2 − 1. Production of labeled structured peptides for capturing molecules
The major reagents used for peptide syntheses including amino acid derivatives, (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), 1-hydroxybenzotriazol (HOBt) and fluorescent dye, 5(6)-carboxyfluoresceine (FAM) and 5(6)-carboxytetramethylrhodamine (TAMRA), e-maleimidocaproic acid (EMCA) were from HiPep Laboratories (Kyoto, Japan). Solid supports used were TentaGel® S-RAM, from Rapp Polymere GmbH, Tübingen, Germany. Other reagents and solvents were purchased from Nacalai Tesque Inc. (Kyoto, Japan) and used as received. Water was prepared by a Milli-Q® apparatus (Merck-Millipore, Tokyo, Japan). On-line LC-MS (LC: Agilent 1100, Agilent Technologies Inc., Tokyo, Japan, MS: HCTultra, Bruker Japan K.K., Yokohama, Japan) was used for quality assessment of peptides used in this study. A 384-well microtiter plate (Thermo Fisher Scientific K.K., Yokohama, Japan) was used to prepare the peptide solution for creating the peptide microarray. Structured peptide libraries used in the present study were synthesized using automated synthesizers, PSSM-8 (Shimadzu Corp., Kyoto, Japan), and/or manual synthesizers, PetiSyzer®s (HiPep Laboratories) by the Fmoc solid-phase strategy and cleaved from resin. After purification on preparative scale columns (50 i.d.×250 mm), one mg of the synthesized fluorescently labeled peptide was dissolved in 180 µL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/H2O/AcOH = 1/1/1 (v/v/v) and dimethyl sulfoxide (DMSO) was added to make a total volume of 400 µL as a stock solution. The concentration of the stock solution was determined by UV measurement using a NanoDrop™ (Thermo Fisher Scientific). The stock solution was diluted 200-fold with methanol and the absorbance at 450–600 nm was measured at 25°C. The concentration of fluorescently labeled peptides in the stock solution was calculated from the absorbance of the maximum absorption wavelength of TAMRA (545 nm) and the absorption coefficient of TAMRA (ε = 91000). The prepared stock solutions were stored at -80°C until used for arraying. Circular dichroism (CD) measurements were performed in 20% trifluoroethanol, 20 mM Tris-HCl containing 0.5 mM CaCl2 (pH 7.4). CD spectroscopy was performed on a Jasco J-720WI spectropolarimeter (JASCO Corp., Tokyo, Japan) with a thermoregulator using a quartz cell with 1 mm pathlength at 25°C.
2–2. Substrate with amino-surface and quantitative determination of surface amino groups and determintion of amino group
A photo-surface processor PL16-110 (Sen Lights Corp., Osaka, Japan) was used as the UV irradiation device. Demension of the amorphous carbon substrate (Nippon Light Metal Co., Ltd., Tokyo, Japan) is width 25.0 x length 75.0 x thickness 1.0 mm, dimensional tolerance: ± 0.1 mm, thickness tolerance: ± 0.03 mm. To increase the hydrophilicity of the amorphous carbon substrate UV irradiation (18.5 mW/cm2, 254 nm) was performed for 15 min. After UV-irradiation carbon substrates were coated with ethanol solution of 1% polyarylamine (PAA) (average molecular weight: 3,000, Nittobo Medical Co. Ltd., Tokyo, Japan) and 1% polyehtyene glycohl (PEG) (average molecular weight: 4000, Sigma-Aldrich K.K., Tokyo, Japan) over the entire surface of the substrate by a Baker applicator (K Paint Applicator, Matsuo Sangyo, Co., Ltd., Osaka Japan), coating thickness was set at 0.076 mm, a coating amount of PAA of 20 µg/cm2. After evaporation of the solvent, the substrate was dried in vacuo for 1 hour, and then under vacuum, 3 min ultraviolet irradiation (18.5 mW/cm2, 254 nm) was performed to immobilize polymer. Further, the substrate was washed with ultrapure water for 1 hour to remove unreacted polymer and then subjected to spin drying to obtain an amino-substrate. The quantification of amino groups on the substrate was carried out by the bromoacetic acid surface quantification method. The resulting polymer coated substrate (12.5x10 mm) was dipped into 0.25 M bromoacetic anhydride-dimethylformamide (DMF) solution and incubated for 1 h at room temperature. It was washed by dip-washing in MeOH, dried under natural conditions, and dipped into 1 mL of 10 mM 2-mercaptoethanol solution. After incubation at room temperature for 1 hour, the dipping solution was collected for bromide ion determination using a personal ion analyzer PIA-1000 (Shimadzu Corp.). In this method, non-specific adsorption to untreated areas on the substrate is also reflected in the quantitative results. Therefore, the same operation was performed on the untreated substrate for accurate quantification. The amount of bromide ions bound to the aminated substrate was calculated according to Eq. 1. The amounts of amino group on the surface were ca. 4–5 nmol/cm2 (commercial aminated glass was 2.6 pmol/mm2).
