Partial characterization of a novel amphibian hemoglobin as a model for graduate student investigation on peptide chemistry, mass spectrometry, and atomic force microscopy

Authors

  • Marcelo P. Bemquerer,

    Corresponding author
    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
    • Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
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  • Jéssica K. A. Macedo,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Ana Carolina J. Ribeiro,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Andréa C. Carvalho,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Débora O. C. Silva,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Juliana M. Braz,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Kelliane A. Medeiros,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Lunalva A. P. Sallet,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Pollyanna F. Campos,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Maura V. Prates,

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • Luciano P. Silva

    1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica- Av. W5 Norte (final), 70770-917 Brasília, DF, Brazil
    2. Programa de Pós-graduação em Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900 Brasília, DF, Brazil
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  • This work is supported by CAPES, CNPq, and FAPDF.

    M.P.B., J.K.A.M, and L.P.S. wrote the paper. All the authors performed the experiments, discussed the results, and commented on the manuscript.

Abstract

Graduate students in chemistry, and in biological and biomedical fields must learn the fundamentals and practices of peptide and protein chemistry as early as possible. A project-oriented approach was conducted by first-year M.Sc and Ph.D students in biological sciences. A blind glass slide containing a cellular smear and an aqueous cellular extract were offered to the students. Qualitative and quantitative cell morphological parameters were analyzed by atomic force microscopy. The fractionation of the aqueous extract was conducted by reversed-phase chromatography followed by analysis of the isolated and partially purified proteins and peptides by mass spectrometry (MS). The proteins were treated by peptidases and the obtained peptide fragments were sequenced by de novo MS/MS, together with peptides already present in the extract. The most abundant protein fractions were identified as the alpha and beta chains of hemoglobin from an amphibian of the Leptodactylus genera. Two of the peptides sequenced by the students were synthesized by the solid-phase methodology, one of those being obtained by the split-and-pool library synthesis method. Thus, the students were able to learn some advanced principles and practices of protein chemistry and bionanotechnology in a 6-weeks project-oriented approach.

Chemistry of peptides and proteins is an important field in basic research and in biotechnology that can be learned from different theoretical and experimental approaches. For the investigations of proteomes, the fundamentals and techniques of two-dimensional electrophoresis combined to mass spectrometry (MS) analysis are a valuable and classical approach [1, 2]. Nevertheless, if the aim is the isolation and identification of biologically active peptides such as neuropeptides and antimicrobial molecules, the fractionation by chromatographic methods is usually more efficient than electrophoretic separations since peptides are usually not within the molecular mass range of gel electrophoresis. Another advantage is that chromatographic methods are compatible with scaling-up to preparative procedures [3, 4]. Also, chromatography is a technique that can be used to furnish relevant physical–chemical data such as a hydrophobicity scale, which may be used as an educational tool in biophysics with the aim of discussing the structural aspects of bimolecular interactions of peptides and proteins [5].

Graduate students in chemistry, biological sciences, and biomedical fields can learn basic and advanced principles of peptide and protein chemistry by working with chromatographic techniques, MS, protein sequencing, and solid-phase peptide synthesis [3, 6–9]. In the same project-oriented approach, we propose that the students may also characterize the details of cellular nanostructure by atomic force microscopy (AFM) [10] and thus they will also learn the fundamentals of the modern field of nanotechnology applied to a biological problem [10, 11]. When considering the most suitable source of a biological material to give rise to experiments involving all of the above mentioned methodologies, apart from microorganisms animal blood appears as the most readily available one. In this respect, in addition to the characterization of peptide and protein content, morphological parameters of red blood cells can also be investigated as an application of nanotechnology to the biological field [10]. Despite the fact that the cells are obviously not in the nanometric scale, morphologic details of its surface are nanostructural and those investigations are relevant for taxonomic aspects in biology [10] or for novel diagnostic methods in clinical sciences [12].

