Protein N-Glycans: Incorporating Glycochemistry into the

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Protein N‑Glycans: Incorporating Glycochemistry into the Undergraduate Laboratory Curriculum Victoria R. Kohout, Zachary J. Wooke, Andrew G. McKee, Megan C. Thielges, Jill K. Robinson,* and Nicola L. B. Pohl* Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States

J. Chem. Educ. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 10/06/18. For personal use only.

S Supporting Information *

ABSTRACT: Glycoscience continues to be an underrepresented topic in current undergraduate-biochemistry-laboratory curricula. Of the educational laboratories present in this subject area, few introduce students to cutting-edge methods and techniques related to carbohydrates. A multiweek series of experiments is described that highlights a recently published bleach-mediated-carbohydrate-cleavage protocol in the context of the isolation and identification of the soybean glycoprotein, βconglycinin. Two different levels of undergraduate biochemistry courses (a first-year introductory course and an upper-level bioanalytical course) completed the experimental set. Although the same sequence of experiments took place for both levels, the teaching approaches to the material varied. The introductory course used an inquiry-type learning approach, whereas the upper-level students were taught with a guided-inquiry approach. Students in both courses were able to successfully isolate glycosylated β-conglycinin from inexpensive soy flour and then analyze the protein with SDS-PAGE and mass spectrometry. Once β-conglycinin was isolated, students were able to effectively adapt a recently published bleach-mediated-carbohydrate-cleavage protocol on this previously untested glycoprotein and quantitate the carbohydrate content with a colorimetric phenol−sulfuric acid assay. The effective execution of this biochemistry-laboratory series demonstrates an alternative undergraduate-laboratory curriculum that introduces students to traditional glycoscience and protein methods along with more cutting-edge techniques in these areas. KEYWORDS: Carbohydrates, Biochemistry, First-Year Undergraduate/General, Upper-Division Undergraduate, Inquiry-Based/Discovery Learning, Proteins/Peptides, Laboratory Instruction, Analytical Chemistry, Hands-On Learning/Manipulatives

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INTRODUCTION Standard undergraduate-biochemistry-laboratory curricula still primarily focus on nucleic acid and protein topics. However, the increasing recognition of the importance of carbohydrates to biochemical functions has led to a call in 2012 by the U.S. National Academy of Sciences to integrate the glycosciences into high-school, undergraduate, and graduate education with both lectures and hands-on activities and experiments.1 As a burgeoning field within biochemistry, the significance and roles of carbohydrates beyond metabolism, including current laboratory approaches to this class of biomolecules, is important for undergraduates to effectively pursue the careers growing in this area.2 At present, instructors have a very limited range of instructional materials available to introduce glycoproteins and carbohydrates into an undergraduate laboratory.3−10 Many of these efforts focus on using wellestablished but dated techniques, such as the iodine test, thinlayer chromatography, or colorimetric assays, for the indication of carbohydrates.3−6 Only a few educational biochemistry experiments published in the last two decades have incorporated more modern methods and techniques.7−10 A 2016 protocol for the isolation of carbohydrates from eukaryotic glycoproteins using bleach emerged as a promising © XXXX American Chemical Society and Division of Chemical Education, Inc.

potential way to introduce modern bioinformatics and glycochemistry within the context of the traditional topics of protein isolation and analysis.11 The low-cost, straightforward nature of this method compared with those of more complex procedures for carbohydrate cleavage from glycoproteins (e.g., requiring expensive enzymes or more toxic or corrosive chemicals) made this new technique attractive for an undergraduate laboratory setting. Plant proteins are an inexpensive source of glycoproteins, and initially wheat proteins were considered; however, wheat gluten is only contaminated with starch and is not glycosylated.7 Therefore, soybeans were chosen an alternative plant-protein source. Soybean (Glycine max (L.) Merr.) is one of the major crops grown worldwide, with over 310 million metrics produced in 2016; it is a protein-rich food source that can replace meat in diets, and it is a material in a number of nonconsumable items.12,13 Many of the commercially beneficial properties of soybeans can be linked to the composition and structure of the two major storage proteins, glycinin (11S) and β-conglycinin Received: July 11, 2018 Revised: September 20, 2018

A

DOI: 10.1021/acs.jchemed.8b00539 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. Experimental outline with learning objectives (in bullet points) corresponding to each experiment. The blue boxes correspond to the protein-based experiments, whereas the red boxes represent the carbohydrate-based experiments.

