exit the skimmer into a differentially pumped mass spectrometer where they are mass analyzed in an orthogonal time-of-flight mass spectrometer. The mobility drift times are several milliseconds typically, while the flight times within the mass spectrometer are 20 µs or less. Therefore, several hundred mass spectra can be obtained after every laser pulse. Mass spectra are acquired after every 30-150 µs, depending on the target mass range, and are each stored individually along with the mobility time at which it was acquired. This process is repeated for several hundred laser shots, until each of the mass spectra contains sufficient intensity to permit analysis. These mass spectra have each been summed over several hundred laser shots, so that the ion mass as a function of mobility can be reconstructed for every desorbed ion. The data are presented as 2D contour plots of ion intensity as a function of ion mobility drift time and mass. All mobility-m/z contour plots were made using IDL software (Research Systems, Boulder, CO). The detection limit of the instrument is in the low-femtomole/ high-attamole range (using angiotensin II as test peptide). Simulations and initial experiments suggest that almost all ions surviving the MALDI event should be transferable from the IM cell to the OTOF MS. However, while there is certainly great potential for the use of this technique, nonspecialists seeking to understand whether this can be practical for their analytical applications shouldsuntil further noticesthink in terms of analyzing picomole loadings of biological materials. RESULTS AND DISCUSSION Spectra were acquired from a mixture of sphingomyelin, dynorphin 1-8, and a short oligonucleotide, as well as from three different lipid types in various combinations with peptides or

peptides and organic compounds. Sphingomyelin, the simplest lipid used, has a phosphocholine headgroup, while cerebroside sulfate adds the complexity of a sulfated sugar as the headgroup, and finally lipid A, which has six fatty acids, phosphate, and two sugars, is the most complex structure of the three. We will discuss and contrast the mobility of the two phospolipids first and then discuss the cerebroside sulfate data to compare the effect of the sulfated headgroup. Examples are also given for the analysis of drug/peptide, drug/lipid, and peptide/lipid noncovalent complexes, as well as the analysis of drugs in saliva. Sphingomyelin/Dynorphin 1-8/CATG. Figure 2 shows a MALDI-IM-OTOF MS two-dimensional contour plot of the mobility-m/z data (and the one-dimensional mass spectrum derived by summing all masses irrespective of mobility) from a mixture of known lipids, peptides, and oligonucleotides. The lipids, peptides, and oligonucleotides are obviously well separated by chemical class in the mobility drift cell due to their differences in collision cross sections. Sphingomyelin contains fatty acid chains varying in length, the major species is seen at MH+ 732.1 amu, and other species are seen at increments of plus (760.1, 788.1, 816.1) or minus (704.1) 28 amu. The lipid trend line with a slope of ∼ 0.18 µs/amu is drawn through the various species of sphingomeylin and those fragments still containing the polar headgroup. The multiple signals in the 2D mobility plot show the variations in the sphingomyelin fatty acid composition (704.1, 732.1, 760.1, 788.1, and 816.1 amu) denoting the heterogeneity present in the extracted brain lipid. Dimer ions formed in the source (not shown) are also present in the 2D plot falling on the same trend line. A second trend line is drawn through the peptide ion signals resulting from dynorphin 1-8 parent ion and its “insource” fragment ions (including the fragment ion corresponding to dynorphin 1-7). The slope of the trend line drawn through the peptide ion signals is ∼0.15 µs/amu, which is typical for this class of molecules under these IM experimental conditions. A third Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

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Figure 2. IM-MS 2D plot of a short oligonucleotide, a peptide, and a lipid. Note the various trend lines along which these compounds and their fragments are aligned.