2–3. Method for evaluation of the distribution of surface amino-group by histogram
The above amino-carbon substrate was reacted with TAMRA and measured by scanner, CRBio II (Hitachi Software Engineering Co., Ltd., Tokyo, Japan). Conditions: Sensitivity of Photomultiplier Tube: 65%, Laser Power: 40%. The resulting fluorescent images were incorporated into Photoshop Ver. 6.0 (Adobe Inc., CA, USA) and inverted the gradation of images were inverted and displayed as histogram of brightness and number of pixels.
2–4. Direct preparation for carboxylate surface
The surface of an amorphous carbon plate polished to a surface roughness of 1 nm was irradiated with 254 nm UV light for 15 min using a PL16-110. The substrate was coated with 2 w/v% polyacrylic acid (average molecular weight 5000, Nippon Shokubai Co., Ltd., Osaka Japan) to 20 µg/cm2 and dried in vacuo for 1 hour. After drying, the substrates were irradiated with 254 nm UV light for 4 min in vacuo to immobilize polyacrylic acid. After immobilization of polyacrylic acid, the substrates were washed with ultrapure water for 1 hour with shaking and spin dried.
2–5. Conversion of carboxyl to amino surface
A carboxyl PepTenChip® (PTC-CA-NN-02, HiPep Laboratories) was derivatized with EMCA (HiPep Laboratories) by the following procedures. Firstly, the substrate was placed in a Slide Fix™ slide jar (Evergreen Scientific, CA, USA) and 10 mM DIPCDI (Sigma-Aldrich K.K.) and 10 mM NHS dissolved in DMF were added and incubated at room temperature for 1 hour. To the resulting mixture 10 mM hexamethylene diamine (Nacalai Tesque Inc.) dissolved in DMF was added and gently stirred at room temp for 1 h, washed 5 times with methanol and dried in vacuo. The chip-surface was thus aminated. A solution of 20 mM EMCA, 10 mM N,N’-diisopropylcarbodiimide (DIPCDI) and N-hydroxysuccinimide (NHS) in dichrolomethane (DCM) was prepared in LibraTube® RT5M (HiPep Laboratories) and shaken at room temperature for 1 hour. After removal of white precipitates by filtration the liquor was added to the above slide jar, diluted with DCM (30 mL) and pyridine was added (10 mM). The resulting mixture was shaken at room temperature for 1 h, washed 5 times with methanol and dried in vacuo.
2–6. Immobilization of capturing molecules on a substrate by manual arraying
Fluorescently labeled peptide probes were manually arrayed on carbon substrates derivatized with EMCA. Fluorescence-labeled peptides were prepared in 1% acetic acid at a concentration of about 100 mM. Since the immobilized probes will be removed by the subsequent washing procedure, there is no need to prepare the concentrations strictly. Substrates, on which 48 spots were derivatized by EMCA, were used for the preparation of peptide arrays and later insertion on an ultrafleXtreme™ (Bruker Japan K.K.). It is also possible to use fully derivatized substrates with an incubation cassette for 24-blocks. Peptide probes were manually arrayed in 0.2 µL by a micropipette. Then, the substrates were incubated in wet and light-shielded environment at room temperature for 30 min. The substrate was washed 5 times with 10 mM mercaptoethanol/2-propanol, spin-dried, and the fluorescence intensity (I0) was measured before applying analytes. After addition of analytes fluorescent intenity (I1) was measured. Fluorescent intensity changes were calculated according to an Eq. 2
2–7. Manufacture of peptide microarray by an arrayer
The microarrayer (NanoPrint LM-60, Arrayit Corp., CA, USA) and printing pin (946MP4, spot diameter 160 µm, Arrayit Corp.) were used for mnufacturing the present peptide microarray according to the manufacture's protocol. Fluorescent labeled peptide stock solution was prepared at 100 µM with 1% acetic acid. Prepared peptide solution was dispensed to a 384- well microtiter plate and set in the NanoPrint LM-60 and arrayed. After arraying, the substrate was washed 3 times with 2-mercaptoethanol solution and ultrapure water and spin-dried. After spin drying, the substrate was observed with a fluorescence detection device (PepTenCam, HiPep Laboratories), and confirmed that the spot state was correct.
