In the Laboratory
Illustrating the Concepts of Isotopes and Mass Spectrometry in Introductory Courses: A MALDI-TOF Mass Spectrometry Laboratory Experiment W Nancy Carter Dopke*,† and Timothy Neal Lovett
Department of Chemistry, Mercer University, Macon, GA 31207; *
[email protected] Mass spectrometry is a widely-used and versatile technique that is increasingly being introduced to undergraduates through laboratory experiments. Most of these experiments involve the mass analysis of volatile samples using GC–MS (1). In the last 20 years a number of techniques have been introduced that expand the usefulness of mass spectrometry to nonvolatile species. Soft ionization techniques allow the mass analysis of nonvolatile species such as biomolecules, polymers, and clusters. Matrixassisted laser desorption/ionization (MALDI) (2, 3) and electrospray ionization (ESI) (4) have been particularly successful soft ionization techniques. The importance of the mass analysis of large proteins using these techniques was highlighted with the awarding of a share of the 2002 Nobel Prize in Chemistry to Koichi Tanaka and John Fenn (5). However, few experiments for undergraduate laboratory students have been published utilizing these techniques (6) and even fewer are designed for first-year students (7). We present a MALDI undergraduate lab experiment for students in an introductory course. In matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry, the analyte is embedded in a solid matrix (usually a small, highly energy-absorbing organic molecule) by depositing the dissolved sample onto a dried spot of matrix on the sample holder, which is called a target. The sample is allowed to air dry before the target is inserted into the instrument, which is under vacuum. The excitation of the matrix by pulsed laser light results in the desorption and ionization of intact molecular ions. The charged species are then propelled into the flight tube where they are separated on the basis of their masses by their mass-to-charge ratio. Soft ionization techniques are used in explorations at the interface of chemical and biological research. Given that the majority of our students are interested in careers at this interface, the incorporation of soft ionization mass spectrometry throughout the curriculum should enhance the education and interest of our students. MALDI mass spectrometry is relatively easy to use and is conceptually accessible to students. The mass spectra can be collected quickly and the experiment has a high tolerance for contaminants. Time-of-flight mass analyzers have been discussed in detail in this Journal (2) and elsewhere (8). The incorporation of a mass spectrometry experiment in the first semester allows students to explore the effect of isotopes on mass spectra that they experimentally collect. They consider the concept of calibration and learn the difference between monoisotopic mass and average mass. In addition, the students are excited about completing an experiment using research instrumentation so early in their academic careers. General chemistry texts often introduce the concept of isotopes and mass spectrometry in the first section of the text. †Current address: Department of Chemistry, Alma College, Alma, MI 48801.
Students learn about the mass spectra of elemental samples, but learn little about the consequences of isotopes on the mass spectra of molecules. Typically the students are only exposed to one type of sample introduction and ionization and one type of mass analyzer. In this laboratory experiment, students prepare a matrix solution, deposit their samples onto a target for the mass spectrometer, collect data using a MALDI–TOF mass spectrometer, and analyze the resulting data. The data analysis includes the determination of the molecular formula of an unknown by utilizing the students’ own mass spectral data and elemental analysis data provided by the instructor. Experimental Procedure Sample Preparation The peptides used were angiotensin(II), substance P, bombesin, neurotensin, and melittin. Prior to the lab, 3 × 10‒6 M peptide solutions are prepared by the instructor. Each student receives two microcentrifuge tubes, one with an unknown peptide and one containing two calibrants, angiotensin(II) and melittin. The solutions may be made in advance and stored in the freezer for over a year until needed. Student Laboratory Procedure In addition to reading the lab procedure prior to the lab, students learn about the general technique and procedure through a pre-laboratory discussion with the instructor. Since we complete this lab early in a first-semester course, our students have limited experience with manipulating volumes and need a tutorial on dealing with the microliter solutions that they will be using. The students prepare their matrix sample by dissolving a microspatula tip full of 