Hemoglobin is the major macromolecular constituent of red blood cells, where its primary function is transport of oxygen bound to the heme prosthetic group and carbon dioxide linked to the N-terminal groups of the alpha and beta chains by forming a carbamate moiety [13]. Hemoglobin is also the classical model protein to teach the relationship between structure and function with phenomena such as cooperativity, transition between the R and T states, oxygen and carbon dioxide binding. Comparison of proteins from different species is interesting to reveal, for example, that amphibians use adenosine triphosphate (ATP) and mammals use 2,3-bisphosphoglycerate (BPG) to stabilize the T-state and thus reduce oxygen affinity [13]. It is also known that the turnover of hemoglobin generates biologically active peptides with antimicrobial and pharmacological activities [14–16]. Also, expression of alpha and beta chains of hemoglobins and of some peptide fragments derived from them has been shown in different organs and tissues [17]. Thus, hemoglobin is still a valuable model for studies in protein and peptide chemistry with an educational focus, especially when a novel molecule from poorly studied animal group is being investigated.

In the experimental approach presented here, the graduate students received an unidentified sample of red blood cells from an amphibian of the Brazilian biodiversity. The students analyzed the qualitative morphology of the cells and some quantitative parameters such as the roughness, radius, surface area, and volume by AFM. The intracellular proteins and peptides of the cells were extracted and fractionated by reversed-phase chromatography and analyzed by MS. The two chains of a novel hemoglobin isoform were obtained and submitted to sequencing by MS furnishing a partial sequence of the protein, whose identity was confirmed by comparison with sequences deposited in databases of primary structures of protein. Peptide fragments of hemoglobin generated by endogenous peptidases were also found. Since the turnover of hemoglobin by specific peptidases can lead to biologically active peptides, such as antimicrobial and neuroactive peptides [14–16], segments of the partially sequenced protein were analyzed in silico and two potential biologically active peptides were selected and synthesized by the solid-phase methodology [12, 18]. Thus, this project-oriented approach during six weeks (60 hours were the total time for the experimental activities) allowed the biological sciences first-year graduate students to learn a plethora of principles and methods related to protein chemistry, cell biology, and nanotechnology and they could obtain preliminary information about a novel molecule. Since hemoglobin is the prototype of an oligomeric protein and for structure–activity relationships in Biochemistry textbooks, it can always lead to relevant discussion in the classroom about protein structure and function.

MATERIAL AND METHODS

Blood Sample

One amphibian specimen of the Leptodactylus genus was obtained from its natural habitat (Goiás State, Central Brazil, Cerrado biome) under IBAMA Permission Number 20692-1. The animal (a photograph is presented in Fig. 1, Supporting Information) was anesthetized with an intracranial injection of lidocaine hydrochloride (2% by weight in water). Then, an incision was done in the ventral part and the heart was exposed. A volume of blood (200 μL) was collected by cardiac punction. One microliter of the blood sample was spread over a circular glass cover slip surface, air-dried for 5 min, and fixed with methanol for 5 min. The remaining sample was centrifuged at 700 × g (4 °C) for 5 min in a bench centrifuge. The blood serum (i.e. the supernatant) was discarded and the rupture of the red blood cells was conducted by addition of 150 μL of Milli-Q® water. The mixture was kept for 5 min at 4 °C to perform an efficient cell lysis followed by centrifugation at 700 × g for 5 min. The aqueous extract (supernatant) of the Leptodactylus sp red blood cell was then freeze-dried and stored at −80 °C until the chromatographic fractionation. The students received the aqueous extracts and the fixed cells as the materials for the course.

Analysis of Red Blood Cells Nanostructural Parameters by Atomic Force Microscopy

AFM imaging was obtained in air on the blood films using a SPM-9600 equipment (Shimadzu, Kyoto, Japan). The images were acquired in contact mode using 200 μm length V-shaped cantilevers (nominal spring constant of ∼0.15 N m−1, resonant frequency of ∼24 kHz) with integrated Si3N4 pyramidal tips (curvature radius < 20 nm). The scanner used has a maximum travel of 125 μm in XY-directions and 7 μm in the Z-direction. All AFM images (100 μm × 100 μm) were captured as 512 × 512 pixels at scan rate of 1 Hz. The images obtained were processed by the SPM-9600 off-line software. The processing consisted of an automatic plane fit leveling of the surface. One-hundred individual red blood cells were manually half-height segmented from the background using the labeling function of the particle analysis software. Maximum diameter, pattern width, mean radius, maximum Z, minimum Z, average Z, perimeter, area excluding hole, area including hole, surface area, and volume were automatically measured. The meaning of these parameters is defined in the Supporting Information and the values obtained are presented in Supporting Information Table 1. Some measurements were compared with those data available on literature for blood of other species which had been investigated by light microscopy and flow cytometry. The same approach was conducted with a human blood sample for comparison. The students conducted the experiments and they organized the data with the assistance of one of the instructors.