(7S), within the raw bean. Of the two, β-conglycinin is a wellknown glycoprotein that composes ∼30−35% of the total soybean-protein content.14 The protein itself consists of three subunits, α′, α, and β, which have molecular masses that are approximately 72, 68, and 52 kDa, respectively.14,15 A crystal structure of β-conglycinin with the glycan structure present has been reported, and the glycan itself has been reported to be a high-mannose N-glycan.16,17 The presence of these covalently linked carbohydrates is critical for understanding the physical properties and biological functions of soy proteins.18,19 Soybeans have a completely sequenced genome, which can allow further insight when using bioinformatics tools such as Protein Prospector and the Basic Local Alignment Search Tool (BLAST).20 A multiweek set of experiments was developed for the isolation, identification, and measurement of the carbohydrate contents of the glycoprotein β-conglycinin from soybean flour. This experiment set utilizes traditional protein- and carbohydrate-laboratory techniques along with a novel, recently published bleach oxidative method for cleaving the sugars from the glycoproteins (Figure 1). Students from two different levels in the undergraduate curriculum, a first-year introductory honors course and an upper-level bioanalytical laboratory, completed this experimental set. At both levels, the glycoprotein β-conglycinin was used as a “novel” substrate because it had not been included in the originally published work. The experiment context was framed as an exercise in how to prepare a vegan meat replacement (“fake meat’’). Therefore, there is a need to identify and characterize not only the proteins but also the attached carbohydrate components of plant proteins that could potentially be used in such products. This gave students a platform to explore the capabilities of the published method on an untested glycoprotein like independent scientists would do in their own research. Incorporation of laboratory experiments that may have real-world relevance has been shown to increase

student engagement in laboratories.21 It should be noted that a large-scale N-glycan extraction from soybeans using the bleach oxidative method was published shortly after these courses were completed, which further highlights the real-world relevance of this work.22 Although the same sequence of experiments took place for both levels, the teaching approaches to the material varied. The lower-level course was taught in an inquiry-type manner that utilized detailed procedures to teach new techniques, topics, and scientific research to introductory-level students.23 The upper-level bioanalytical course used a guided-inquiry-based approach to the glycoprotein exploration. The more advanced students were not provided with step-by-step procedures but instead were given research papers describing the protocols. They interpreted the papers and adapted procedures on the basis of time and equipment limitations. This learning style more closely mimics real scientific work and has been shown to help students engage in higher-level cognitive processes.24

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EXPERIMENTAL OVERVIEW The newly developed set of experiments took place over six 3 h laboratory periods. Students from two different levels in the undergraduate curriculum, a first-year introductory course (15 students) and an upper-level bioanalytical laboratory (45 students), completed this experimental set. The introductorycourse students worked individually, whereas the upper-level laboratory students worked in groups of two to three to complete the different experiments. Students carried out the extraction of β-conglycinin over two 3 h laboratory periods and utilized a modified procedure based on the work of Liu et al.25 The modified procedures lacked the repetition of steps found in the original work to accommodate laboratory time constraints, and the lower-level-course protocol can be found in the Supporting Information (SI, p S4). Students assessed the purity of the isolated 7S glycoprotein using gel electrophoresis during week 3. All of the students performed protein B




Figure 2. Gel electrophoresis. (a) Student gel stained with Coomassie Blue. The molecular weight ladder can be found in lane 1 (weights labeled), and soybean-protein samples of various concentrations can be found in the other lanes. The bands in lane 10 have been labeled with the corresponding protein names and their estimated masses from the standard plot. (b) Standard plot of log(MW) vs retention factor (Rf). Points calculated from the pictured gel and the data can be found in the SI (p S56).

possible allergic reactions in soybean-sensitive individuals without proper use of PPE. Isolated proteins were stored in a 4 °C refrigerator after being freeze-dried and ultimately disposed of in normal trash. Experimental waste created should be properly disposed according to local chemical-wastedisposal guidelines.

