In the Laboratory
Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 1: Identifying Proteins Based on Molecular Mass
W
Isaac J. Arnquist and Douglas J. Beussman Department of Chemistry, St. Olaf College, Northfield, MN 55057; *
[email protected] Proteomics, the analysis of all proteins in an organism at a given point in time, is a major area of research, both in academic and industrial settings. Students need to learn about this important field to be better prepared to enter the workforce or undertake advanced research at the biology–chemistry interface. Mass spectrometry (MS) is one of the most widely used analytical techniques in proteomics, as well as other fields. Electrospray ionization (ESI) (1, 2) and matrix-assisted laser desorption ionization (MALDI) (3–5) allow large biological molecules to be ionized and analyzed by mass spectrometers. ESI–MS, often coupled with liquid chromatography (LC– MS), can be used to analyze a wide variety of molecules, including oligonucleotides, lipids, small organic molecules, peptides, and proteins. Although it is a crucial research tool, LC–MS has yet to be routinely incorporated into the undergraduate curriculum. A search of publications in this Journal dealing with LC–MS techniques provided only nine 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 three discuss the lab analysis of biological molecules (12–14). Of these three lab experiments, one presents the analysis of several peptides and small proteins (12), one presents a method for the identification of two proteins based on in-solution enzymatic digestion (13), and one characterizes metmyoglobin (14). We have created several experiments for undergraduates that demonstrate how ESI–MS can be applied to the analysis of peptides and proteins.1 These experiments are part of a bioanalytical chemistry course developed at this college, but they could be used in a variety of lab courses, including biochemistry or instrumental analysis courses. In the lab experiment presented here, we discuss the analysis of several medium-sized proteins by ESI–MS, focusing on the analysis of highly charged ions. Students are given an unknown protein and must complete the analysis and identify their sample from a list of 17 potential proteins. Background Since ESI often produces multiply charged ions, the correct charge state of an ion signal must be determined before an accurate molecular mass can be assigned to the analyte. For biological molecules in general, the larger the analyte molecule, the more highly charged the resulting ions often are. For small charge states, the charge on the ion can be determined by observing the isotopic distribution, but this becomes more difficult for more highly charged ions since the mass-to-charge ratio (m∙z) spacing between isotopes becomes smaller as the charge state increases. In general, for most current benchtop mass spectrom
eters, charge states above 4 cannot be determined by looking at the isotope patterns, but instead a mathematical treatment of adjacent charge state ion signals must be used to assign the correct number of charges and thus allow the correct molecular mass to be calculated. Since individual isotopes cannot be distinguished for these large biomolecules, monoisotopic molecular masses are not calculated but rather average molecular masses are obtained, corresponding to the sum of the average atomic masses for all of the atoms in the analyte molecule. A discussion of isotopic effects in analyzing large biomolecules has previously been published (15). Molecular mass of a biomolecule is often a crucial piece of data. Several methods exist for obtaining molecular mass information, including size-exclusion chromatography, gel electrophoresis, light scattering spectroscopy, and mass spectrometry. Of these, mass spectrometry provides the most accurate value, but also requires the most sophisticated and expensive instrument. We allow our students to obtain molecular mass information for a protein by several of these methods, comparing ease of use, accuracy of data, and overall cost of analysis; however, a detailed description of these comparisons is beyond the scope of this article. Experimental Procedure Mass spectra were obtained using an ESI Bruker Daltonics esquire 3000+ ion-trap mass spectrometer and accompanying software, although any ESI-equipped mass spectrometer should be capable of providing comparable spectra. All peptide samples were purchased from Sigma and were either stored in the freezer (‒20 °C) or refrigerated (0 °C), following given guidelines. For each protein, a 100 pmol∙μL lab solution was prepared from stock using nanopure (18 MΩ) water with 0.1% formic acid. The solutions were stored in 1.5 mL microcentrifuge tubes under the same conditions cited for the stock samples. Stock solutions were stored for up to three years in the freezer (‒20 °C), although longer-term storage could be accomplished by preparing the stock solutions, dividing them into appropriate aliquots, lyophilizing them, and storing the aliquots in the freezer. This would also limit the degree of hydrolysis that can occur with proteins over time. Injection of the 100 pmol∙μL 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 positive-ion mode with the capillary voltage set at ‒4500 V and an end plate offset of ‒500 V. Source parameters were initially set with a N2 nebulizing gas pressure of 10 psi, a N2 dry gas flow rate of 4.0 L/min, and a drying temperature of 250 °C. These initial parameters yielded
www.JCE.DivCHED.org • Vol. 84 No. 12 December 2007 • Journal of Chemical Education 1971
In the Laboratory
signals for all proteins, although students were allowed to optimize the source parameters for each individual sample. The instrument was set to scan from 50 to 3000 m∙z. Spectra were saved and analyzed using Bruker DataAnalysis software. The needle, end plate, injector tube, and syringe were cleaned between runs using acetonitrile followed by isopro panol.