Reversed-Phase High Performance Chromatography

The red blood cell aqueous extract was fractionated by reversed-phase chromatography (Source 5 RPC ST column, 150 × 4.6 mm, GE Healthcare, Cardiff, UK). The sample was dissolved in 350 μL of 0.1% aqueous trifluoroacetic acid (Solvent A). The analytical conditions were as follows: Source 5RPC column, flow rate at 1.0 mL min−1, room temperature; gradient program: H2O:ACN:TFA (98:02:0.1; v:v:v) for 5 min, then mobile phase composition varied from H2O:ACN:TFA (98:02:0.1; v:v:v) to H2O:ACN:TFA (20:80:0.1; v:v:v) in 80 min, where TFA is trifluoroacetic acid. The chromatographer was composed of a LC-10 pump, a SPD-10AV UV–vis detector, a FCV-10AL solvent mixer, a DGU-14A degasser, a Rheodyne injector, all these accessories coupled to a SCL-10A System Controller (Shimadzu, Kyoto, Japan). Re-chromatography when necessary was performed on a Shim-pack XR ODS column (30 × 2.0 mm) at a flow rate of 0.4 mL min−1 and at 50 °C, coupled to a Proeminence UFLC 20 AD, Shimadzu (Kyoto, Japan). Detection was conducted at 216 and at 280 nm for both steps. Linear variations of the percentage of acetonitrile (ACN) over H2O (both ACN and water contained 0.1% TFA by volume) were optimized by the students since the total chromatographic runs were up to 20 min. For the analysis of synthetic peptides the students also optimized the gradient program with the supervision of one of the instructors. More detailed experimental conditions of the chromatography can be found in the Supporting Information.

MALDI Mass Spectrometry Analysis and Sequence Comparison to Databases

MALDI MS was employed to attest the purity of the compounds isolated by chromatography and for preliminary characterization of the partially purified fractions. In a 1.5 mL polypropylene tube, the matrix solution was prepared by weighting 5.0 mg of α-cyano-4-hydroxy-cinnamic acid (HCCA) which was then solubilized with 250 μL of ACN, 200 μL of Milli-Q® water, and with 50 μL of an aqueous TFA solution (at 3% by volume). Then, 1 μL of the sample solution was mixed with 3 μL of the saturated matrix solution and the mixture was spotted on each well of the 384-wells steel plate leaving it to dry at room temperature (20 °C) for about 20 min. Proteins with molecular masses of 15.6 kDa and 16.4 kDa, corresponding to the alpha and beta chains of hemoglobin, were more fully characterized. They were reduced with 100 mmol L−1 1,4-bis-sulfanylbutane-2,3-diol (dithiothreitol) in 100 mmol L−1 ammonium bicarbonate and alkylated by 50 mmol L−1 2-iodoacetamide in 100 mmol L−1 ammonium bicarbonate. The proteins were then digested with immobilized trypsin (Pierce) in 100 mmol L−1 ammonium bicarbonate, pH 8.0, for 40 min. The peptides obtained through enzymatic digestion were sequenced by MALDI-TOF/TOF MS. All the MS analyses were performed in a Bruker Daltonics Ultraflex III (Billerica, MA) with the external calibrations performed accordingly to Bruker Daltonics instructions. Fragmentations for manual denovo peptide sequencing were performed by MALDI-TOF/TOF MS using the LIFT™ method [19]. Sequencing was manually performed by using the PepSeq software (MassLynx 4.0, Waters, Milford, MA). It is important to point out that the students performed all these experimental procedures, with assistance of the instructor especially for the equipment calibration and for the peptide fragmentations. Database (Swiss-Prot) searches for sequence similarities were performed using protein BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) with default parameters.