separations using commercially available SDS-PAGE gels with a 4−15% gradient in Tris running buffer, and protein bands were detected using Coomassie Blue stain. The stained gels were further analyzed by mass spectrometry with the Indiana University (IU) Biological Mass Spectrometry Facility by excising individual gel bands, digesting the proteins to peptides with trypsin, and analyzing the samples by LC-MS/MS.7 The resulting mass-spectrometry data provided information for band identification, and peptide information can be found in the SI (p S28). During week 4, students carried out the small-scale (10 mg glycoprotein) bleach oxidative-carbohydrate-cleavage method from the recently published work without any modifications.11 Sample quantification and analysis of the extracted carbohydrates was achieved in week 5 with the phenol−sulfuric acid (PSA) assay.26 D-(+)-Mannose was selected as the representative sugar for the PSA calibration curve because it is the primary monosaccharide component from the reported oligosaccharide for the 7S protein.17 The final week involved a soybean bioinformatics unit for the first-year introductory course, whereas the upper-level course undertook a unit on mass spectrometry of proteins. Each class worked with a guided handout the final week (pp S23 and S65). The introductory-course students were assessed through formal written journal-style reports, whereas the upper-level course completed a poster presentation about the work completed. The SI includes prelab assignments, experiment handouts, and assessments for both class levels.

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RESULTS AND DISCUSSION

β-Conglycinin Glycoprotein Extraction

The set of laboratory experiments was designed to introduce carbohydrates apart from their role in metabolism into a biochemistry laboratory course in the context of the more standard process of teaching students about protein isolation and analysis. The isolation of β-conglycinin was found to be extremely robust even when utilizing a variety of modified protocols. Nearly all students (>95%) were able to isolate more than enough freeze-dried glycoprotein to complete gel electrophoresis and the bleach oxidative-carbohydrate-cleavage experiments. The modified protocols saw some degradation of the glycoprotein purity compared with that of the original work; however, on the basis of prior work completed with soybean glycoprotein extractions, this difference did not greatly affect the desired outcome of this laboratory set.25 The 11S protein was isolated by an associate instructor for purificationcomparison purposes by gel electrophoresis; however, students can also easily complete this isolation concurrently with the glycoprotein isolation and identify the gel electrophoresis bands based on literature precedent. The protein isolation can also be easily done on a larger scale by a single person to directly provide material for the next set of experiments if pedagogical goals do not include student experience with protein isolation.

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HAZARDS The bioinformatics unit has no associated hazards. Appropriate personal-protective equipment (PPE; goggles, protective lenses, etc.) was worn at all times. Students were instructed to review all material safety data sheets for the chemicals handled during each laboratory section. All necessary solutions were prepared by an instructor prior to each laboratory. nHexane is a neurotoxin and should be handled carefully. Concentrated sulfuric acid used for the PSA assay should be handled with caution because of its extremely corrosive nature. Household bleach can spot colored clothing, cause skin and eye irritation with direct contact, and react violently with ammonia-containing chemicals to form chloramines that are highly toxic. There are no other known special hazards except

Gel Electrophoresis

To gain practical knowledge of current methods for analyzing protein samples, students assessed the purity of the isolated 7S glycoprotein using gel electrophoresis. The 7S and 11S protein fractions were prepared in varying concentrations ranging from 1 to 3 mg/mL and separated using commercially available SDS-PAGE gels with a 4−15% gradient in Tris running buffer, and protein bands were visualized with Coomassie Blue stain. Alternatively, a periodic acid−Schiff (PAS) glycoprotein stain that turns any protein containing carbohydrates a magenta or C




identified, and the locations and bands agreed with previously reported results.13 To introduce students to the basics of protein-based bioinformatics analysis, a unit was implemented with the soybean-peptide mass-spectrometry data; some representative peptide data from LC-MS/MS analysis are provided in Table 2, and a more complete data set can be found in the SI (p S28) to allow instructors without access to a mass-spectrometry facility to still continue with the bioinformatics lesson. Soybeans have a fully sequenced genome, making it possible to easily run searches with the commonly used BLAST algorithm and database. Students completed a step-by-step worksheet to identify where a particular peptide fragment was found in the larger protein sequence, understand how to find a homologue, and how to transform a protein amino acid sequence into its DNA base pairs. The unit was, in part, motivated by the desire to search for potential replacements to soy proteins in the development of new meat substitutes. Other legume sources could also serve as potential protein sources for another implementation of this entire set of laboratory exercises as more of these genome sequences become available.

