In the Laboratory
Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 2: Peptide Identification via Molecular Mass Determination Isaac J. Arnquist and Douglas J. Beussman* Department of Chemistry, St. Olaf College, Northfield, MN 55057; *
[email protected] Protein analysis has become a major research area in many laboratories. With the growing field of proteomics, it is important for students to be exposed to this area. One of the major analytical techniques used in proteomics, as well as other areas of drug discovery, is mass spectrometry. Electrospray ionization (ESI) (1, 2) and matrix-assisted laser desorption ionization (MALDI) (3–5) allow biological molecules to be analyzed by mass spectrometers. While this is routinely done in research laboratories, students get little exposure to these types of analyses, especially at the undergraduate level. Hopefully the incorporation of these techniques into the undergraduate curriculum will parallel similar trends in GC–MS and NMR that have occurred previously. In the laboratory experiment presented here, which is part two of a four-part set of experiments, we describe how mass spectrometry can be integrated into the curriculum to determine the molecular mass and identity of an unknown peptide. This experiment is part of a bioanalytical chemistry course developed at this college, but could be used in a variety of laboratory courses, including biochemistry or instrumental analysis courses. To allow anyone who wishes to incorporate biological mass spectrometry into their curriculum, even if they do not currently have access to a mass spectrometer, we have included several sets of complete data in the online material for this article, which could be used to demonstrate the concepts discussed below as classroom exercises or as “mock” laboratory experiments. Although most ESI analyses are done using the coupled liquid chromatography–mass spectrometry (LC–MS) technique, ESI can be used on a stand-alone mass spectrometer with relatively pure samples, without the prior LC separation technique, as is presented in this article. A search of previous publications in this Journal dealing with LC–MS or ESI techniques provided only 10 examples. Of these, one discusses the 2002 Nobel Prize in Chemistry being awarded in part for the development of ESI (6), three discuss the fundamentals and applications of ESI (7–9), one discusses the analysis of small organic molecules (10), one presents a characterization of organometallic compounds (11), and the final four discuss the analysis of biological molecules (12–15). Of these four papers, one presents a characterization of metmyoglobin (12), one presents a method for the identification of two specific proteins (13), one presents a general method for the analysis of proteins (14), and one presents the analysis of several peptides and small proteins (15). In this last manuscript, the authors described using charge state deconvolution to obtain the charge and thus the molecular mass of a set of peptide peaks. This technique is almost never done with small peptides and is not even possible if a single charge state is observed. In the laboratory experiment presented here, we discuss the analysis of relatively small peptides by ESI, including the determination of charge state and thus molecular mass using isotopic patterns. Students are given an unknown peptide and must complete the analysis and identify 382
the peptide from a list of 18 potential peptides. The online material contains the spectra of seven of the listed peptides, and can be used as instructional material if an electrospray mass spectrometer is not available. Unlike other mass spectrometry ionization methods, which primarily produce singly charged ions, ESI often produces significant quantities of multiply charged ions. Since mass spectrometers measure mass-to-charge (m∙z) ratios, to correctly determine the molecular mass of an analyte, the charge on the ion signal must first be determined. One of the easiest ways to do this is to observe the isotopic distribution of the ions of interest. The ability to observe individual isotopes is related to the resolving power of the mass spectrometer being used. Thus, a low resolution quadrupole mass spectrometer (i.e., with resolution ~1 m∙z), might not be useful for this sort of analysis. A quadrupole ion-trap instrument, with a resolution of 0.1 m∙z and a range of 0–3000 m∙z, would be sufficient for the analysis of most molecules, including peptides, up to a mass of ~3000 Da. Mass spectrometers with higher resolving