tein, students were given a list of 17 possible proteins to choose from, which included the five proteins actually used in the lab, as shown in Table 1. The amino acid sequence of each protein is given in the Supplemental Material.W Instructors could choose to provide these sequences instead of the values in Table 1 and have students calculate the molecular mass of each protein if time permits. Since mass spectrometers measure the m∙z, to correctly determine the molecular mass of a species using mass spectrometry, the charge state of the ion signal must be known. Most ionization techniques create primarily singly charged ions, but the ESI process routinely creates multiply charged ions. In general, the larger the molecular mass of the analyte, the more charges it will obtain. While this fact allows large molecular mass molecules to be analyzed using relatively inexpensive mass spectrometers that have upper m∙z ranges of a few thousand Daltons, it also makes the determination of the molecular mass more difficult, since the correct charge state must first be determined. Once the charge state of an ion signal is known, along with the observed m∙z, the correct molecular mass of the analyte can easily be calculated. The isotopic pattern for small peptides can usually be observed to determine the correct charge state, based on carbon isotope separation. For ions with more than three or four charges, which is generally observed for large analytes including proteins, the isotopic patterns cannot be distinguished without using expensive high-resolution mass spectrometers. Instead, the average m∙z is observed, which does not directly indicate the charge state of the ion. In positive-ion ESI–MS, analyte molecules acquire one or more positive charges by the addition of protons (H+). This not only adds a charge to the analyte (A), but also adds a mass of approximately 1 to the analyte, yielding a charged species (A + H)+ that can be analyzed by the mass spectrometer. Multiply charged ions that may form during the ionization process are due to the addition of multiple protons to the analyte (A + nH)+n, where n is the number of protons added and thus also the charge state on the resulting ion. If two adjacent peaks are analyzed, then the higher m∙z peak (m2) is due to the (A + nH)+n ion, and the lower m/z peak (m1) is due to the (A + {n + 1}H)+(n+1) ion. The charge (n) on m2 can be determined by
Hazards
Table 1. List of Possible Proteins and Their Corresponding Average Molecular Mass Protein
Average Molecular Mass/Da
β-Galactosidase, E. coli
116,352
Phosphorylase b, rabbit
97,158
Protein Kinase C alpha, human
76,633
SGK2, human
47,604
Enolase, baker’s yeast
46,802
Chymotrypsinogen A, bovine
25,666
Trypsin inhibitor B, soybean
20,041
Apomyoglobin, horse
16,951
Lysozyme, chicken
14,313
α-Lactalbumin, bovine
14,186
Ribonuclease A, bovine
13,742
Cytochrome c, horse
12,360
Thioredoxin1, E. coli
11,675
Elastin, sheep
8,662
Ubiquitin, bovine
8,565
Aprotinin, bovine
6,511
Metallothionein 1A, human
6,133
Note: Proteins analyzed in the lab are shown in bold.
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 hot gas or hot metal source components may exist. Skin contact with the proteins and solvents should be avoided by wearing gloves. Goggles should be worn when working with the samples, which should be prepared in a well-ventilated area such as a hood. Results and Discussion Five proteins (apomyoglobin, aprotinin, cytochrome c, lysozyme, and ubiquitin), ranging in molecular mass from 6.5 to 16.9 kDa, were analyzed. The proteins and their corresponding molecular masses are listed in Table 1. The spectra for all five species were obtained using the protocol given in the experimental section and are available in the Supplemental Material.W,2 When attempting to identify their unknown pro-
n =
m1 − 1. 008 m 2 − m1
(1)
Therefore, the molecular mass (M) of the analyte can be determined by (2) M = n m 2 − n 1. 008 or (3) M = n + 1 m1 − n + 1 1.008 Either peak (m1 or m2) can be used to calculate the molecular mass of the analyte, but averaging the calculated molecular masses from several of the multiply charged ion signals, assuming they have good signal-to-noise ratios, may be more useful. If several signals are used, a standard deviation of the calculated molecular mass can be obtained, which can provide some measure of error in the calculated average value. The mass spectrum for cytochrome c is shown in Figure 1. Looking at the m∙z 1030.88 peak (m1) and the m∙z 1124.47 peak (m2) and using eq 1, the charge on m2 is 11, corresponding
1972 Journal of Chemical Education • Vol. 84 No. 12 December 2007 • www.JCE.DivCHED.org
Intensity / 10 5
In the Laboratory
1030.88
2.0 1.5
0.0
1236.84 1374.05
883.81 824.79 800
900
1000
1100
1200
1300
1400
1500
1600
m/z Figure 1. Mass spectrum of cytochrome c (molecular mass 12,360 Da) showing charge states +9 to +15.
to a molecular mass for cytochrome c of 12,358 Da, with the m1 peak also indicating a molecular mass of 12,358 Da. The average molecular mass calculated from all seven ion signals observed in Figure 1 is 12,358 Da, which is a mass error of ~0.02% compared to the actual molecular mass of 12,360 Da. With proper calibration, mass errors of less than 1% are common for larger proteins, even if relatively inexpensive mass spectrometers are used for the analysis. Conclusion We have described a straightforward lab designed to determine the molecular mass of medium-sized proteins 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. Since isotope distributions are often not resolved for highly charged large molecules such as proteins, the spacing between adjacent ion signals, separated by a single charge, can be used to determine the charge state and thus the correct molecular mass of the protein by a simple set of equations. We have presented five proteins that can be given as unknowns to students to analyze, determine the charge state and correct molecular mass, and identify based on a list of possible proteins if desired. Spectra for all five proteins analyzed are presented in the Supplemental Material.W Acknowledgments 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 IJA) from an institutional grant awarded to St. Olaf College by the Howard Hughes Medical Institute.
Material
Detailed instructor notes, instructions for students, copies of all five protein spectra suitable for using in classroom exercises for those without direct student access to a mass spectrometer, and the amino acid sequences of all proteins listed in Table 1 are available in JCE Online.
951.68
1.0 0.5
WSupplemental
1124.47
Notes 1. This is the first part of a four-part series of lab experiments that we have developed to teach students how peptides and proteins can be analyzed. The next lab examines the molecular mass determination of peptides by observing the isotopic distributions using ESI–MS. The final two labs will describe the sequencing of peptides and the analysis and identification of proteins separated by gel electrophoresis. 2. The spectra of the five proteins can be used in a classroom or as previously collected lab data if direct access to an ESI–MS is not available.
Literature Cited 1. Yamashita, Masamichi; Fenn, John B. J. Phys. Chem. 1984, 88, 4451–4459. 2. Fenn, John B.; Mann, Matthias; Meng, Chin K.; Wong, Shek F.; Whitehouse, Craig M. Science 1989, 246, 64–71. 3. Karas, Michael; Buchman, Doris; Hillenkamp, Franz. Anal. Chem. 1985, 57, 2935–2939. 4. Tanaka, Koichi; Waki, Hiroaki; Ido, Yutaka; Akita, Satoshi; Yoshida, Yoshikazu; Yohida, Tamio. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. 5. Karas, Michael; Hillenkamp, Franz. Anal. Chem. 1988, 60, 2299–2301. 6. Vestling, Martha M. J. Chem. Educ. 2003, 80, 122–125. 7. Hofstadler, Steven A.; Bakhtiar, Ray; Smith, Richard D. J. Chem. Educ. 1996, 73, A82–A88. 8. Bakhtiar, Ray; Hofstadler, Steven A.; Smith, Richard D. J. Chem. Educ. 1996, 73, A118–A123. 9. Hop, Cornelis E. C. A.; Bakhtiar, Ray. J. Chem. Educ. 1996, 73, A162, A164–A169. 10. Bergen, Robert H., III; Benson, Linda M.; Naylor, Stephen. J. Chem. Educ. 2000, 77, 1325–1326. 11. Coe, Benjamin J. J. Chem. Educ. 2004, 81, 718–721. 12. Weinecke, Andrea; Ryzhov, Victor J. Chem. Educ. 2005, 82, 99–102. 13. Reimann, Curt T.; Mie, Axel; Nilsson, Carina; Cohen, Arieh. J. Chem. Educ. 2005, 82, 1215–1218. 14. Stynes, Helen C.; Layo, Araceli; Smith, Richard W. J. Chem. Educ. 2004, 81, 266–269. 15. Dopke, Nancy C.; Treichel, Paul M.; Vestling, Martha M. J. Chem. Educ. 2000, 77, 1065–1069.
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