pink color could be used (pp S11−15). Although the PAS stain is useful for detecting glycoproteins, the PAS-staining protocol is time-consuming because of the fixing, oxidizing, reducing, and several repeated wash steps. Students were able to estimate the molecular weights of the isolated protein subunits in the gel stained with Coomassie Blue by using a set of molecularweight standards and preparing a standard plot of log(MW) versus the retention factor (Rf, Figure 2b). Mass-Spectrometry Analysis and Bioinformatics

The identities of the proteins in the gel bands were determined by submitting the bands for analysis at the IU Biological Mass Spectrometry Facility. In brief, the gel bands were destained, and the proteins were digested with trypsin. The tryptic digests were analyzed by liquid chromatography−tandem mass spectrometry (LC-MS/MS)7 and the peptide masses were searched in ProteinProspector to identify the proteins in each gel band, as described in the SI (p S68). The gel bands submitted for analysis (Figure 3) are identified in Table 1 (see

Carbohydrate Extraction Utilizing the Bleach Oxidative Method and Phenol−Sulfuric Acid Assay

Once the isolation of β-conglycinin was complete, all of the students were able to complete the bleach oxidativecarbohydrate-cleavage procedure on this lyophilized protein fraction. The PSA assay for carbohydrate quantification and confirmation was found to work well as a qualitative test, but it proved more problematic as a quantitative test. Nearly all of the students’ extracted samples (>90%) had higher absorbance than the blank on the calibration curve, and one could visually see the solution turn a yellow color indicating sugar was present (Figure 4b). However, many students (∼70%) had calibration curves that had R2 ≤ 95% instead of the desired value of R2 ≥ 95% for quantitation of the extracted glycan from the protein sample. The PSA assay is used for its ability to accurately quantify small amounts of sugar present and appears to be very responsive to small changes in reagent concentrations and mixing conditions. This procedure was found to rely on following the precise protocol; changes in the order of reagent addition and pipetting inconsistently caused issues in making a reliable calibration curve. More practice with pipetting viscous liquids prior to preparing this calibration curve is suggested to increase student success for the PSA assay. The carbohydrate analysis could be further expanded to include carbohydrate-structural determination involving techniques such as MALDI-TOF mass spectrometry. Work by Zhu et al. discusses the isolation and characterization of the

Figure 3. Another gel submitted for mass-spectrometry analysis with the bands labeled.

p S76 for the full table). The isolated sample was a mixture of proteins because the sample preparation did not involve extensive purification, and the sample had a relatively high concentration of protein. Although the proteins appeared as distinct bands in the gel, the detection of multiple proteins in each band shows that the gel bands are actually Gaussian peaks with the maximum protein concentration at the center of the band. The low detection limits of the LC-MS/MS technique led to the detection of multiple proteins within a given band. The identity of each protein band was determined by the most abundant protein. The different subunits of β-conglycinin and glycinin from the 7S and 11S fractions, respectively, were

Table 1. ProteinProspector Analysis of Mass-Spectrometry Data for the Most Abundant Proteins from Gel Electrophoresis Protein Abundance in Each Gel Banda Protein Name Lipoxygenase β-Conglycinin, α chain β-Conglycinin, β subunit Glycinin G2 Glycinin G4 Glycinin G1

01 200 69 44 23 24 23

b

02

03

04

05

06

07

145 272a 80 34 31 26

91 585b 118 34 27 26

47 307 541b 29 42 35

33 113 157 205b 145 152

55 74 74 105b 106b 87

13 30 37 177 229b 296b

a

The gel bands are shown in Figure 3. bThe highest number represents the most abundant protein in the band. D




Table 2. Representative Mass-Spectrometry Data from ProteinProspector for Peptide Sequences Previous Amino Acid

Database Peptide Sequence Between Previous and Next Amino Acids

Next Amino Acid

R R R K K K R K R R K R

AILTLVNNDDRDSYNLHPGDAQR QVQELAFPGSAQDVER VLFGEEEEQRQQEGVIVELSK FFEITPEKNPQLR QIVTVEGGLSVISPK HFLAQSFNTNEDIAEKLQSPDDER VFYLAGNPDIEYPETMQQQQQQK QIVTVEGGLSVISPK YSVEMSAVVYK IYDYDVYNDLGDPDK DWVFTDQALPADLIK EFDSFDEVHGLYSGGIK