power and higher m∙z ranges (such as time-of-flight, ion cyclotron resonance, or certain hybrid instruments) could analyze even larger peptides and small proteins. When analyzing a molecule using mass spectrometry, the difference between the average molecular mass and the monoisotopic molecular mass must be considered. Discussions of isotopic effects in analyzing large biomolecules have previously been published (16, 17). If isotopic resolution is obtained, then not only will the monoisotopic molecular mass be observed, but the isotope pattern arising from the isotopic distribution of the various atoms comprising the molecule will also be observable and can yield useful information (18). Although carbon is not the only atomic species that contributes to the isotopic distribution of the molecule, it is the most important isotope for mass spectral analysis using ESI and will allow the determination of the number of charges on an ion and thus the correct molecular mass of this ion. Complete isotope patterns can be used to confirm the molecular formula of molecules as well, and programs exist that will calculate the theoretical isotopic distribution for a given molecular formula (19). Experimental Procedure The experimental procedure was developed for one laboratory period. Quality spectra were obtained using an ESI Bruker Daltonics esquire 3000+ ion-trap mass spectrometer and accompanying software, although any ESI–equipped mass spectrometer with high enough resolving power should be capable of providing comparable spectra. All peptide samples were purchased from Sigma and were either stored as obtained in the freezer (‒20 °C) or refrigerated (4 °C), following given guidelines until ready to be diluted. A 100 pmol∙μL (100 μM) concentration was prepared from stock using nanopure water (18 MΩ cm, Barnstead Nanopure series 550). The solutions were stored in 1.5 mL microfuge tubes in the freezer (‒20 oC).
Journal of Chemical Education • Vol. 86 No. 3 March 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Bombesin RHYNSGHFWFLGP
C71H110N24O18S C78H100N22O17
Substance P RPKPQQFFGLK Angiotensin 1 GDFYWPFR Arg8-Vasopressin RPPGFSVFR Bradykinin ACTH fragment 4-10 RKRSRKE RERAERE RKRARKE EARSRER
C63H98N18O13S C64H100N18O14 C62H89N17O14 C55H66N12O12 C46H65N15O12S2 C50H75N15O11 C50H73N15O11 C44H59N13O10S C38H74N18O11 C36H64N16O14 C38H74N18O10 C34H62N16O13
RKRSRAE TVFGLR HLGLAR YIGSR
C35H67N17O11 C32H53N9O8 C29H51N11O7 C26H42N8O8
Monoisotopic Molecular Mass/Da 1618.82 1616.76 1346.73 1344.77 1295.68 1086.49 1083.44 1061.58 1059.56 961.42 958.58 944.48 942.58 902.47 901.52 691.40 665.40 594.31
Note: Peptides analyzed in the laboratory are shown in bold.
While the authors have observed no peptide degradation or hydrolysis with samples kept in this manner for over a year, a better long-term storage solution is to prepare 100 μL aliquots of the diluted samples and then freeze dry each aliquot. Storing these dried samples at ‒20 °C will allow samples to be kept for several years. Freeze dried samples can be reconstituted in 100 μL of nanopure water prior to laboratory use. Direct injection of the 100 μM sample solutions into the mass spectrometer was accomplished using a direct infusion syringe pump and a 250 μL syringe. The flow rate was set at 240 μL∙hour (4 μL∙min) and typically less than 100 μL was needed in the syringe to complete the experiment. All samples were analyzed in the positive ion mode, with the capillary voltage set at ‒4500 V, an end plate offset of ‒500 V, a N2 nebulizing gas pressure of 10 psi, a N2 dry gas flow rate of 1.0 L∙min, and a drying temperature of 300 °C. The instrument was optimized for 650 m∙z, which yielded all spectra shown here and in the online material. The instrument was set to scan from 50 to 2000 m∙z with a scan rate of 13,000 m∙z per second (the “normal resolution” mode). Spectral data were saved and analyzed using Bruker DataAnalysis software. The needle, end plate, injector tube, and syringe were cleaned between runs using acetonitrile followed by 2-propanol. Hazards Commercial mass spectrometers have built-in safety interlocks, so there is low risk associated with the operation of the instrument. Since electrospray sources use a heated drying gas, care should be taken when cleaning the source region, as there may be hot gas or hot metal source components. Skin contact with the peptides and solvents, especially acetonitrile, should be avoided by wearing gloves. Acetonitrile may cause irritation to skin, eyes, and respiratory tract. It is a flammable liquid.