2–8. Washing protocol for re-use
As a control, 10 µL of phosphate buffered saline (PBS) was dispensed on to chip and a cover glass was put on it to acquire a fluorescence image, which was set as I0. Next, 10 µL of the sample was used and a cover glass was placed on it, and the mixture was incubated at room temperature for 30 minutes to obtain a fluorescent image according to the procedure of a normal assay, which was designated as I1. The operation of removing the cover glass, immersing it in the substrate cleaning solution and cleaning was repeated 5 times, and spin drying was performed. The sample (10 µL) was applied on the dried substrate again, covered with a cover-glass, and incubated for 30 min. at room temperature to obtain a fluorescent image. In this way, the assay-process a fluorescent image and washing was repeated 5 times. This operation was repeated up to 20 times (total number of washings 100 times), and data was acquired. After removal of analyte by washing the chip can be stored for several months in an aluminum coated-bag (Ramizip® AL9, Seisannipponnsha Co.,Ltd., Tokyo, Japan).
2–9. Chip and MS experiments with manual operation
For this assay a 24 block chamber were used. Preliminary 150 of fluorescent labeled a-helical peptides were tested with hemoglobin and four peptides (A0394, A0535, A0536 and A0537) were found to be interacted with hemoglobin. These peptides, 0.2 µL each, were placed by manual array on the EMCA-derivatized carbon substrate surface and incubated at room temperature for 30 min for immobilization. 30 min later, the substrate surface was washed five times with 10 mM mercaptoethanol isopropanol. And then, 50 µL of PBS was placed in the wells and incubated for 30 min at room temperature. After 30 minutes, PBS was removed from the wells, spin dried, and fluorescence intensity was measured(I0). Next, 50 µL of hemoglobin solution as a captured molecule (10 µg/mL) was placed in the wells and incubated for 30 min at room temperature. After 30 min, the protein solution was removed and wells were washed 3 times with 0.1% Tween 20 in PBS, PBS, ultrapure water, and finally spin-dried followed by fluorescence intensity measurement (I1). Sinapinic acid solution consisting of 1 µL of 0.1% TFA/30% acetonitrile was applied as matrix at the position where the peptides was spotted and dried spontaneously. Protein Calibration Standard I (Bruker Japan K.K.) was spotted at the position where the peptide was not immobilized, and Hemoglobin 1 mg/mL was spotted as a positive control. The presnt chip was mounted on an adapter (P/N 8235380, Burker Japan K.K.), and inserted into the mass spectrometer.
2–10. Chip and MS experiments for PepTenChip® prepared by an arrayer
Fluorescently labeled peptides (48 α-helix, 100 β-sheet, 100 β-loop) were arrayed in one block on a carbon substrate whose surface was derivatized by EMCA using LM-60. After arraying, the substrate was washed three times with 2-Propanol/H2O = 1/1. The peptide-arrayed substrates were incubated with 10 µL of BSA solution (1 mg/mL in 50 mM NH4HCO3) for 1 hour at room temperature, covered with a cover glass, and incubated in the dark. The cover glass was removed and the substrate surface was rinsed with running water and spin dried. The Protein Calibration Standard I was spotted as above and BSA solution (0.2 µL) as a positive control was spotted followed by spraying with trypsin (Sigma-Aldrich K.K.) using TM-Sprayer™ M3 (HTX Technologies, LLC., NC, USA). The spraying conditions are shown in the Online resource 1. After trypsin spraying, substrates were incubated with NH4HCO3 for 2 h at 37 ºC in a humidified environment. The matrix CHCA (Sigma-Aldrich K.K.) was sprayed using the above TM-Sprayer and Imaging Mass spectrometry (IMS) measurements were performed by a Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS).