Laboratory Experiment pubs.acs.org/jchemeduc
Protein Quantification by Elemental Mass Spectrometry: An Experiment for Graduate Students Gunnar Schwarz,†,§ Stefanie Ickert,† Nina Wegner,† Andreas Nehring,† Sebastian Beck,† Ruediger Tiemann,† and Michael W. Linscheid*,† †
Department of Chemistry, Humboldt-Universitaet zu Berlin, 12489 Berlin, Germany S Supporting Information *
ABSTRACT: A multiday laboratory experiment was designed to integrate inductively coupled plasma−mass spectrometry (ICP−MS) in the context of protein quantification into an advanced practical course in analytical and environmental chemistry. Graduate students were familiar with the analytical methods employed, whereas the combination of bioanalytical assays with ICP−MS is rare. Small groups of graduate students quantified ovalbumin in hen egg white using metal-coded affinity tagging (MeCAT). Proteins were covalently labeled with lanthanide chelate complexes and quantified according to the lanthanide content by ICP−MS using internal and external standards. The results were in good agreement with reference values. As an alternative approach, a Bradford assay was used for determination of the ovalbumin content of the internal standard. The chosen workflow provides hands-on experiences for the students in principles of analytical chemistry, quantitative protein analyses, gel electrophoresis, ICP−MS, calibration, and data handling. The experiment constitutes a research-oriented approach as students apply their knowledge and skills in new contexts. KEYWORDS: Graduate Education/Research, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Mass Spectrometry, Proteins/Peptides, Quantitative Analysis, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Environmental Chemistry
■
INTRODUCTION Protein quantification plays an important role in biological and medical research. There are several methods ranging from simple UV-absorption1 and densitometric analyses of gels subsequent to electrophoretic separation of proteins2 to mass spectrometry (MS)-based methods.3 Inductively coupled plasma mass spectrometry (ICP−MS)4 as a typical detector for metals is not common for protein quantification, as most proteins contain only elements such as carbon, hydrogen, oxygen, nitrogen, and sulfur. These elements provide rather poor sensitivity and high background in elemental MS. Chemical protein labeling using metal complexes is currently under investigation in order to quantify proteins according to the metal amount via ICP−MS.5−7 Potential advantages of this approach are high sensitivity, a large dynamic measuring range and low limits of detection.7 One promising method is metalcoded affinity tagging (MeCAT).8 MeCAT uses 1,4,7,10tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and its lanthanide chelate complexes (Figure 1A) that are covalently bound via a reactive iodoacetamide (IA) group to proteins (Figure 1B). ICP−MS is used to quantify the labeled protein by the amount of metal in the sample (Figure 1C). It appears to be useful to support theoretical principles by practical exercises and thereby involve students in current research.9−13 Students get to know ICP-MS mainly as an © 2014 American Chemical Society and Division of Chemical Education, Inc.
advanced instrument with outstanding properties for elemental quantitative analyses. A new experiment was developed for advanced analytical and environmental chemistry students, in order to include ICP-MS in our lab course to demonstrate the importance of ICP-MS for biosciences. In this experiment, quantitative analysis of proteins using MeCAT was introduced along with the processing of bioanalytical samples and protein separation by gel electrophoresis. This enabled hands-on experience in this new approach merged with the application of generic principles and methods in analytical and bioanalytical chemistry. The experiment was performed by 27 students during the winter term 2012−2013.
■
EXPERIMENTAL OVERVIEW The analytical aim of this experiment was to quantify ovalbumin in hen egg white. Commercially available ovalbumin served as an internal standard. The thiol groups of cysteine residues in proteins were labeled using a lanthanide chelate complex. The protein content was determined from the lanthanide amount in ICP−MS analyses (Figures 1 and 2) via external calibration using standard salt solutions or via the internal ovalbumin standard, which was labeled using a different lanthanide. Furthermore, the protein content of the ovalbumin Published: October 10, 2014 2167
dx.doi.org/10.1021/ed500339c | J. Chem. Educ. 2014, 91, 2167−2170
Journal of Chemical Education
Laboratory Experiment
discussed advantages and drawbacks with students. Furthermore, the wider context of proteomics in research was also discussed. During the lab work, one instructor was with the students at all times to guide and assist the experimental procedure and discuss the methods and alternatives. Due to organizational reasons, the experiment was split into three parts of 3 to 6 h that took place in the afternoon of three consecutive days and consumed about 13 hours in total, including some breaks (see Supporting Information for details).