I L E D W K S W D G R L

Protein Name β subunit of β-conglycinin β subunit of β-conglycinin β subunit of β-conglycinin β subunit of β-conglycinin glycinin G4 subunit glycinin G4 subunit glycinin G4 subunit glycinin G4 subunit lipoxygenase lipoxygenase lipoxygenase lipoxygenase

(fragment) (fragment) (fragment) (fragment)

Figure 4. Phenol−sulfuric acid assay. (a) Representative N-glycan structure for β-conglycinin.17,27 (b) PSA assay with D-(+)-mannose standards and 7S fraction samples. The D-(+)-mannose standards were run in duplicate in the two rows, whereas the extracted 7S samples (A,B,C) were run in triplicate in the first row. (c) Successful D-(+)-mannose calibration curve. More examples of student calibration curves can be found in the SI (pp S20 and S64).

soybean N-glycans using a variety of analytical techniques, which could be applied to carbohydrate-structural analysis.22

described the biochemical relevance of carbohydrates, the theory of the experiments conducted, and the interpretation of results. The students were able to successfully link the experiments performed to one or two larger scientific topics (such as meat replacement) to underlay the importance of the work that they completed. Several individuals were even able to expand upon the work by proposing pertinent future experiments involving glycoproteins. These experiments included MALDI mass spectrometry of the isolated glycans and procedural modifications to increase yields of the extracted biomolecules.

Student Assessment

Introductory-student learning was assessed using in-class quizzes (pp S30−37) and a final written formal laboratory report. All of the students in the course gained understanding of the theory behind the laboratory techniques on the basis of their performances on quizzes, with students scoring primarily >80% on each quiz. The knowledge gained through the quizzes and the experimental set was highlighted in the quality of the journal-style laboratory reports. The reports accurately E




Facility for introducing the students to the wonders of protein mass spectrometry. This material is based in part upon work supported by the National Science Foundation under CHE1362213 and by Indiana University.

The upper-level bioanalytical course gave a poster presentation about the results of their experimental set. This gave the students the opportunity to present their findings in a written and oral manner, mimicking scientific communication at a conference. Each group of students gave a brief oral presentation about the work the group completed and answered questions posed by the main instructor and a small group of teaching assistants. The completed posters were wellorganized and clearly highlighted the scientific work conducted. Students were excited to share their knowledge and discuss the science behind their work. Most of the students were able to successfully answer questions about their work and give explanations about the importance of glycoproteins. Also, students provided insight about future experiments on which to expand on what they completed, such as protocol improvements and further analysis of both the protein and carbohydrate components. This format showed that the students had a solid understanding of the underlying material while revealing their critical thinking about the field of glycoscience.

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CONCLUSION A multiweek experimental set was successfully implemented in a first-year undergraduate course and an upper-level bioanalytical laboratory course in which proteins and glycoproteins were isolated, characterized, and analyzed using soybean β-conglycinin as the model. This experimental set was used to effectively introduce students to carbohydrates and traditional protein-isolation techniques while also incorporating a very current cutting-edge bleach-mediated-carbohydrate-cleavage technique. The inquiry-based-learning models and real-world context successfully triggered student interest in the experiments conducted.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00539. Student handouts consisting of the student laboratory procedures, examples of the course quizzes, laboratoryreport and poster guidelines, and notes for the instructor (PDF, DOCX)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Email: [email protected] (J.K.R.). *E-mail: [email protected] (N.L.B.P.). ORCID

Zachary J. Wooke: 0000-0002-0728-4806 Megan C. Thielges: 0000-0002-4520-6673 Nicola L. B. Pohl: 0000-0001-7747-8983 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the students in the Fall 2016− Spring 2018 introductory course and bioanalytical laboratory for their participation in this experiment set. We would especially like to thank Jonathan Trinidad and the Indiana University Laboratory for Biological Mass Spectrometry F




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Protein N-Glycans: Incorporating Glycochemistry into the

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