Solid-Phase Synthesis of the Peptides

The peptides H-LIVFPWTQRY-OH and H-A(S)(T)A(E)(S)K(R)YR-OH (where the residues of a combinatorial synthesis are indicated in alphabetical order after the first choice) were synthesized manually (in polypropylene vessels with frits) by the Fmoc/t-butyl solid-phase methodology using the Wang resin [9]. The resins employed (mass corresponding to 0.1 mmol) were Fmoc-Tyr(tBu)-Wang-Resin (0.44 mmol g−1, Peptides International) and Fmoc-Arg(Pmc)-Nova Syn (0.09 mmol g−1, NovaBiochem). Couplings of the Fmoc-amino acids (0.4 mmol) were conducted with [benzotriazol-1-yloxy(dimethylamino)- methylidene]-dimethylazanium tetrafluoroborate/N-ethyl-N-propan-2-ylpropan-2-amine (TBTU:DIPEA; 0.4 mmol:0.8 mmol) in N,N-dimethylformamide (DMF) for 60–90 min. Deprotections occurred with 25% (by volume) 4-methylpiperidine in DMF (2 × 15 min). The resins were washed with 2-propanol and with DMF after the coupling and deprotection steps and these washing procedure were repeated more three times. The peptides were cleaved from the resins by TFA in the presence of carbocation scavengers that the students themselves selected [9]. For the split-and-pool synthesis of H-A(S)(T)A(E)(S)K(R)YR-OH, the idea was to prepare the peptides whose most frequent sequences were found by the students when using protein BLAST tool for sequence comparisons. First, the mass corresponding to 0.1 mmol of Fmoc-Arg(Pmc)-Nova Syn (0.09 mmol g−1, NovaBiochem) was weighed and the amino-group deprotection was conducted with 25% (by volume) 4-methylpiperidine in DMF (2 × 15 min). Coupling of Fmoc-Tyr(tBu)-OH (0.4 mmol) was conducted with TBTU/DIPEA (0.4 mmol/0.8 mmol) in DMF for 60 min. After one more deprotection step, the peptidyl-resin was then dried under reduced pressure, separated into two equal parts and transferred to two reaction vessels. Couplings of Fmoc-Lys(Boc)-OH (0.4 mmol) and Fmoc-Arg(Pbf)-OH (0.4 mmol) were conducted separately in DMF for 60 min. Peptidyl-resins were then pooled. After other deprotecion step, the peptidyl-resin was dried under reduced pressure, then three equal parts were transferred to reaction vessels and couplings of Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)OH (0.4 mmol each) were conducted (TBTU/DIPEA, 0.4 mmol/0.8 mmol in DMF). The peptidyl resins were pooled and deprotection (with 25% 4-methylpiperidine in DMF) was conducted. A final split was performed (three equal parts) and couplings of Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH and Fmoc-Thr(tBu)-OH (0.4 mmol each) were conducted (TBTU/DIPEA, 0.4 mmol/0.8 mmol in DMF), followed by pooling the peptidyl-reins before the final deprotection. Detailed synthesis protocols are presented in the Supporting Information. Crude peptides were purified by reversed-phase chromatography and analyzed by MALDI-TOF MS as described in the previous section.

Chemical and Physical Hazards

Acetonitrile and trifluoroacetic acid used in the chromatographic runs were manipulated with gloves and in a fume hood since the reagents are specially toxic causing skin burns and eye damage. All of the procedures employed for manual peptide synthesis were also conducted in a fume hood, since most of the employed reagents are toxic to the eyes and skin. Students must be particularly advised about the toxicity of the following chemicals: phenol causes severe skin burns and irritation of the lungs, potassium cyanide inhibits cellular respiration, N,N-diisopropylethlyamine is a moderately strong organic base, the coupling reagent TBTU can be irritating to the skin, and ninhydrin reacts with skin proteins and amines. There are no special recommendations concerning the laser hazard of the UltraFlex III mass spectrometer but direct exposure to laser must always be avoided. A list of the chemical compounds used with their CAS numbers can be found in Supporting Information.

