Examination and Manipulation of Protein Surface Charge in Solution

Laboratory Experiment pubs.acs.org/jchemeduc

Examination and Manipulation of Protein Surface Charge in Solution with Electrospray Ionization Mass Spectrometry Deborah S. Gross*,† and Hal Van Ryswyk‡ †

Department of Chemistry, Carleton College, Northfield, Minnesota 55057, United States Department of Chemistry, Harvey Mudd College, Claremont, California 91711, United States

‡

S Supporting Information *

ABSTRACT: Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for examining the charge of proteins in solution. The charge can be manipulated through choice of solvent and pH. Furthermore, solution-accessible, protonated lysine side chains can be specifically tagged with 18-crown-6 ether to form noncovalent adducts. Chemical derivatization of a protein in this fashion followed by “soft” ionization can provide a wealth of specific information regarding side chain identity, location, and surface chemistry. A variety of proteins are examined with ESI-MS under a range of conditions in this self-guided experiment, and the results are compared with those predicted from structural information available in the protein data bank. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Biochemistry, Laboratory Instruction, Inquiry-Based/Discovery Learning, Hands-On Learning/Manipulatives, Instrumental Methods, Mass Spectrometry, Proteins/Peptides “Soft” ionization techniques for mass spectrometry (MS) have revolutionized the analysis of large, nonvolatile biomolecules. Electrospray ionization (ESI) allows one to transfer large molecules from solution into the gas phase as ions for mass spectral analysis with minimal (or no) fragmentation. There is a wide range of introductory papers and experiments exploiting ESI and allied soft ionization techniques with biomolecules to determine protein molecular weight,1−8 protein sequence,9,10 and the composition of mixtures.11,12 A recent pair of experiments extend ESI-MS to probe the solution-phase chemistry of biomolecules.13,14 Noncovalent protein/adduct complexes formed in solution can, under proper instrumental conditions, remain intact as a protein is transferred to the gas phase using ESI. Chemical derivatization of a protein followed by “soft” ionization can provide a wealth of specific information regarding side chain identity, location, and surface chemistry.15 There is a detailed research literature of such experiments, many of which, such as the particularly powerful method of proton/deuteron exchange, are used to probe the solvent-accessible surface of proteins to obtain solution-phase structural information through mass spectrometry experiments.16 Ly and Julian recently developed a related technique to probe solvent-accessible protein surface sites, which they christened selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS).17 In the original version of SNAPP, 18-crown-6 ether (18C6, 264 Da) was added to protein or peptide solutions, and it complexed specifically with protonated lysines on the solvent-accessible surface of the © 2014 American Chemical Society and Division of Chemical Education, Inc.

protein; the 18C6 cavity was too small to incorporate protonated arginines present. This specific 18C6/Lys + interaction allowed the solution structure of each charge state of the protein to be probed independently through observation of the unique pattern of multiple 18C6s that attached to the various charge states of the protein. Few experiments in the chemical education literature illustrate the power of ESI to probe solution-phase protein secondary and/or tertiary structure. This experiment has been used as an opportunity for students to read a recent literature paper deeply, discuss experimental goals, design their own experiment to explore the behavior of an unknown protein, use the research paper as a template for their data analysis, and, finally, compare their results with those expected from published Protein Data Bank (PDB) structures. The specific goals of this experiment were to have upper-level students (1) use ESI-MS to determine the molecular weight of a protein and (2) explore the solution structure of a protein as a function of solvent conditions directly and through SNAPP-MS, such that the number of lysines on the protein surface can be probed and compared to the number in the whole protein, as well as the number on the surface as determined by the protein’s crystal structure. These goals were achieved in a self-guided, 4-h experiment that used a recent research paper17 provided to students in advance of the laboratory as an experimental guide. Published: July 15, 2014 1240

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Laboratory Experiment

EXPERIMENT OVERVIEW

This experiment was performed by students working individually or in pairs in three different upper-level electives at Carleton and Harvey Mudd Colleges, including Bioanalytical Chemistry (Carleton, one offering, 8 students), Spectroscopic Characterization of Chemical Compounds (Carleton, 3 offerings, 58 students), and Instrumental Analysis Laboratory (Harvey Mudd, 3 offerings, 24 students). Three ESI-MS instruments utilizing ion trap mass analyzers were employed successfully, but any ESI-equipped mass spectrometer that allows sample to be directly infused through a syringe pump can be used, although the MSn extension requires an instrument capable of these experiments. Students prepare solutions of an unknown protein (ubiquitin, myoglobin, or cytochrome c) in three solvents, both with and without 18C6 added. The six samples are infused into an ESI mass spectrometer. Students calculate the molecular weight of the protein, observe trends in protein solution structure based upon solvent and complexing agent, and correlate these trends to the number of lysines in the primary sequence and on the protein surface as determined by crystal structure. These results are compared with those obtained by students working with another protein, then presented as a laboratory report in the style of a journal article or oral presentation.