333.67
100
200
300
400
500
600
700
800
900
1000
m/z B
333.67 334.11 334.66 333
334
335
m/z
336
666.33
Intensity
Molecular Formula
Intensity
Peptide
A
666.33
Intensity
Table 1. List of Possible Peptides to Choose from and Their Corresponding Molecular Masses
C 667.23 668.28
665
666
667
668
669
m/z
Figure 1. (A) Mass spectrum of HLGLAR showing +1 and +2 charge peaks. (B) The +2 peak is enlarged to verify the charge state due to the isotope effect. (C) The +1 peak is enlarged to verify the charge state due to the isotope effect.
Results and Discussion Seven peptides, listed in Table 1, ranging in molecular mass from 665 to 1619 Da, were analyzed. All spectra were obtained using the protocol given in the experimental section and are provided in the online material. When attempting to identify their unknown peptide, students were given the entire list of 18 possible peptides shown in Table 1 to choose from. Because of the natural occurring isotope 13C, there will be peaks of increasing m∙z (Δ m = 1) from the lowest m∙z molecular ion peak, which represents the ion corresponding to all carbon atoms being 12C. For small molecules (less than about 1300 Da), the highest intensity isotopic peak will correspond to all carbon atoms being 12C (16, 17). As the number of carbon atoms in an ion increases, in general yielding a larger mass, so does the probability that the ion will contain a 13C isotope. Thus, for ions with mass larger than about 1300 Da, the 13C isotope peaks may be larger than the peak corresponding to all carbon atoms being 12C. Charge state determination can be obtained based on the m∙z differences between consecutive 13C peaks: 1 z m (1) % z observed A mass spectrum of the peptide HLGLAR, with a monoisotopic molecular mass of 665.40 Da, is shown in Figure 1. Two peaks corresponding to a charge of +1 and +2 are observed. The magnified spectrum of the +1 isotope cluster shows the isotope effect. The isotopes create a distribution of peaks separated by 1 m/z unit when the charge is +1. Furthermore, if the charge were +2, +3, or +4, the isotope peaks would be evenly distributed by 0.50, 0.33, and 0.25 m∙z units, respectively. For positive-ion ESI, the normal mode of ionization is the addition of a proton (H+) to the analyte, A, thereby creating a singly charged positive ion (A + H)+. Thus, singly charged positive ions appear at an m∙z value that is 1 unit higher than the actual molecular mass of the molecule being studied. Thus, in Figure 1, HLGLAR is observed at 666.33 m∙z, corresponding to a molecular mass of 665.33 Da, or a mass error of ~0.01% compared to the theoretical molecular mass of 665.40 Da.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 3 March 2009 • Journal of Chemical Education
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In the Laboratory
Acknowledgments
530.71
Intensity
Intensity
530.71 531.19 531.68 530
300
400
500
600
531
700
m/z
800
532.15
532
533
900
1000
1100
m/z Figure 2. Mass spectrum of bradykinin (showing only a +2 charge peak. The peak is enlarged to show the separation of approximately 0.5 m/z units owing to 13C isotope effect.