RESULTS

The students were divided in two groups of four and they started the experimental work by the morphological nanoscale analysis of the red blood cells by AFM [10, 11]. The students immediately suspected that the glass slide contained a blood smear due to the red color; however, they were surprised by the fact that the cells had an elongated instead of discoid form as they imagined from the largely known mammalian red blood cell structure. In fact, the results confirmed and reinforced the well-established fact that amphibians have elliptical and larger red blood cells [20] when compared to the mammalian cells (Figs. 1a and 1b). The students also measured several dimensional features of the amphibian red blood cells at a nanoscale and compared these measurements with those obtained for the red blood cells of one of them (Supporting Information, Table 1) and also with available database (http://www.genomesize.com/cellsize/). A photograph of the amphibian species is available (Fig. 1, Supporting Information).

Figure 1.

Contact mode AFM images of red blood cells from Leptodactylus sp (a) and human (b). The sample was scanned in constant force using 200-μm-length V-shaped cantilever with integrated pyramidal silicon nitride tip. The original acquired areas were 125 μm side perfect squares and images were acquired as 512 × 512 pixels at scan rate of 1 Hz. The histograms at the right side of the panels represent the height distributions along the AFM images.

The biochemical experiments were initiated by the chromatographic fractionation of the red blood cell crude extract. Figure 2 shows a typical reversed-phase separation of the aqueous–organic extract (i.e. a 50%, by volume, aqueous ACN solution) of the cells. MS analysis of the fractions suggested that products with retention times of 50.8 min and 55.1 min, with (M+H)+ values of 15,577 and 16,463, respectively, correspond to the alpha and beta chains of hemoglobin (Figs. 6 and 7 of Supporting Information show the mass spectra of the alpha and beta chains, respectively. A possible isoform for the beta chain is also presented). The students then conducted the reduction, alkylation, and peptide bond hydrolysis with trypsin and with other peptidases (elastase, cathepsin C, chymotrypsin, Asp-N and Glu-C endopeptidases, and leucyl aminopeptidase) of the hemoglobin chains. The obtained peptides were submitted to MS/MS fragmentation and the students were stimulated to obtain peptide sequences by the “de novo” MS/MS sequencing approach. Thus, the students could learn the principles and practices of a sophisticated methodology that is very useful in protein chemistry. Some of the obtained sequences are listed in Table I and those sequences allowed the students to confirm the identities of the hemoglobin chains by comparison with other amphibian hemoglobin sequences [6, 7, 21]. The students were able to elucidate more peptide sequences and some examples of those spectra are in Figs. 4 and 5 of Supporting Information.

Figure 2.

Reversed-phase chromatography of the acidic aqueous extract obtained from the red blood cells of Leptodactylus sp. The sample was solubilized in 350 μL of 0.1% aqueous trifluoroacetic acid (Solvent A). Analytical conditions: Source 5RPC, flow rate at 1.0 mL min−1, room temperature; gradient program: H2O:ACN:TFA (98:02:0.1; v:v:v) during 5 min, then mobile phase composition varied from H2O:ACN:TFA (98:02:0.1; v:v:v) to H2O:ACN:TFA (20:80:0.1; v:v:v) in 80 min. The straight line indicates variation of ACN whose percentage (by volume) is indicated in right y-axis; the absorbance was monitored at 216 nm (upper trace) and at 280 nm (lower trace). Some of the molecules identified are indicated.

Table I. List of the ions ([M+H]+) observed for peptides obtained by the hydrolysis of Leptodactylus sp. hemoglobin by trypsin or by the action of endogenous peptidases
[M+H]+SequenceChainRun time (min)
  1. It is worth pointing out that the ion observed at [M+H]+ = 2805.60 corresponds to a peptide that was linked by disulfide bond to free cysteine present in the medium. All the ions were in the singly-charged state (z = 1). The authors decided between leucine and isoleucine from comparison to sequences deposited on protein databanks.

1323.02LIVFPWTQRYBeta41.8
2686.52FTPELQASFEKAFCG VADAIGKGYHBeta44.6
2805.53FTPELQASFEKAFC(-C)GVADAIGKGYHBeta44.1
1425.90EFTPELQASFEKBeta44.9
1296.99FTPELQASFEKBeta43.8
1586.80LAAHAQEYGGEALTRAlpha42.4
1524.78ASGHSEAIGAEILNRAlpha43.4
1769.20DKFLVAVAASLTSKYRAlphaNot assigned