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HAZARDS 18-Crown-6 ether is harmful if swallowed and acts as an irritant to the eyes, respiratory system, and skin. There are no hazards associated with the proteins discussed in this article. Certain commonly electrosprayed peptides can be bioactive, and instructors should consider them more carefully before use (e.g., melittin, which is extracted from honeybee venom). Goggles, protective clothing, and gloves should be worn at all times during sample preparation steps.

Figure 1. Typical student results of relative intensity as a function of mass-to-charge ratio (m/z) for ubiquitin electrosprayed from three different solvents: (A) water, (B) 50:50 water:methanol, and (C) 49.5:49.5:1 water:methanol:acetic acid. The labels denote the charge state (z) associated with each peak. Asterisks indicate peaks due to chemical interferences that are ignored in the students’ analysis.

RESULTS The charge-state distribution of most proteins, as observed in ESI mass spectra, is sensitive to solvent. A typical student ESI mass spectrum of ubiquitin in water is shown in Figure 1A. Six separate charge states, (M + 5H)5+ through (M + 10H)10+, were observed. These charges corresponded to protonation of the surface-accessible arginines and/or lysines in the native crystal structure. As expected, addition of methanol (Figure 1B) had a very small effect on ubiquitin’s solution structure, as can be seen by the increased relative abundance of the 8+ and 9+ charge states, although it induced larger structural changes in some other proteins, as discussed in the instructor’s notes. This was consistent with the stability of the ubiquitin structure and with Ly and Julian’s results indicating that addition of methanol induced a portion of the protein to adopt a slightly more open structure.17 As a result of the contribution of this more open structure, the number of solvent-accessible charges was larger with methanol present. Acid is often used to denature proteins;18 addition of sufficient acetic acid to decrease the solution pH to ∼2 led to denaturation of ubiquitin. As a result, more basic residues were accessible to this low pH solvent, leading to a higher average charge on the gas-phase ions observed from the denatured protein (Figure 1C).19,20 The average molecular weight of the protein was calculated using any or all of these spectra.

Examination of the crystal structure of ubiquitin (PDB ID = 1UBQ) showed that all seven of the lysine residues present are on the outer surface of the protein, typical for residues that are charged. Addition of 18C6 to the solution formed a range of adducts between 18C6 and protonated lysine side chains (Figure 2) wherein the observed mass-to-charge ratio of the ion (m/z) increased by 264/z (the mass of neutral 18C6 divided by the charge of the ion complex) for each 18C6 coordinated. The number of adducts was charge-state-dependent (Figure 3). Generally, as more sites from the inside of the protein are available for coordination−either because they are accessible due to denaturation of the native protein structure or because they are charged−the total degree of coordination increases. This was especially notable in the 13+ charge state, which was visible only in the solution containing acetic acid, and in which the highest intensity peak was coordinated with three 18C6 molecules (13-3). In other charge states and other solutions, the highest intensity peak in any distribution corresponded to zero, one, or two 18C6 molecules attached. The uses of tandem MS (MS/MS or MSn) to probe the relative stability of ions and “supercharging” reagents to enhance protein charging were undertaken as optional extensions of these experiments in a second 4 h laboratory period. Typical student results are shown in the instructor’s notes.

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Figure 2. Typical student results for ubiquitin electrosprayed in 49.5:49.5:1 water:methanol:acetic acid with 18-crown-6. Selected peaks are labeled with charge (z) and number of 18-crown-6 adducts (n) as (z-n).

Figure 3. Typical student results for 18-crown-6 adduct distribution of ubiquitin at four different charge states, in each solvent system (black bars are water, gray bars are 50:50 water:methanol, and white bars are 49.5:49.5:1 water:methanol:acetic acid). Each peak is normalized to the base peak in its respective distribution. The x-axis labels indicate the charge state (z) and the number of 18-crown-6 adducts (n) as (z-n). The 13+ charge state was only observed in the 49.5:49.5:1 water:methanol:acetic acid system.

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ASSESSMENT Students made 12 min oral presentations of their results or wrote lab reports in the format of a journal article that were assessed by the instructor with a rubric. Students exhibited (i) uniform success in the ability to deconvolute ESI mass spectra and obtain a molecular weight for a protein; (ii) a high ability to differentiate between the total number of lysines in the protein, the number on the surface of the protein as illustrated by the crystal structure, and the number of solvent-accessible basic

sites as controlled by solvent conditions; and (iii) a satisfactory ability to discuss how solution-phase chemistry could be examined by ESI-MS in the gas phase. In exit interviews at the end of the courses, students indicated an appreciation for the open-ended nature of the experiment and a general sense of accomplishment on being able to read a research paper and translate its procedure and findings into an experiment of their own design. 1242