Multiply charged ions, (A + nH)+n, are formed primarily by the addition of multiple protons where n is the number of protons added and thus also the charge on the ion. For multiply charged ions, the molecular mass, M, can be calculated by m z
observed
MA
MA nMH n
m n z
(2)
nMH
observed
The doubly charged peak for HLGLAR is observed at 333.67 m∙z, yielding a molecular mass for HLGLAR of 665.34 Da, corresponding to a mass error of ~0.01%. Since two charge states are observed in Figure 1, both can be used to determine a better molecular mass by averaging the two individually calculated molecular masses. Thus, in this case, the reported molecular mass would be (665.33 + 665.34)∙2 = 665.34 Da. The spectrum of bradykinin, a peptide with a monoisotopic molecular mass of 1059.56 Da, is shown in Figure 2. If the molecular mass is known, it is easy to determine that the peak shown must have a charge of +2. However, if the identity of the peptide is unknown, analyzing the 13C effect would help determine that the charge state of the 530.71 m∙z peak is indeed +2, which corresponds to a molecular mass of 1059.42 Da, or a mass error of 0.013%, since the 13C peaks average ~0.5 m∙z and so eq 1 gives z = 2. Assigning a different charge state would result in an incorrect molecular mass determination. The effects of additional charge states can be observed in the spectra included in the online material. Conclusion We have presented a relatively simple method for the determination of the molecular mass of peptides using an electrospray ionization mass spectrometer. Since electrospray ionization often creates multiply charged ions, the charge state of an observed ion must first be determined before the correct molecular mass can be calculated. This can often be done based on the apparent m/z spacing of the isotopic peaks. We have presented seven peptides (spectra given in the online material) that can be given as unknowns to students for them to analyze, determine the charge state and correct molecular mass, and identify based on a list of possible peptides if desired. 384
We gratefully acknowledge funding support for the purchase of the electrospray ionization mass spectrometer from the National Science Foundation (NSF-MRI grant #0320738). We also wish to acknowledge funding support (summer salary for I.J.A.) from an institutional grant awarded to St. Olaf College by the Howard Hughes Medical Institute. The authors would also like to thank Anna E. Larson for collecting some of the spectra shown in the online material section. Literature Cited 1. Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451–4459. 2. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. 3. Karas, M.; Buchman, D.; Hillenkamp, F. Anal. Chem. 1985, 57, 2935–2939. 4. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yohida, T. Rapid Commun. Mass Sp. 1988, 2, 151–153. 5. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. 6. Vestling, M. M. J. Chem. Educ. 2003, 80, 122–124. 7. Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. J. Chem. Educ. 1996, 73, A82, A84–A88. 8. Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. J. Chem. Educ. 1996, 73, A118–A123. 9. Hop, C. E. C. A.; Bakhtiar, R. J. Chem. Educ. 1996, 73, A162, A164–A169. 10. Bergen, R. H., III; Benson, L. M.; Naylor, S. J. Chem. Educ. 2000, 77, 1325–1326. 11. Coe, B. J. J. Chem. Educ. 2004, 81, 718–721. 12. Stynes, H. C.; Layo, A.; Smith, R. W. J. Chem. Educ. 2004, 81, 266–269. 13. Reimann, C. T.; Mie, A.; Nilsson, C.; Cohen, A. J. Chem. Educ. 2005, 82, 1215–1218. 14. Arnquist, I. J.; Beussman, D. J. J. Chem. Educ. 2007, 84, 1971–1973. 15. Weinecke, A.; Ryzhov, V. J. Chem. Educ. 2005, 82, 99–102. 16. Yergey, J.; Heller, D.; Hansen, G.; Cotter, R. J.; Fenselau, C. Anal. Chem. 1983, 55, 353–356. 17. Dopke, N. C.; Treichel, P. M.; Vestling, M. M. J. Chem. Educ. 2000, 77, 1065–1069. 18. Watson, J. T.; Sparkman, O. D. Introduction to Mass Spectrometry, 4th ed.; John Wiley: Chichester, U.K., 2007; pp 273–278. 19. For a PC freeware software program, see, for example, Molecular Weight Calculator at http://ncrr.pnl.gov/software/; for a Mac freeware software program, see http://home.datacomm.ch/marvin/ iMass/; for Web-based calculators, see, for example, http://www. cem.msu.edu/~reusch/OrgPage/mass.htm or http://www.ch.cam. ac.uk/magnus/MolWeight.html or http://www.geocities.com/junhuayan/pattern.htm (all accessed Oct 2008).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Mar/abs382.html Abstract and keywords Full text (PDF) Links to cited URLs and JCE articles Supplement Detailed instructor notes, instructions for students, copies of seven peptide spectra, including expanded isotope regions
Journal of Chemical Education • Vol. 86 No. 3 March 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