2,5-dihydroxybenzoic acid (DHB) in 20 μL of 33% acetonitrile in ultra pure water (18 MΩ) containing 0.1% trifluoroacetic acid. Each student spots 1 μL of the matrix solution on the instrument target, a stainless steel plate containing marked flat wells. After the matrix spot has dried, 1 μL of the calibrant mixture and 1 μL of their unknown sample is spotted on top of their matrix spot. After the target with all students’ samples has dried, the target is inserted into the instrument, and each student collects mass spectral data on their unknown. Students learn more about the instrumentation from discussions while the target is inserted into the vacuum system and while data are being collected. The Bruker Daltonics Omniflex mass spectrometer has a camera built into the system so that users can see their sample spots while they collect their data. This provides students with a visualization of what is really happening to their sample and decreases the likelihood that a student views the instrument as a “black box”. As students collect their spectra, they are encouraged to sample from different parts of
1968 Journal of Chemical Education • Vol. 84 No. 12 December 2007 • www.JCE.DivCHED.org
Hazards Precautions to prevent skin contact, inhalation, and ingestion should be taken during the preparation of the samples and completion of the laboratory experiment. Acetonitrile is an irritant and is toxic by inhalation, ingestion, and skin absorption. Trifluoroacetic acid has a pungent odor and can cause severe burns. Thus, the use of gloves is recommended during sample preparation. The use of fume hoods and protective eyewear is particularly recommended when handling the solvents acetonitrile and trifluoroacetic acid since these chemicals can cause serious damage to the eyes. The matrix 2,5-dihydroxybenzoic acid (DHB) is also an irritant. The peptides used in the experiment are bioactive peptides and instructors should prepare the dilute samples of each peptide to limit the possibility of exposure for the students. The peptides are not hazardous at the concentrations used by the students in the experiment, though their toxicological properties have not been thoroughly investigated and caution should still be exercised. Student Results and Discussion Each student acquires a mass spectrum of an unknown peptide and two peptide calibrants during the lab. Three peptides have been used as unknowns: substance P, bombesin, and neurotensin. A sample mass spectrum of an unknown (substance P) with two calibrants, angiotensin(II) and melittin, collected by a student is shown in Figure 1. Each peptide is detected as the molecular species plus a proton, [M + H]+, and gives rise to an isotope envelope consisting of multiple peaks owing to the presence of the less abundant isotopes of carbon, nitrogen, oxygen, and hydrogen. The first peak in the isotope envelope is the monoisotopic peak: the peak that is the result of the detection of the single isotope combination of the lowest mass isotope of each element present. For substance P, which has a molecular formula of C63H98N18O13S, the monoisotopic peak is from the ion with the formula [12C631H9914N1816O1332S]+. The monoisotopic peak is easily distinguished in the isotope envelope for peptides with masses up to 3000 Da. The students use their experimentally determined mass spectral data and provided elemental analysis data to determine the molecular formula of their unknown. The molecular mass is determined from their mass spectra using the mass of the monoisotopic peak minus the mass of one hydrogen atom.
1349.73 Da
1347.74 Da
1046.54 Da
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1350.71 Da
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2845.76 Da
Elemental Percentage Data The students complete the experimental part of the lab by receiving elemental analysis data on their unknown from the instructor. Instructors can use the ChemPuter found online (9) to determine elemental percentages from a molecular formula.
3000
Absolute Intensity
their spot and to vary the laser power to improve their signal. Students calibrate their spectrum using the monisotopic peaks of their calibrants, angiotensin(II) (1046.5 Da) and melittin (2845.8 Da). They then print an up-close view of their unknown signal for analysis. Each student needs between 4 and 9 minutes with the instrument, making throughput an important issue to consider. A class size of 12–16 students can complete the lab in a three-hour lab period. For larger classes, the students may need to be split into groups for the data acquisition.
1347.74 Da 1348.72 Da
In the Laboratory
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Figure 1. Student collected mass spectrum of unknown (substance P, 1347.74 Da) with calibrants angiotensin II (1046.54 Da) and melittin (2845.76 Da). The inset is the isotope envelope for the unknown.