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(8) Weinecke, A.; Ryzhov, V. Fundamentals of Biomolecule Analysis by Electrospray Ionization Mass Spectrometry. An Instrumental Analysis Laboratory Experiment. J. Chem. Educ. 2005, 82 (1), 99−102. (9) Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 3: De Novo Peptide Sequencing Using Electrospray Tandem Mass Spectrometry. J. Chem. Educ. 2009, 86 (8), 966−968. (10) Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. Electrospray Ionization Mass Spectrometry: Part II: Applications in Characterization of Peptides and Proteins. J. Chem. Educ. 1996, 73 (6), A118− A123. (11) Eibisch, M.; Fuchs, B.; Schiller, J.; Suess, R.; Teuber, K. Analysis of Phospholipid Mixtures from Biological Tissues by Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS): A Laboratory Experiment. J. Chem. Educ. 2011, 88 (4), 503−507. (12) Stynes, H. C.; Layo, A.; Smith, R. W. LC-MS of Metmyoglobin at pH = 2. Separation and Characterization of Apomyoglobin and Heme by ESI-MS and UV-Vis. J. Chem. Educ. 2004, 81 (2), 266−269. (13) Kim, D. H.; Eckhert, C. D.; Faull, K. Utilization of Negative Ion ESI-MS and Tandem Mass Spectrometry To Detect and Confirm the NADH−Boric Acid Complex. F. J. Chem. Educ. 2010, 88 (1), 106− 110. (14) Ryzhov, V.; Sunderlin, L. S.; Keller, L. M. M.; Gaillard, E. R. Measuring Gas-Phase Basicities of Amino Acids Using an Ion Trap Mass Spectrometer. A Physical Chemistry Laboratory Experiment. J. Chem. Educ. 2005, 82 (7), 1071−1073. (15) Zaikin, V. G.; Halket, J. M. Derivatization in Mass Spectrometry–8. Soft Ionization Mass Spectrometry of Small Molecules. Eur. J. Mass Spectrom. 2006, 12 (2), 79−115. (16) Konermann, L.; Tong, X.; Pan, Y. Protein Structure and Dynamics Studied by Mass Spectrometry: H/D Exchange, Hydroxyl Radical Labeling, and Related Approaches. J. Mass. Spectrom. 2008, 43 (8), 1021−1036. (17) Ly, T.; Julian, R. R. Using ESI-MS to Probe Protein Structure by Site-Specific Noncovalent Attachment of 18-Crown-6. J. Am. Soc. Mass Spectrom. 2006, 17 (9), 1209−1215. (18) Dill, K. A.; Shortle, D. Denatured States of Proteins. Annu. Rev. Biochem. 1991, 60, 795−825. (19) Iavarone, A. T.; Jurchen, J. C.; Williams, E. R. Effects of Solvent on the Maximum Charge State and Charge State Distribution of Protein Ions Produced by Electrospray Ionization. J. Am. Soc. Mass Spectrom. 2000, 11 (11), 976−985. (20) Schnier, P. D.; Gross, D. S.; Williams, E. R. On the Maximum Charge State and Proton Transfer Reactivity of Peptide and Protein Ions Formed by Electrospray Ionization. J. Am. Soc. Mass Spectrom. 1995, 6 (11), 1086−1097.

CONCLUSION ESI-MS effectively probes the solution charge state of proteins. Protein charge states can be manipulated by changing the solvent, the pH, and by the addition of reagents that can form noncovalent adducts with solvent-accessible residues. This experiment allowed students to investigate the impact of protein tertiary structure on complexation, with changes in protein tertiary structure having a dominant effect on the charge state distribution and changes in the secondary structure influencing the number of 18C6 adducts observed for a given charge state. Students also learned about the important technique of electrospray ionization mass spectrometry while having an opportunity to use a model from the chemical literature as the basis of their experimental design and approach to data analysis.

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ASSOCIATED CONTENT

S Supporting Information *

Student handout and instructor notes with a description of two extensions. This material is available via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support by the National Science Foundation (awards 0077540 and 0922481). The students in Chemistry 306 and 335 at Carleton College and Chemistry 112 at Harvey Mudd College are gratefully acknowledged for their participation in the development of these experiments. Data shown in Figures 1−3 were obtained by Ross Hamilton and Jing Jing Ling, students in the winter 2010 offering of Chemistry 306 at Carleton College.

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REFERENCES

(1) Vestling, M. M. Using Mass Spectrometry for Proteins. J. Chem. Educ. 2003, 80 (2), 122−124. (2) Hofstadler, S. A.; Bakhtiar, R.; Smith, R. D. Electrospray Ionization Mass Spectroscopy: Part I. Instrumentation and Spectral Interpretation. J. Chem. Educ. 1996, 73 (4), A82−A88. (3) Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A.; Smith, R. D. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Instrumentation and Applications. J. Chem. Educ. 1997, 74 (11), 1288−1292. (4) Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry Into Undergraduate Teaching Labs, Part 1: Identifying Proteins Based on Molecular Mass. J. Chem. Educ. 2007, 84 (12), 1971−1973. (5) Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 2: Peptide Identification via Molecular Mass Determination. J. Chem. Educ. 2009, 86 (3), 382−384. (6) Cohen, A.; Reimann, C. T.; Mie, A.; Nilsson, C. Introduction to Biological Mass Spectrometry: Determining Identity and Species of Origin of Two Proteins. J. Chem. Educ. 2005, 82 (8), 1215−1218. (7) Dopke, N. C.; Lovett, T. N. Illustrating the Concepts of Isotopes and Mass Spectrometry in Introductory Courses: A MALDI-TOF Mass Spectrometry Laboratory Experiment. J. Chem. Educ. 2007, 84 (12), 1968−1970. 1243

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