One of the groups performed a more thorough collection of the fractions than the other without neglecting the minor (i.e. less abundant) components of the chromatographic separation. An interesting finding is that one of the less abundant peptides was sequenced by the de novo MS/MS approach (Fig. 3) and it was identified as a hemorphin analogue, a molecule of the opioid class [22]. Also, some other peptides were generated by the possible action of peptidases present in the blood sample and a potential antimicrobial peptide with sequence H-FTPELQASFEKAFCGVADAIGKGYH-OH were found, which was probably released from the amino-terminus of the alpha chain by the action of a Glu-C-like peptidase. Partial sequencing of the enzymatic digestion of the alpha-chain revealed a peptide containing as a subfragment, the pentapeptide H-TSKYR-OH, which the students identified as a neokyotorphin, an antitumorigenic molecule [23]. Thus, the multifunctional role of the globin genes could be clearly shown to the students, who were also able to identify, with instructor assistance, the hemoglobin prosthetic group (protoporphyrin IX, mass spectrum is shown in Fig. 8 of Supporting Information).

Figure 3.

De novo sequencing of the hemorphin peptide obtained by reversed-phase fractionation of the acidic aqueous extract obtained from the red blood cells of Leptodactylus sp. The spectrum was recorded by MALDI-MS/MS (Bruker Daltonics Ultraflex III) in the positive mode.

The peptide corresponding to the hemorphin analogue, whose sequence was elucidated by the students, was manually synthesized by the solid-phase methodology with the Fmoc/t-butyl approach. By stepwise synthesizing the peptides, the students learnt the fundamentals of the solid-phase methodology [9, 18]. It must be reported that the students made a mistake in the choice of one of the Fmoc-amino acids and the peptide H-LIVPPWTQRY-OH was synthesized instead of the desired peptide H-LIVFPWTQRY-OH. By analyzing the mass spectrum (MS and MS/MS) of the crude peptide, the error was detected by them and the correct hemorphin peptide was then synthesized. Furthermore, the neokyotorphin pentapeptide was synthesized by the students by the split-and-pool approach. Hence, they were also introduced to the basic principles and practices of the synthesis of peptide libraries. The chromatographic separations of the synthetic peptides were optimized by the students with the previous experience (chromatograms are shown as Figs. 2 and 3 of Supporting Information). MS analysis of the crude synthetic products shown in Fig. 4 allowed the students to identify the 18 peptides of the library and a careful analysis of the m/z signal intensity could be related to the presence or absence of more basic amino acids, especially the arginine residue that has a high proton affinity in the gas phase [24]. The biological assays of the synthetic peptides were not performed by the students during the course schedule, but these experiments will be conducted by the senior authors and the results will be published elsewhere together with the complete hemoglobin sequence.

Figure 4.

Mass spectrum of the crude product H-A(S)(T)A(E)(S)K(R)YR-OH (where the possible residues of a combinatorial synthesis are indicated in alphabetical order after the first choice) obtained by the Fmoc/t-butyl solid-phase methodology. The spectrum was recorded by MALDI-MS/MS (Bruker Daltonics Ultraflex III) in the positive mode.

DISCUSSION

In this project-oriented approach, the students were able to conduct the analysis of the cell-surface nanostructure by AFM and the isolation and characterization of peptides and proteins from the red blood cell by chromatography, mass spectrometry and peptide synthesis. Despite being micrometer-sized objects, analysis of the cell nanostructural properties is a valuable tool for taxonomical [10] and diagnosis [11] applications. Thus, application of a classical nanotechnology tool to cell biology can give rise to relevant biological data. To acquire the structural parameters (presented in Table 1 of Supporting Information) the students learnt the principles of the atomic force microscope operation. On the other hand, the methodological approaches employed for the biochemical characterization of the red blood cell components are keystones in peptide chemistry and proteome investigations. Thus, in a period of 6 weeks when the graduate students had free access to the laboratory equipments and when they could interact with the instructors, they were able to obtain valuable data and to generate information for the partial sequencing of a novel amphibian hemoglobin.