trend line has been drawn through the CATG parent ion and corresponding in-source fragments having a slope of ∼0.13 µs/ amu. Note the isobaric separation of the [ATG]+ ion and the [dynorphin 1-8 - H2O]+ ion at m/z 964.2. Another mixture using entirely different molecules comprising oleyl semi-lysobisphosphatidic acid, dynorphin 1-9, and CATG yields the same pattern of separation and the same trend line slopes between the different molecular types and the same average trend line slopes (data not shown). The overall result to come from these comparisons is that although mobility separations within a class of molecules will vary on the order of 2% (because of slight variations in the mobility of isobaric conformers or because of alkali or matrix adduct formation), the mobility differences between isobaric ions from the three different biomolecular types of lipid, peptide, and oligonucleotide are ∼15-20% with a difference in mobility drift time between oligonucleotides and lipids of over 30%. The implications of these results are, first, that mixtures of these molecules in a MALDI sample can be routinely separated during the millisecond “dead time” between laser pulses and, second, that the occurrence of a molecular ion along a particular trend line identifies it as one of a particular molecular type without resorting to more complicated structural analysis techniques such as MS/MS. Sphingomyelin/Dynorphin 1-7/Chlorisondamine: Figure 3 shows the mobility-m/z separation of the lipid, peptide, and organic (drug) molecule mixture. The dynorphin 1-7 parent ion and its fragment ions lie along a trend line, which intersects with the mobility axis on the right at 1290 µs. The dynorphin 1-7 parent ion at m/z 869.0 has a mobility drift time of 750 µs. The second trend line is drawn through the lipid parent ion and those fragments still containing the polar headgroup. The multiple peaks show the variations in the sphingomyelin fatty acid composition 2190

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(704.1, 732.1, 760.1, 788.1, and 816.1 amu, which denotes the heterogeneity present in the extracted brain lipid). The dimers of sphingomyelin are seen at 1463, 1519, 1575, and 1631 amu, respectively. A fragment ion of the lipid dimer also appears on the same trend line, which can be assigned to a loss from the dimer ion of one of the C16H38O fatty acid tails. A 20% separation in the average mobility trend lines for the lipid and peptide is calculated by comparing the mobility drift times of the dynorphin parent ion (750 µs for m/z 869.0) to that of the matrix adducted sphingomyelin parent ion [ML + matrix + Na]+ (890 µs for m/z 877). The 140-µs difference between these two different but nearly isobaric ions is a specific illustration of the dependence of the molecular analyte type on drift time. Additional deviations from the average trend line are seen within each molecular class as well. Another line to guide the eye has been constructed through a set of ions at masses higher than the parent lipid, which span the range between 900 and 1300 amu. These ions are noncovalent complexes of chlorisondamine with sphingomyelin (and its biologically derived minority fatty acid chain length variations of 2CH2). Chlorisondamine is a nicotinic antagonist, contains two quaternary ammonium groups and a tetrachloroisoindoline ring, and has a hydrophobic head and a hydrophilic body and tail.25 The complexes formed by the parent lipid lie along a line that slopes toward faster mobility drift times than the unadducted lipid but are still slower than isobaric peptides. Sodium adducts of the various sphingomyelins are also seen. The major lipid MH+ (m/z 732.1) forms a noncovalent complex with chlorisondamine and one matrix molecule (m/z 1232.6). This complex has nearly the (24) Ruotolo, B. T.; Gillig, K. J.; Woods, A. S.; Egan, T. F.; Ugarov, M. V.; Schultz, J. A.; Russell, D. H., submitted to Anal. Chem.. (25) Woods, A. S.; Moyer, S. C., Wang, H. Y.; Wise, R. Y. J. Proteome Res. 2003, 2, 207-212.

Figure 3. IM-MS 2D plot of a mixture of sphingomyelin (ML), dynorphin 1-7 (MP) and chlorisondamine (Chl).

Figure 4. IM-MS 2D plot of a mixture of lipid A and the gastric peptide sulfated gastrin. The mobility plots of the lipid and its fragments and the peptide and its fragments lie on two different trend lines.

same mass but almost a 10% slower drift time compared to the lipid dimer fragment at m/z 1226 (which has lost one of the C16H38O fatty acid tails). Another subtlety is the effect on drift time of retaining or losing the phosphate headgroup as seen in the mass region around m/z 500. The lipid fragment ions having lost a headgroup are significantly slower than those fragments, which retain the headgroup but lose either of the fatty acid tails. The [M + H]+ ion of chlorisondamine m/z 358.2 has a mobility of 390 µs.

