Intact cell MALDI-TOF mass spectrometry on single bovine ...
Quartz Crystal Microbalance for On-line Investigations of … · MALDI Mass Spectrometry MALDI...
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MALDI Mass Spectrometry MALDI experiments were carried out on a time-of-flight (TOF) mass spectrometer Voyager DE-PRO (Applied Biosystems, Foster City, CA, USA) equipped with a pulsed nitrogen laser. The samples collected from the QCM experiments were concentrated using ZipTips C18 units (Millipore, Schwalbach, Germany). As matrix a solution of 10 mg/mL CHCA in acetonitrile/0.1% TFA (1:1, v/v) was used. The resultant solution was mixed with the matrix solution 1:1 (v/v) on the sample plate. Then, the samples were dried in a gentle stream of air at ambient temperature (24 °C). Measurements were per- formed operating in the positive-ion reflector mode. Survey spectra of the peptide mixtures were obtained by accumulating data from 500 to 2000 laser shots. CONCLUSIONS • The described approach allows on-line investigations of proteolytic reactions under certain conditions. • Additional information, such as peptide sequences or enzyme cleavage sites can be obtained by employing mass spectrometry. ⇒ Combining QCM and mass spectrometry provides a better insight in enzymatic degradation of proteins. EXPERIMENTAL Immobilization of β-casein The immobilization of bovine β-casein was performed in a three stage process at room temperature. After every stage the quartz surface was dried under a stream of nitrogen. At first, a cleaned quartz crystal was sub- merged in a 1 mM ethanolic solution of THDA for two hours to obtain a self assembled monolayer (SAM), then douched with ethanol. In the second step the carboxylic groups of THDA were activated by covering the upper electrode with a 10 % (m/v) solution of EDC in diluted hydrochloric acid (pH 3.5). Finally, the quartz crystal was submerged in a 5 mg/mL solution of β-casein in a buffer containing borate (pH 8.8) for two hours. Washing with water and drying under nitrogen finalized the immobili- zation procedure. A schema of surface coating can be seen in Fig. 1. Atomic force microscopy shows the sur- face texture of immobilized β-casein (Fig. 2). Corresponding author: [email protected] REFERENCES [1] K.A. Marx, Biomacromolecules 4 (5), (2003), 1099-1120 [2] G. Sauerbrey, Zeitschrift für Physik 155, (1959), 206-222 INTRODUCTION In recent decades quartz crystal microbalance (QCM) has become a valuable tool for studies of biomolecular interactions, especially in liquid state [1]. A quartz crystal sandwiched between a pair of electrodes produces bulk acoustic waves when an AC voltage is applied. Typical oscillation frequencies are in the range of 5 to 10 MHz. Changes at the solid/liquid interface cause a frequency shift. The linear relationship between frequency shift (∆f) and adsorbed mass (∆m) is described by Sauerbreys equation [2]: where f is the fundamental frequency, A the area of the 0 electrode, G the shear modulus and ρ the density of the q q quartz crystal. Proteins are, besides fats and carbohydrates, the main nutrients in human food. They show an amazing struc- tural variety, and among their breakdown products, the peptides, are numerous compounds with miscellaneous biological effects. These bioactive peptides can be gen- erated in vivo through gastrointestinal processes or released in vitro by hydrolysis reactions using various proteases. Real-time investigation of proteolytic digestion is a suit- able strategy to identify the influence of certain condi- tions on protein digestion. Therefore, in this study, β- casein from bovine milk, which is an important source of bioactive peptides, was chosen as a model protein to investigate the influence of temperature and pH during its digestion by pepsin or trypsin. In further experiments, QCM results were verified by mass spectrometry (MS) and peptide sequencing tools. MATERIALS • β-casein from bovine milk, pepsin from porcine gastric mucosa, and trypsin from bovine pancreas (Sigma Aldrich, Taufkirchen, Germany) • 16-thiol-hexadecanoic acid (THDA) (Sigma Aldrich) • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma Aldrich) • α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Aldrich) RESULTS AND DISCUSSION Both the peptic and tryptic digests of bovine β-casein were performed at the sensor surface and the degrada- tion process was monitored in real-time. Fig. 4A shows the ∆f vs. time plot for peptic digestion. The rapid increase in frequency within the first 50 minutes indi- cates a fast enzymatic digestion, whereas the further curve progression represents only little mass decrease at the sensor surface. The immobilization of β-casein results in incomplete degradation by the enzyme as the orientation of the protein impedes enzyme binding. The influence of pH on the activity of pepsin and trypsin is shown in Fig. 4B and 5A. The decrease in the activity of pepsin between pH 2, 3, and 4 results in frequency shifts of 130 Hz, 80 Hz, and 15 Hz respectively