Sequencing of peptides by MS is almost widespread, thus learning its fundamentals and practices has to become straightforward for students and researchers interested in protein chemistry. By obtaining sequence information by MS/MS spectra interpretation, the students were introduced to the basic principles of chemical fragmentation of peptides in the gas phase and also to the biological value of the comparison of the sequences [6, 7, 24]. It must be pointed out that not all the students acquired expertise in the analysis of MS/MS spectra, but the elucidation of the sequences of the peptides presented here (Fig. 3, Table I and also in Supporting Information) were conducted by some of the students assisted by the instructors. Those students could surely become capable of analyzing MS/MS spectra of peptides with some more training. The sequences obtained were analyzed by the students that initially could identify whether the segment obtained belonged to the alpha or to the beta chain. Considering that globins are one of the most studied protein families [25], they could also compare the sequences with those of other amphibian and fish hemoglobins that are easily found in protein databanks and they could evaluate the phylogenetic relationships of the novel protein to that of related organisms [21].

Hemoglobin is a source of biologically active peptides that are generated by the protein turnover catalyzed by specific peptidases. Hemorphins are produced by the hydrolysis of hemoglobin beta chain with the acidic peptidase cathepsin D as catalyst [22]. Cathepsin D was probably activated in the acidic milieu in which the red blood cell extract was diluted (i.e. 0.1% aqueous TFA) and in which it remained for 1–2 hours before the chromatographic run. Thus, one of the sequenced peptides could be identified as a hemorphin. Also, a neokyotorphin was sequenced after the students had performed digestion of hemoglobin alpha chain with trypsin and since it is a short chain peptide with some variation in its sequence, it was a good model for introducing the idea of solid-phase synthesis of peptide libraries.

The variations in the sequence of the pentapeptides were selected by the students (with some help from the instructors) according to the most frequent sequences found in protein sequence databases. The advantage of synthesizing a pool of peptides, apart from the possibilities of studying additive and synergistic effects in biological assays, was that the students were able to evaluate the difficulties of separation and analysis of very similar molecules by conventional chromatographic methods. Another important aspect was that the analysis by MS of the mixture present in the crude peptide was illustrative of the principles of peptide ionization because the signal intensity of the arginine-containing peptides was always higher than those observed for the lysine-containing peptides. The ionization of peptides containing arginine when compared to peptides without this amino acid residue has important implications for the peptide fragmentation accordingly to the well-accepted mobile proton model [24].

To sum up, the present work was a project-oriented approach in the fields of biochemistry and cell biology with investigations of methodologies applied to nanoscience and protein chemistry. Despite the plethora of advanced techniques, the students themselves were able to conduct all the experimental steps of the study. They performed the AFM analysis of the red blood cell surfaces, chromatographic separations, MS data acquisition, and analyses and syntheses of the peptides. A final report of the project was presented by groups of four or five students. More instructor interventions were required in the mass spectra interpretation when compared to the other methodologies and it was clear that not all the students could learn how to sequence a peptide by MS.

There are some descriptions of projected-oriented approaches applied to advanced undergraduate students or to early graduate students. Those course proposals are partially [26] or completely [27] based on real research projects. Especially for undergraduate students there are some problems related to the course time schedule but a well planed research project has been successfully conducted [27]. One of us had also a successful project-oriented course for second-year undergraduates in which the students had some instructor guidance to develop a project in enzymology allowing the students to introduce themselves to a relevant research problem in biotechnology, i.e., the effect of organic solvents on enzymatic catalysis [28, 29]. Thus, it is clear that project-oriented approaches to graduate and even to undergraduate students must include an innovative research activity. For graduate students the course time schedule is not a major problem, even though some students and other researchers complain about the excessive time the students spent at the laboratory.

CONCLUSIONS

From a red blood cell extract of an Amphibian from the Brazilian biodiversity, a project-oriented approach was conducted by first-year graduate students. Chromatographic fractionation led to the separation of the alpha and beta chains of hemoglobin and to isolation of other peptides. Sequencing of those peptides and of products of treatment of globin chains with peptidases were conducted by de novo MALDI MS/MS. Two biologically active peptides, corresponding to hemorphin and neokyotorphin, were identified as segments of hemoglobin and they were synthesized by the solid-phase method, one of those by the split-and-pool strategy. Also, nanostructural characterization of the surface of red blood cells was conducted by AFM. Thus, a simple biological material was very useful for graduate students to learn a plethora of fundamentals and methods in protein chemistry, biochemistry, and nanobiotechnology. Assays of biological activity of hemoglobin and of the peptides obtained by action of endogenous or exogenous peptidases were beyond the scope of the course, but another course focusing on this functional characterization could be interesting for biochemical pharmacology approach.