Lipid A/Sulfated Gastrin. Figure 4 shows a 2D plot of ion mobility versus m/z of a lipid A/sulfated gastrin mixture illustrating a large separation in mobility drift time for lipid/peptide ions. The differences in mobility between the isobars of the two molecular classes is similar to the 20% differences seen in Figure 3 (sphingomyelin, dynorphin, chlorisondamine mixture). The trend line drawn through the peaks for sulfated gastrin and its major fragments demonstrates that even a modified peptide has a higher mobility (smaller collision cross section) than a lipid as Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

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Figure 5. (a) IM-MS 2D plot of cerebroside sulfate extracted from brain. Note the presence of a lipid dimer in the upper right-hand corner. (b) IM-MS 2D plot of a mixture of dynorphin 1-7 and cerebroside sulfate. Note the presence of a lipid/peptide complex in the upper right-hand corner and the difference in mobility time between the dimer and the complex.

shown by the trend line connecting the lipid A sodiated ion at m/z 1741 with its fragments. The major lipid fragments can be explained either by loss of fatty acid moieties of 183 or 210 amu or by fragmentation at the ether linkage between the two (sugar backbones) followed by losses of fatty acid side chains. An interesting subtlety in the mobility plot of the fragment ions can be seen in Figure 4 data within each of the lipid fragment ion signals, which have lost one or the other of the fatty acid side chains and some of which have also lost the attached alkali cations. Although the fragment masses may differ by 17, depending on which fatty acid is lost, the overall mobility time is roughly the same as evidenced by a general trend within each fragment group, which is roughly horizontal in the 2D plot. The change in mass through loss of a side chain does not produce a proportional change in the overall mobility drift time within each of the lipid fragment families. We interpret this result to mean that the collision cross section of lipid A fragments is determined by the steric packing of the lipid tails (fatty acids), which does not vary much with the loss of one or the other type of fatty acid. Alkali cation adduction to the headgroup of the various fragments also does not change the mobility drift time significantly from that of the unadducted ion. Both of these effects cause the fragment groupings to appear along near-constant mobility drift times. A third fragmentation process yields a low-mass series of fragments of ∼300 amu, which retain only part of the sugar headgroup with lipids attached. These fragments are seen to be slower and well resolved by mobility from any of the almost isobaric peptide and matrix ions. This is an important result for future work in which MALDI-IM-OTOF MS may be useful in analyzing complex mixtures of biomolecules with smaller organic molecules such as drugs. 2192

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Cerebroside Sulfate/Dynorphin 1-7 and Their Lipid/ Peptide Noncovalent Complex. Figure 5a shows a 2D plot of ion mobility drift time versus m/z of brain cerebroside sulfate ions. A plot obtained using similar conditions of a lipid/peptide mixture is shown in Figure 5b. The spectra were acquired in the same experiment with a higher resolution performance OTOF MS. The mass assignments are complicated somewhat by heterogeneity in the fatty acid composition, resulting in two forms of the lipid. In the higher mass heteromer, an OH group is at C14 of the C24 chain fatty acid, whereas the OH is absent and a double bond is at the same site in the other heteromer of the lipid. The signal for doubly sodiated cerebroside sulfate parent ion is the highest intensity peak in the spectrum. At high salt concentrations, the doubly sodiated lipid parent ions are exclusively formed instead of singly sodiated lipid parent ions, which might have been expected to be present in the mass spectrum. Within experimental error, the slope of the trend lines corresponding to cerebroside sulfate ions (and its fragments) and that of sphingomyelin ions in Figure 2 are roughly the same. The dynorphin fragments can be overlapped to normalize the two data sets for this comparison. Thus, the differences in the size of the two different headgroups do not create a difference in the mobility of the two types of lipids, probably because both headgroups are of similar geometric size and density. However, the lipid fragment ions near m/z 800, which result from loss of some or all of the sodiated sulfate functional headgroups, have mobilities that are significantly less and are situated above the trend line connecting parent ions with lower mass fragments that retain intact headgroups. This shift to longer drift times upon losing the sulfate headgroup is again of a magnitude similar to that observed in Figure 2 for the sphingomyelin fragments that have lost their phosphatidylcholine head-

Figure 6. (a) IM-MS 2D plot of a mixture of peptide SVLpYTAVQPNE (P10) and acetylcholine (Ach). Note the drift generated by the formation of noncovalent complexes between acetylcholine and the aromatic residues and the acetylcholine and the phosphate group. (b) IM-MS 2D plot of a mixture of peptide SVLpYTAVQPNE (P10) and chlorisondamine (CHL), which is fairly hydrophilic. Thus, no drift was seen when it formed a noncovalent complex with P10.

group. The shift direction is not unexpected considering that the headgroups are the hydrophilic components of both lipids. The effect on mobility drift time of the different lipid headgroup types is an important question that will be studied more systematically in the future as the stability of our instrument is improved. However, the overall lipid mobility does not seem to be a strong function of headgroup type. The plot in Figure 5a also shows the signal of the lipid dimer (m/z 1820). Compared to the ion signal visible in the same mass region in Figure 5b for the mixture of peptide and lipid, the dimer mobility drift time is significantly longer (1580 µs compared to 1470 µs). Both data acquisitions were performed using the same sample plate, which guarantees high

correlation of the mobility time. Therefore, although an accurate mass assignment is difficult, one can conclude that the signal in Figure 5b indeed corresponds to the complex between peptide and lipid rather than the dimer of either peptide or lipid. The dimer signal is suppressed in the spectrum of the mixture; however, one can also observe a reduced intensity of the lipid monomer signal. Not only do different classes of biomolecular ions have different mobility drift times, but the complexes that they form between each other may also show similar trends. Peptide-Small Molecule interactions. Figure 6a is a 2D plot of mobility versus m/z of the phosphorylated peptide SVLpYTAVQPNE (P10) in the presence of acetylcholine. Signals Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

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Figure 7. (a) IM-MS 2D plot of morphine in saliva. (b) IM-MS 2D data of morphine in saliva from (a) after any ion signal of two ion counts or less has been subtracted.

for the protonated peptide and noncovalent complexes corresponding to the addition of one, two, and three acetylcholine molecules are present. Addition of one molecule of acetylcholine has no effect on the mobility of P10. However, the addition of two or three acetylcholines shifts the mobility drift time away from the peptide average trend line by 5%. A trend line can be drawn through the peaks of p10 at m/z 1301.4, its fragments, and the P10/single acetylcholine complex at m/z 1446.6. A second trend line can be drawn through p10 plus two acetylcholine complex and p10 plus three acetylcholine complex. 2194

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We had previously shown that quaternary amines form noncovalent complexes with aromatic residues and with phosphate groups.15 The differences in the drift time-m/z relationship between phosphorylated and nonphosphorylated peptides are currently of interest because of the possibility of using IM-MS as a rapid method to screen for the presence of phosphopeptides in a complex protein digest mixture.24 The chlorisondamine complex with P10 (Figure 6b) has a mobility drift time that places it along the average trend line of the peptides in contrast to the behavior observed for the second and third acetylcholine additions (shown

in Figure 6). The chlorisondamine must, therefore, attach to the P10 peptide in a configuration that produces an ion volume little different from that of the same m/z random-coiled peptide. This is a behavior completely different from that for the complexation of small aromatics (either drugs of matrix) with sphingomyelin (Figure 3). Any modeling of the geometric configuration of either this drug/peptide complex or of the drug/sphingomyelin complex will have to account for these data. We also see in Figure 6b only one chlorisondamine molecule forming a complex with P10 in contrast with up to three acetylcholines. Presumably, steric exclusion by the larger and bulkier chlorisondamine may partly explain these different behaviors. Morphine in Saliva. We have compared two techniques, highvacuum MALDI and MALDI-IM-OTOF MS, for the analysis of morphine in saliva prepared according to NIDA protocols at concentrations of 5 ng/µL. MALDI (along with other mass spectrometric techniques) has not traditionally been used for drug analysis in physiological fluids without chemical or chromatographic preseparation to eliminate interferences from isobaric biomolecular ions. The relative sensitivities of the MALDI-IMOTOF MS and of the high-vacuum MALDI instrument are difficult to compare since we do not know how much of the sample was consumed in either case (CHCA was used as a matrix). The number of laser shots was 50 for the MALDI MS analysis (data not shown) and ∼3000 for the MALDI-IM-OTOF MS. Acquisition time for the MALDI-IM-OTOF MS spectra was a few minutes at a laser pulse rate of 10 Hz. Figure 7 demonstrates the utility of MALDI-IM-OTOF MS for separation and differentiation of the complex mixture of biological ions present as interferences in saliva samples. Figure 7a is a 2D plot of ion mobility versus m/z for a 3000 laser shot acquisition of morphine in saliva in ATT matrix. ATT matrix was intentionally used to provide a worst case test since it is not as efficient as CHCA for the analysis of morphine; i.e., more background ions are present in Figure 7a than for morphine in CHCA (data not shown). We also note that the morphine molecular ion is not present in the matrix saliva blank. An important feature of the data in Figure 7a is the distribution of ion counts in 2D mobility/mass space. Since the lower right corner and the upper left corner of the 2D plot are free of ion counts, the “background” ions are not random instrumental noise, which would be spread more nearly uniformly over 2D space. Instead, these ions represent “chemical noise” and, with the addition of ion mobility information, offer a significant advantage in noise reduction not available in a one-dimensional MALDI spectrum. Biological interfering ions are widely distributed above and below the diagonal “trend line” containing the morphine (m/z 285.3) and ATT matrix monomer and dimer ions (m/z 144.3 an 287.3). Figure 2 has illustrated that ions on trend lines above the drug mobility time are most likely lipid and liposaccharide ions, ions

slightly above are likely to be of peptide origin, and ions considerably below are likely to be of oligonucleotide origin. Figure 7a also shows a 1D derived mass spectrum along the top “x-axis”, which is the sum of all ions at each mass irrespective of their mobility time. This derived mass spectrum is complex and “noisy”, illustrating why MALDI is often rejected for small molecule analysis. Figure 7b on the other hand shows a replotting of the 2D data excluding any ion intensity of two ions or less. By using the mobility, this 1D mass spectrum now produces a very high signal/noise ratio, even for a relatively low total ion count in each mass bin. This experiment demonstrates the ability to directly detect the presence of drugs without chemical extraction from saliva and without the chemical derivatization step necessary for detection by GC/MS. The detection limit is comparable to that of GC/MS and will increase as additional instrumental improvements are made to MALDI-IM-oTOF mass spectrometers. CONCLUSIONS MALDI-IM-OTOF MS is emerging as a technique with a detection limit and mass accuracy approaching conventional MALDI instrumentation. In addition, MALDI-IM-OTOF MS separates biomolecules according to their composition, hence allowing assignments of ion chemical type based simply on their specific mass and mobility drift time. The data presented support the use of IM-MS as a fast preseparation technique for complex mixtures of biomolecules as it separates peptides, lipids, and oligonucleotides along distinct trend lines prior to mass analysis within the “dead time” inherent in a modern high laser pulse rate MALDI TOFMS experiment. Within molecular class, smaller deviations in collision cross section may provide a large enough difference in mobility drift time to rapidly screen biomolecules for the presence of specific peptide, lipid, or oligonucleotide modifications. For some applications, this may eliminate the need for more complicated MS/MS techniques or time-consuming preseparation steps prior to mass analysis. These last properties make it a powerful tool in fingerprinting bioorganisms and could make cataloging all types of organisms a much easier task. Furthermore, the use of MALDI-IM-OTOF MS as a research tool for tissue imaging and its use as a clinical tool for rapid analysis of drug levels in saliva are suggested. Both of these potential applications deserve attention soon. ACKNOWLEDGMENT Ionwerks is grateful for NIH phase II SBIR support (2 R44 GM57736-02) and NIDA contract N44DA-3-7727.

Received for review November 20, 2003. Accepted February 19, 2004. AC035376K

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