■
MATERIALS AND INSTRUMENTATION The reagent based on DOTA harboring a trivalent lanthanide, which was linked to an iodoacetamide group for protein reactivity14 (MeCAT-IA, Figure 1A) was provided. Details of the synthesis of the MeCAT-IA reagent were published earlier.15 Seven different sets (one set per supplier) of chicken eggs were purchased at local supermarkets or provided from local poultry keepers. From every set of eggs, two egg whites were combined, lyophilized, and homogenized with a mortar and pestle prior to an experiment. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and ICP−MS were carried out. Further details can be found in the Supporting Information.
Figure 1. (A) Structure of MeCAT-IA. The chelate complex harbors a lanthanide ion, indicated as Ln3+. (B) Reaction of MeCAT-IA with proteins or peptides. Thiol groups react with MeCAT-IA by elimination of hydrogen iodide and formation of a thioether. (C) Principle of quantification. After purification, the labeled protein is quantified by ICP-MS. The amount of protein is determined according to the lanthanide amount in the sample.
■
EXPERIMENTAL PROCEDURE Students worked in groups of two. After dissolving and reducing the proteins in the samples (Figure 2), proteins in reduced lyophilized hen egg white were labeled in aqueous buffer solution using MeCAT-IA reagents. The ovalbumin standard was labeled separately using a different lanthanide within the reagent. Both labeled hen egg white and ovalbumin standard were then mixed and submitted to gel electrophoresis. The protein bands of labeled ovalbumin were excised from the gel and dissolved in nitric acid. The resulting solutions were analyzed by ICP-MS and the ovalbumin content of the hen egg white was determined by the lanthanide content in the sample using the internal standard. Furthermore, the ovalbumin content was determined according to external calibration using lanthanide standard solutions. A general workflow is
standard was characterized by a Bradford assay as an example of a simple alternative method.1 Although students were familiar with the basic analytical concepts and methods, a hand-out introduced MeCAT in particular. To ensure that students are familiar with the basic principles and procedures, it was expected that they prepared themselves primarily via self-study for this experiment. The status of the preparation was verified during a dialogue with the instructor at the beginning of the experiment. The instructors emphasized established methods for protein quantification, such as stable isotope labeling with amino acids (SILAC), and
Figure 2. Workflow for quantification of ovalbumin in hen egg whites using MeCAT. After separate reduction and differential labeling, using different lanthanide ions within the labeling reagent, the standard and sample were combined and proteins were separated by gel electrophoresis. Redissolved residues of mineralized gel bands were analyzed by ICP-MS. The amount of ovalbumin within the hen egg white was then determined by external calibration using standard salt solutions of lanthanides or by comparison to the internal standard. 2168
dx.doi.org/10.1021/ed500339c | J. Chem. Educ. 2014, 91, 2167−2170
Journal of Chemical Education
Laboratory Experiment
displayed in Figure 2. The Bradford assay followed a standard protocol.1 Every student group processed and analyzed two or three lyophilized samples and delivered a written report, which contained a general introduction to the field as well as a description of the concept of MeCAT and general experimental procedures during the experiment. Furthermore, students gave a detailed discussion of the experimental data.
■
HAZARDS Safety goggles and lab coats should always be worn when working in the laboratory. Components of hen egg white, N,N,N′,N′-tetramethylethylenediamine (TEMED), acetone, and a solution of acrylamide/bis(acrylamide), tris(2-carboxyethyl) phosphine, sodium dodecyl sulfate and Coomassie Brilliant Blue R are considered to be irritants. TEMED and solutions of acrylamide/bis(acrylamide), sodium tetraborate, Coomassie Brilliant Blue R and hydrogen peroxide are considered toxic. Nitric acid and hydrogen peroxide solution are highly corrosive. Acetone is extremely flammable. Although the dangers of DOTA and its lanthanide complexes have not been evaluated so far, MeCAT reagent solutions were considered to be harmful and irritant. The use of laboratory gloves is mandatory when working with all substances given above. To avoid injuries, special caution is required when excising pieces of gels using scalpels.
Figure 3. Coomassie-stained SDS-PAGE of three aliquots of mixtures of labeled hen egg white (labeled with lutetium-MeCAT-IA) and ovalbumin standard (labeled with holmium-MeCAT-IA). Left lane: protein molecular weight marker.
Two students per group were found to be best considering the workload for the students, and more than three was inadequate. Most of the students recommended continuation of this experiment in the following years. According to their comments, students especially appreciated gaining experiences with the gel electrophoresis and ICP-MS. Students mainly reported “good” to “great” gains in techniques, manual, and practical skills (see Supporting Information for details). The discussions with students during the experiment provided fruitful opportunities to reflect on further research questions and problems, such as the requirements for protein quantification in general and for MeCAT in particular.7
■
RESULTS AND DISCUSSION A general characterization of the protein content of the ovalbumin standard was carried out using a Bradford assay. Bradford assays performed by students determined a protein amount of the ovalbumin standard of 53% with a standard deviation of ±5% (N = 11), despite the suppliers statement of a content of >98% based on agarose electrophoresis. However, this can be interpreted in a way that >98% of the total protein content is ovalbumin, while not considering nonprotein contents. The results from the Bradford assay not only demonstrated the importance of a well-characterized standard in quantitative analyses to students but also encouraged their critical thinking toward validity of analytical results and their declaration. It has to be stated that the supplier should not sell this protein as “standard” without a detailed declaration. Hen egg white was labeled with lutetium-MeCAT-IA and ovalbumin standard was labeled with holmium-MeCAT-IA. The samples were combined and separated by electrophoresis (Figure 3). Using MeCAT, the ovalbumin content determined by students of the lyophilized egg white samples ranged from 29.8% to 77.3% (Table S1, Supporting Information) with no significant differences between external or internal calibration. However, most of the values were lower than contents found in the literature.16,17 Results from student analyses that were checked by the instructors are displayed in Table 1 and Table S1 (Supporting Information). Considering the biological variance, possible inhomogeneity of the samples, various sample preparation steps, and a general unfamiliarity of students with the procedures, the results were in good agreement with values determined by the instructors in triplicate using the same protocol (Supporting Information Table S1). All results were presented to and discussed with the participating students during an extra meeting after completion of the experiments.
■
CONCLUSIONS Students analyzed ovalbumin in hen egg white using MeCAT for quantification, which involved covalent labeling of proteins with lanthanide ions within a chelate complex and quantification by ICP-MS gaining hands-on experiences in principles of analytical chemistry. Thereby, students became familiar with the basic concepts of protein research and learned to apply generic and advanced elements of analytical chemistry, such as calibration using different internal and external standards, in a broader and new context. In addition, by applying a rather simple Bradford assay and the complex MeCAT, a method still being further improved, students were able to compare the methods on the basis of hands-on experiences and discuss advantages and drawbacks. Furthermore, the experiment combined modern bioanalytical chemistry with elemental mass spectrometry and highlighted the potential of research in progress for advance teaching. Thus, the described example of implementing a rather new tool in analytical chemistry into an advanced practical course should encourage researchers to use new methods for advanced teaching as well. If the theoretical and practical background is sufficient, this experiment could also be appropriate for upper-level undergraduate students in an advanced analytical lab. The hands-on experience of students was superior for learning reflection on basic techniques and on using advanced strategies as compared to 2169
dx.doi.org/10.1021/ed500339c | J. Chem. Educ. 2014, 91, 2167−2170
Journal of Chemical Education
Laboratory Experiment
Table 1. Analytical Results of Ovalbumin Quantification in Lyophilized Hen Egg Whites via External Calibration and Internal Standarda Ovalbumin Quantification in % (w/w) Source
Sample 1 (N = 4)
Sample 2 (N = 2)
Sample 3 (N = 3)
Sample 4 (N = 4)
Sample 5 (N = 3)
Sample 6 (N = 4)
Sample 7 (N = 2)
External Calibration Internal Standard
41.0 ± 5.8 43.8 ± 8.0
43.1 ± 22.0 51.4 ± 21.2
48.6 ± 2.6 53.8 ± 2.0
40.5 ± 7.3 46.1 ± 10.4
53.6 ± 7.7 59.6 ± 15.8
38.1 ± 7.5 39.5 ± 8.0
34.3 ± 1.4 33.0 ± 4.9
a
Note: Further result details can be found in the Supporting Information (Table S1). Standard deviations from N independent replicates are given. W. A metal-coded affinity tag approach to quantitative proteomics. Mol. Cell. Proteomics 2007, 6 (11), 1907−1916. (9) Healey, M. Linking research and teaching to benefit student learning. J. Geogr. Higher Educ. 2005, 29 (2), 183−201. (10) Xie, Q.; Li, Z.; Deng, C.; Liu, M.; Zhang, Y.; Ma, M.; Xia, S.; Xiao, X.; Yin, D.; Yao, S. Electrochemical quartz crystal microbalance monitoring of the cyclic voltammetric deposition of polyaniline - A laboratory experiment for undergraduates. J. Chem. Educ. 2007, 84 (4), 681−684. (11) Kennedy, B. J. Integrating advanced high school chemistry research with organic chemistry and instrumental methods of analysis. J. Chem. Educ. 2008, 85 (3), 393−395. (12) Chamberlain, J. S. Teaching and research. J. Chem. Educ. 1929, 6 (4), 710. (13) Tomasik, J. H.; Cottone, K. E.; Heethuis, M. T.; Mueller, A. Development and Preliminary Impacts of the Implementation of an Authentic Research-Based Experiment in General Chemistry. J. Chem. Educ. 2013, 90 (9), 1155−1161. (14) Schwarz, G.; Beck, S.; Weller, M. G.; Linscheid, M. W. MeCATnew iodoacetamide reagents for metal labeling of proteins and peptides. Anal. Bioanal. Chem. 2011, 401 (4), 1203−1209. (15) Benda, D.; Schwarz, G.; Beck, S.; Linscheid, M. W. Quantification of intact covalently metal labeled proteins using ESIMS/MS. J. Mass Spectrom. 2014, 49 (1), 13−18. (16) Csonka, F. A.; Jones, M. A. Egg White Proteins. Fed. Proc. 1951, 10 (1), 176−176. (17) Mine, Y. Recent Advances in the Understanding of Egg-White Protein Functionality. Trends Food Sci. Technol. 1995, 6 (7), 225−232.
lectures and seminars. This might not only trigger interest of young researchers but also enhance motivation and creativity.
■
ASSOCIATED CONTENT
S Supporting Information *
Information on experimental details, evaluation of student’s feedback, experimental hazards, student hand-out, exemplary data analysis, and instructor notes. This material is available via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address §
Laboratory of Inorganic Chemistry, ETH Zurich, 8093 Zurich, Switzerland. This author’s affiliation has changed during manuscript revison. However, the contribution to the manuscript was solely related to the affiliation mentioned at the top of this document. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank Georg Kubsch for continuous support in organization. Furthermore, we gratefully acknowledge all students that participated in this practical course, and Proteome Factory AG for technical support.
■
REFERENCES
(1) Bradford, M. M. Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72 (1−2), 248−254. (2) Carter, J.; Petersen, B. P.; Printz, S. A.; Sorey, T. L.; Kroll, T. T. Quantitative Application for SDS-PAGE in a Biochemistry Lab. J. Chem. Educ. 2013, 90 (9), 1255−1256. (3) Bantscheff, M.; Lemeer, S.; Savitski, M. M.; Kuster, B. Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present. Anal. Bioanal. Chem. 2012, 404 (4), 939− 965. (4) Houk, R. S. Inductively coupled plasma-mass spectrometry and the European discovery of America. J. Chem. Educ. 2000, 77 (5), 598− 607. (5) Tholey, A.; Schaumloffel, D. Metal labeling for quantitative protein and proteome analysis using inductively-coupled plasma mass spectrometry. Trends Anal. Chem. 2010, 29 (5), 399−408. (6) Sanz-Medel, A.; Montes-Bayon, M.; Bettmer, J.; FernandezSanchez, M. L.; Encinar, J. R. ICP-MS for absolute quantification of proteins for heteroatom-tagged, targeted proteomics. Trends Anal. Chem. 2012, 40, 52−63. (7) Schwarz, G.; Mueller, L.; Beck, S.; Linscheid, M. W. DOTA based metal labels for protein quantification: a review. J. Anal. At. Spectrom. 2014, 29 (2), 221−233. (8) Ahrends, R.; Pieper, S.; Kuhn, A.; Weisshoff, H.; Hamester, M.; Lindemann, T.; Scheler, C.; Lehmann, K.; Taubner, K.; Linscheid, M. 2170
dx.doi.org/10.1021/ed500339c | J. Chem. Educ. 2014, 91, 2167−2170