The elemental analysis data are used to determine the empirical formula, which is then compared to the molecular mass to determine the molecular formula of their unknown. The students are instructed to discuss why they use the monoisotopic peak in their analysis of the unknown as part of their discussion in their lab writeup. In each of the three classes that have completed this experiment, some of the students have been unsuccessful at detecting their unknown in the spot that they themselves deposited on the target. This problem is easily remedied by having these students collect data on a spot containing an unknown and the calibrants deposited on the target by the instructor prior to the start of the student’s laboratory time. Summary Students completing this lab gain hands-on experience using a mass spectrometer and learn about the requirements for mass spectral analysis and the consequences of isotopes. The data analysis reinforces concepts often taught early in general chemistry courses. We continue to expose students to mass spectrometry throughout their undergraduate careers with the goal of providing them the opportunity to assimilate the chemistry, usefulness and beauty of MALDI mass spectrometry. The lab could be adapted to any of the soft ionization techniques, depending upon what is available or preferred. Changes to the sample preparation and data collection procedure could easily be made to accommodate a different technique. Acknowledgments We gratefully acknowledge the sources of funding used in the completion of this project. The purchase of the Bruker Daltonics Omniflex mass spectrometer was made possible through funds from National Science Foundation DUE Award #0126930 with matching funds from Mercer University. Funds for the development of the lab, including student and faculty
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In the Laboratory
summer salaries, were provided by the Dean of the College of Liberal Arts at Mercer University. We would also like to thank the students of the 2002, 2003, and 2004 Chemistry 115 Advanced General Chemistry classes who tested and performed this experiment. WSupplemental
Material
The student handout and instructor notes are available in this issue of JCE Online. Literature Cited 1. For example: (a) Sobel, R. M.; Ballantine, D. S.; Ryzhov, V. J. Chem. Educ. 2005, 82, 601–603. (b) Solow, M. J. Chem. Educ. 2004, 81, 1172–1173. (c) Mowery, K. A.; Blanchard, D. E.; Smith, S.; Betts, T. A. J. Chem. Educ. 2004, 81, 87–89. (d) Fong, L. K. J. Chem. Educ. 2004, 81, 103–105. (e) Wood, W. F.; Price, D. Chem. Educator 2002, 7, 226–232. (f ) Witter, A. E.; Klinger, D. M.; Fan, X.; Lam, M.; Mathers, D. T.; Mabury, S. A. J. Chem. Educ. 2002, 79, 1257–1260. (g) Smith, D. C.; Forland, S.; Bachanos, E.; Matejka, M.; Barrett, V. Chem. Educator 2001, 6, 28–31. 2. Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. J. Chem. Educ. 1997, 74, 1288–1292. 3. (a) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (b) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A–1203A. (c) Karas, M.; Bahr, U.; Giebmann, U. Mass
Spectrom. Rev. 1991, 10, 335–357. (d) Karas, M. Biochemical Mass Spectrom. 1996, 24, 897–900. (e) Stump, M. J.; Fleming, R. C.; Gong, W.-H.; Jaber, A. J.; Jones, J. J.; Surber, C. W.; Wilkins, C. L. Appl. Spectrosc. Rev. 2002, 37, 275–303. 4. (a) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (b) Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. J. Chem. Educ. 1996, 73, A82–A88. (c) Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. J. Chem. Educ. 1996, 73, A118–A123. (d) Hop, C. E. C. A.; Bakhtiar, R. J. Chem. Educ. 1996, 73, A162–A169. 5. Vestling, M. M. J. Chem. Educ. 2003, 80, 122–124. 6. (a) Reimann, C. T.; Mie, A.; Nilsson, C.; Cohen, A. J. Chem. Educ. 2005, 82, 1215–1218. (b) Sunderlin, L. S.; Ryzhov, V.; Keller, L. M. M.; Gaillard, E. R. J. Chem Educ. 2005, 82, 1071–1073. (c) Weinecke, A.; Ryzhov, V. J. Chem. Educ. 2005, 82, 99–102. (d) Stynes, H. C.; Layo, A.; Smith, R. W. J. Chem. Educ. 2004, 81, 266–269. (e) Moe, O. A.; Patton, W. A.; Kwon, Y. K.; Kedney, M. G. Chem. Educator 2004, 9, 272–275. (f ) Bergen, H. R.; Benson, L. M.; Naylor, S. J. Chem. Educ. 2000, 77, 1325–1326. 7. Counterman, A. E.; Thompson, M. S.; Clemmer, D. E. J. Chem. Educ. 2003, 80, 177–180. 8. (a) Cotter, R. J. Anal. Chem. 1992, 64, 1027A–1039A. (b) Cotter, R. J. Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research; American Chemical Society: Washington, DC, 1997. (c) Downard, K. Mass Spectrometry: A Foundation Course; Royal Society of Chemistry: Cambridge, 2004; pp 40–43. 9. Sheffield ChemPuter. http://www.shef.ac.uk/chemistry/chemputer/ (accessed Sep 2007).
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