COURSE COMPLETION

This graduate course has a moderate time frame, but as any upper-level course it needs to have a stringent pedagogic evaluation. Therefore, a combination of attendance, laboratory practice, final report, and oral presentation of the results has been used as assessment tools.

Attendance

Because of the moderate time frame of the course, attendance was a critical part to successful completion. All students fulfilled more than 95% of attendance to the activities.

Laboratory Practice

The behavior of the students in the laboratory was respectful, motivated, and efficient, but there were significant differences between the two workgroups in terms of the level of such attributes. As previously reported, one of the groups performed a more careful chromatographic separation than the other and they advanced more in the biochemical characterization of the obtained peptides.

Final Report

The students were required to prepare a printed final report describing all the results obtained during the course. The report had a typical scientific format with the following parts: title, introduction, aims, experimental section, results, discussions, conclusions, and references.

Oral Presentation

At least one of the students was required to expose to professors and other colleagues the studies developed by her group following a class discussion. Both groups opted by the format for which all students presented some part of the developed activities. All students were required to be present.

Theoretical Background

Since this graduate course was project-oriented, presentations of theoretical matters were reduced to the minimal necessary and as requested by the students. Nevertheless, the students attend at classes about fundamentals of nanobiotechnology, peptide synthesis, chromatography of peptides and proteins, analysis and sequencing of peptides by MS, and about the concept of fragments of functional proteins as biologically active peptides. Discussion of some research papers was also conducted with the students. There was no required prerequisite but one semester of organic chemistry and one semester of basic biochemistry are highly recommended.

NOTES

We list below some notes that could be useful for the instructors.

  • 1The instructor may stimulate the students to be aware that every piece of experimental results is valuable. For instance the students could identify hemoglobin by MS analysis of the chains isolated by reversed-phase chromatography (Figs. 6 and 7 of Supporting Information) and by sequencing some hemoglobin fragments by MS/MS. Also, the students were able to identify the heme group of hemoglobin as a protoheme IX ion (with iron in its ferric state) with m/z at 616.4 (Fe(III)C34H32N4O4). The MS/MS spectra of this ion led to the loss of the one or two carboxymethyl groups (Fig. 8 of Supporting Information), in agreement with the literature [29].
  • 2One interesting subject discussed with the students is the existence of biologically active peptides that are endogenously generated from proteins by enzymatic hydrolysis. This important biological phenomenon is well described in the literature [14–16] leading to potential economy of genome space and to the possibility of biochemical regulation of peptidase activity. One of the peptides found by the students in the chromatographic separation of red blood cell extract was a hemorphin analogue. Hemorphin is a segment of hemoglobin possibly generated by the action of the aspartic peptidase Cathepsin D [22] whose specificity has already been studied [30]. The students aligned and compared the sequences of hemoglobins available in databanks to try to find some specificity clues that lead to hemorphin release. Also, the activation of a cathepsin-like enzyme in acidic milieu (used for the preparation of red blood cell extracts) was discussed.
  • 3Alternative approaches can be suggested to introduce the students to the field of nanotechnology as applied to a biochemical problem. Peptidase enzymes may be immobilized in nanostructured materials such as silica [31]. The action of those immobilized enzymes upon natural or synthetic substrates can be evaluated by spectrophotometry, but also by chromatography followed by MS. Other aspects such as the effect of organic solvents over the activity of immobilized (or free) biocatalysts can also be investigated by the students [28, 32].
  • 4From the physiological chemistry viewpoint some aspects can be discussed with the students. Hemopexin is a 435-residues protein found in plasma, whose function is the binding of heme moiety released from hemoglobin and myoglobin turnover [33, 34]. Thus, hemopexin scavengers free heme that can cause toxic effects due to the generation of radical species. The search for hemopexin by monitoring the Soret bands of protoporphyrin (by absorbance spectroscopy) may be conducted as a project-oriented approach.

Acknowledgements

The authors thank to Eder Alves Barbosa for helping with animal collection and preparation of the red blood cell extract.

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