within 50 minutes. In our experiments trypsin was most active at pH 7.4. Reduced activity with decreased pH was dem- onstrated by frequency shifts of 73 Hz, 46 Hz, and 15 Hz. The effect of different temperatures on the digestive pro- cess is shown in Fig. 5B. It can be seen that a tempera- ture rise of 5 K clearly leads to faster enzymatic degrada- tion. Applying mass spectrometry and sequencing software, several peptide sequences from bovine β-casein were unambiguously identified in the samples collected dur- ing the QCM experiments (see Fig. 6). ACKNOWLEDGMENTS The authors would like to thank Dr. A. Hauser from the Institute of Physical Chemistry for performing the AFM experiments. Martin Luther University Halle-Wittenberg Institute of Pharmaceutics and Biopharmaceutics, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany A. Huenerbein, C.E.H. Schmelzer, and R.H.H. Neubert Quartz Crystal Microbalance for On-line Investigations of Enzymatic Reactions on Dietary Proteins 1 2 3 Quartz Crystal Microbalance QCM experiments were carried out on a LiquiLab 21 (ifak e.V., Barleben, Germany). The device was modified in terms of cell design and valve control. Gold plated 10 MHz AT-cut quartz crystals were placed in the measur- ing cells. A temperature stability of 0.1K was maintained. All experiments were performed under flow conditions with a flow of 0.15 mL/min. Fig. 3 shows the experimen- tal setup. Diluted hydrochloric acid and phosphate buffered saline (PBS) were used as solvents for pepsin and trypsin, respectively. Initially the cells were rinsed with solvent for 10 minutes. Subsequently a 0.1 μmol enzyme solu- tion was applied to the sensor over the next 230 minutes. The enzyme was rinsed out by repeating the solvent flow for another 60 minutes. , Figure 3: Experimental setup (1: measuring cells; 2: bubble trap; 3: pump; 4: valves; 5: silicon tubes; 6: data acquisition and control) Figure 5: ∆f vs. time for tryptic digests; A: development of frequency at different pH-values; B: tem- perature dependence of tryptic digest Figure 6: MALDI-TOF survey spectrum of a peptic β- casein digest; proteolyses was performed in a QCM experiment at pH 3 Figure 2: An image of β-casein on the quartz electrode, captured by an atomic force microscope in non-contact mode 1 3 4 5 2 6 Solvent Solvent Enzyme Enzyme Sample collection MS Figure 4: ∆f vs. time for peptic digests; A: entire fre- quency shift over 300 min; B: development of frequency at different pH-values 0 50 100 150 200 250 300 0 20 40 60 80 100 120 140 160 0 20 30 40 50 0 20 40 60 80 100 120 140 ∆ f / Hz t / min ∆ f / Hz t / min A B 0 20 30 40 50 0 10 20 30 40 50 60 70 80 ∆ f / Hz t / min A 0 20 30 40 50 0 20 40 60 80 100 ∆ f / Hz t / min B pH 6.4 pH 6.4 pH 7.4 pH 7.4 30 °C 30 °C pH 3 pH 3 pH 2 pH 2 pH 4 pH 4 25 °C 25 °C pH 5.4 pH 5.4 Figure 1: Schema of immobilization (1: β-casein; 2: SAM of THDA; 3: quartz crystal)
Transcript of Quartz Crystal Microbalance for On-line Investigations of … · MALDI Mass Spectrometry MALDI...
MALDI Mass Spectrometry
MALDI experiments were carried out on a time-of-flight (TOF) mass spectrometer Voyager DE-PRO (Applied Biosystems, Foster City, CA, USA) equipped with a pulsed nitrogen laser. The samples collected from the QCM experiments were concentrated using ZipTips C18 units (Millipore, Schwalbach, Germany). As matrix a solution of 10 mg/mL CHCA in acetonitrile/0.1% TFA (1:1, v/v) was used. The resultant solution was mixed with the matrix solution 1:1 (v/v) on the sample plate. Then, the samples were dried in a gentle stream of air at ambient temperature (24 °C). Measurements were per-formed operating in the positive-ion reflector mode. Survey spectra of the peptide mixtures were obtained by accumulating data from 500 to 2000 laser shots.
CONCLUSIONS
• The described approach allows on-line investigations of proteolytic reactions under certain conditions.
• Additional information, such as peptide sequences or enzyme cleavage sites can be obtained by employing mass spectrometry.
⇒ Combining QCM and mass spectrometry provides a better insight in enzymatic degradation of proteins.
EXPERIMENTAL
Immobilization of β-casein
The immobilization of bovine β-casein was performed in a three stage process at room temperature. After every stage the quartz surface was dried under a stream of nitrogen. At first, a cleaned quartz crystal was sub-merged in a 1 mM ethanolic solution of THDA for two hours to obtain a self assembled monolayer (SAM), then douched with ethanol. In the second step the carboxylic groups of THDA were activated by covering the upper electrode with a 10 % (m/v) solution of EDC in diluted hydrochloric acid (pH 3.5). Finally, the quartz crystal was submerged in a 5 mg/mL solution of β-casein in a buffer containing borate (pH 8.8) for two hours. Washing with water and drying under nitrogen finalized the immobili-zation procedure. A schema of surface coating can be seen in Fig. 1. Atomic force microscopy shows the sur-face texture of immobilized β-casein (Fig. 2).
Corresponding author: [email protected]
REFERENCES
[1] K.A. Marx, Biomacromolecules 4 (5), (2003), 1099-1120
[2] G. Sauerbrey, Zeitschrift für Physik 155, (1959), 206-222
INTRODUCTION
In recent decades quartz crystal microbalance (QCM) has become a valuable tool for studies of biomolecular interactions, especially in liquid state [1]. A quartz crystal sandwiched between a pair of electrodes produces bulk acoustic waves when an AC voltage is applied. Typical oscillation frequencies are in the range of 5 to 10 MHz. Changes at the solid/liquid interface cause a frequency shift. The linear relationship between frequency shift (∆f) and adsorbed mass (∆m) is described by Sauerbreys equation [2]:
where f is the fundamental frequency, A the area of the 0
electrode, G the shear modulus and ρ the density of the q q
quartz crystal.
Proteins are, besides fats and carbohydrates, the main nutrients in human food. They show an amazing struc-tural variety, and among their breakdown products, the peptides, are numerous compounds with miscellaneous biological effects. These bioactive peptides can be gen-erated in vivo through gastrointestinal processes or released in vitro by hydrolysis reactions using various proteases.Real-time investigation of proteolytic digestion is a suit-able strategy to identify the influence of certain condi-tions on protein digestion. Therefore, in this study, β-casein from bovine milk, which is an important source of bioactive peptides, was chosen as a model protein to investigate the influence of temperature and pH during its digestion by pepsin or trypsin. In further experiments, QCM results were verified by mass spectrometry (MS) and peptide sequencing tools.
MATERIALS
• β-casein from bovine milk, pepsin from porcine gastric mucosa, and trypsin from bovine pancreas (Sigma Aldrich, Taufkirchen, Germany)
• 16-thiol-hexadecanoic acid (THDA) (Sigma Aldrich)
• 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma Aldrich)
• α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Aldrich)
RESULTS AND DISCUSSION
Both the peptic and tryptic digests of bovine β-casein were performed at the sensor surface and the degrada-tion process was monitored in real-time. Fig. 4A shows the ∆f vs. time plot for peptic digestion. The rapid increase in frequency within the first 50 minutes indi-cates a fast enzymatic digestion, whereas the further curve progression represents only little mass decrease at the sensor surface. The immobilization of β-casein results in incomplete degradation by the enzyme as the orientation of the protein impedes enzyme binding. The influence of pH on the activity of pepsin and trypsin is shown in Fig. 4B and 5A. The decrease in the activity of pepsin between pH 2, 3, and 4 results in frequency shifts of 130 Hz, 80 Hz, and 15 Hz respectively within 50 minutes. In our experiments trypsin was most active at pH 7.4. Reduced activity with decreased pH was dem-onstrated by frequency shifts of 73 Hz, 46 Hz, and 15 Hz.The effect of different temperatures on the digestive pro-cess is shown in Fig. 5B. It can be seen that a tempera-ture rise of 5 K clearly leads to faster enzymatic degrada-tion.Applying mass spectrometry and sequencing software, several peptide sequences from bovine β-casein were unambiguously identified in the samples collected dur-ing the QCM experiments (see Fig. 6).
ACKNOWLEDGMENTS
The authors would like to thank Dr. A. Hauser from the Institute of Physical Chemistry for performing the AFM experiments.
Martin Luther UniversityHalle-WittenbergInstitute of Pharmaceutics and Biopharmaceutics, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
A. Huenerbein, C.E.H. Schmelzer, and R.H.H. Neubert
Quartz Crystal Microbalance for On-line Investigations of Enzymatic Reactions on Dietary Proteins
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Quartz Crystal Microbalance
QCM experiments were carried out on a LiquiLab 21 (ifak e.V., Barleben, Germany). The device was modified in terms of cell design and valve control. Gold plated 10 MHz AT-cut quartz crystals were placed in the measur-ing cells. A temperature stability of 0.1K was maintained. All experiments were performed under flow conditions with a flow of 0.15 mL/min. Fig. 3 shows the experimen-tal setup. Diluted hydrochloric acid and phosphate buffered saline (PBS) were used as solvents for pepsin and trypsin, respectively. Initially the cells were rinsed with solvent for 10 minutes. Subsequently a 0.1 µmol enzyme solu-tion was applied to the sensor over the next 230 minutes. The enzyme was rinsed out by repeating the solvent flow for another 60 minutes.
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Figure 3: Experimental setup (1: measuring cells; 2: bubble trap; 3: pump; 4: valves; 5: silicon tubes; 6: data acquisition and control)
Figure 5: ∆f vs. time for tryptic digests; A: development of frequency at different pH-values; B: tem-perature dependence of tryptic digest
Figure 6: MALDI-TOF survey spectrum of a peptic β-casein digest; proteolyses was performed in a QCM experiment at pH 3
Figure 2: An image of β-casein on the quartz electrode, captured by an atomic force microscope in non-contact mode
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Figure 4: ∆f vs. time for peptic digests; A: entire fre-quency shift over 300 min; B: development of frequency at different pH-values
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Figure 1: Schema of immobilization (1: β-casein; 2: SAM of THDA; 3: quartz crystal)
