MATERIALS AND METHODS

SECTION II: Chapter IV

SECTION II: Chapter IV

Acrylamide/Bis solution (40%), N,N,N’,N’-tetramethylethylenediamine, acrylamide powder, ammonium persulfate as well as the linear IPGs were obtained from Bio-Rad Laboratories (Hercules, CA).

4.2.2. SAMPLE PREPARATION

4.2.2.1. Mab purification

Mab 1 was purified by hydrophobic charge induction chromatography (HCIC) on a MEP HyperCel column containing 4-mercaptoethyl-pyridine (MEP) [22]. Briefly, the filtered cell culture supernatant was directly loaded onto a MEP HyperCel column pre-equilibrated with physiological PBS. Elution of antibody was performed by lowering the pH with 50 mM acetate buffer pH 4.0. The collected IgG fraction was then stored frozen for further analysis. The purity was estimated as 96% by HPLC on a TSK G4000 SWXL analytical column.

Mab 2 preparation was purified by cation exchange chromatography on DEAE Ceramic HyperD (CM HyperD) [23]. Briefly, the filtered cell culture supernatant was adjusted to pH 4.5 and the ionic strength to a concentration of 0.12 M NaCl.

The column was also equilibrated with 50 mM acetate buffer pH 4.5, 0.12 M sodium chloride. Elution was performed by 50 mM acetate buffer pH 4.5 containing 1 M sodium chloride. The purity was estimated of 89%.

4.2.3. POLISHING METHODS

4.2.3.1. Purified sperm whale myoglobin

Commercial myoglobin from sperm whale was first purified by anion-exchange chromatography for removing a number of impurities naturally present in this preparation; after that it was mixed either with human serum proteins (at a ratio of 1.8 or 3.7 mg of protein per 100 mg of pure myoglobin) or with an E. coli protein whole extract (at a ratio of 5 mg of protein per 100 mg of pure myoglobin). The buffer used was 25 mM phosphate, pH 7.0. Subsequently, 400 mL of each solution was mixed with 80 mL of Equalizer Beads and incubated under gentle shaking for 20 min at room temperature. The mixture was then centrifuged in a spin column to drain out and collect the liquid phase containing polished 120

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myoglobin. The collected polished myoglobin was then analyzed as described in the following paragraphs.

4.2.3.2. Recombinant HSA, Protein A and injectable HSA

A solution of the purified protein to be polished was prepared in PBS. A column of solid-phase combinatorial peptide library was packed separately (1 mL volume), equilibrated with PBS and connected to a chromatographic setup comprising a pumping system and UV/pH detection unit for recording both events at the column outlet. The column was loaded continuously with the protein solution to be polished at a linear flow rate of 50 cm/h. The flowthrough was collected in fractions of a few millilitres each to analyze the capability of the solid phase to remove protein impurities, likewise a frontal analysis applied for the determination of the binding capacity of sorbents [24]. Once the load was terminated, a PBS solution was introduced for washing out the excess of proteins. Eventually, the captured proteins were desorbed from the beads by a solution of 9 M urea-citric acid pH 3.5, containing 2% CHAPS. In the case of injectable HSA, the recovery before electrophoresis was done using Laemmli buffer at 100°C for 5 minutes.

The protein concentration of each loaded solution was 15 mg/mL for Protein A, 10 mg/mL for recombinant HSA, and 50 mg/mL for injectable HSA; the total volumes loaded were 200, 100, and 10 mL, respectively. The desorbed proteins were collected as a single fraction.

4.2.3.3. Mab preparation

One hundred and fifty mg of each purified and lyophilized Mabs were dissolved in PBS at a concentration of 3 mg/mL. Separately, a small chromatographic column was filled with 0.5 mL of solid phase combinatorial peptide library and then equilibrated with PBS. The column was connected to a chromatographic system comprising a pump and a UV/pH detection unit for recording both events at the column outlet. The column was loaded continuously with protein solutions at a linear flow rate of 50 cm/hour. The flow-through was eliminated and, once the loading was completed, a PBS solution was introduced for washing out the proteins in excess. The captured proteins were finally desorbed using a solution of 9 M urea-citric acid, pH 3.3, containing 2% CHAPS. The fractions collected from 121

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both columns, containing 7 mg of amplified impurities, were then dialyzed and lyophilized prior to analysis.

4.2.4. ELECTROPHORETIC ANALYSIS

4.2.4.1. SDS gel electrophoresis

Mono-dimensional SDS-PAGE of collected protein impurities was performed by using 10-well pre-cast 1-mm-thick 16% polyacrylamide Tricine gel plates.

Samples of appropriate protein concentration were diluted 2-fold in sample buffer.

Thirty µL of diluted sample were loaded per lane and electrophoretic migration was carried out at 150 volts for about 90 minutes. Staining and de-staining were performed with Colloidal Coomassie Blue and 7% acetic acid water solution, respectively.

4.2.4.2. 2-DE analysis

The same protein amount (i.e. 150 µg) was used for all the samples investigated (starting material and Equalizer Bead eluates). The protein samples, after being dialyzed against ammonium acetate and subsequently lyophilized, were directly solubilized in IEF buffer (7 M urea, 2 M thiourea, 3% CHAPS, 40 mM Tris, 5 mM TBP and 10 mM acrylamide) and allowed to be alkylated at room temperature for 60 minutes. In order to stop the alkylation reaction, 10 mM DTT was added to the solution, followed by 0.5% Ampholine and a trace amount of bromophenol blue.

Seven-cm long IPG strips pH 3-10 (for myoglobin and recombinant HSA) or pH 4-7 (for recombinant protein A) were rehydrated with 150 μL of protein solution for 4 hours. IEF was carried out with an initial voltage gradient from 100 up to 1000 V, followed by 1000 volts constant for 5 hours. The voltage was then increased again rapidly up to 5000 volts in 30 min, and kept at such a value until reaching 30 000 Vh. For the second dimension the IPG strips were laid on a 10-20% acrylamide gradient gel, and the electrophoretic run was performed by setting a current of 5 mA/gel for 1h, followed by 10 mA/gel for 1h and 20 mA/gel until the dye front reached the bottom of the gel. Gels were then immediately stained with colloidal Coomassie Blue. Destaining was performed in 7% acetic acid. The 2-DE gels were scanned with a Versa-Doc Imaging System (Model 3000, Bio-Rad, Hercules 122

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CA), and the images were treated with the software PDQuest version 8.0 (Bio-Rad, Hercules CA).

4.2.5. SELDI-MS ANALYSIS

This analysis, based on both protein interaction with a chemically modified surface and MS for the discrimination of masses captured by the surface, was performed according to a previously described method [25]. Briefly, each spot of a ProteinChip® Array (referred to as biochip throughout the text) was equilibrated twice with 5 μL of the indicated array-specific binding buffer for 5 min according to the manufacturer’s instructions. Each spot was then loaded with 6 μL of the sample previously diluted with the array binding buffer so as to have the appropriate final protein concentration (i.e. 20 ng/μL). After an incubation period of 30 min with constant shaking, the sample was carefully removed and each spot was washed three times with 5 μL of the binding buffer for 5 min to eliminate non-adsorbed proteins, followed by a quick rinse with deionized water. All the surfaces were air-dried followed by two applications of a saturated solution of sinapinic acid in a mixture of 50% acetonitrile (ACN) in water containing 0.5% aqueous trifluoroacetic acid (TFA) (1 μL of this solution per application with air drying in between). The biochips were then analyzed by SELDI-TOF MS using a Ciphergen PCS 4000 ProteinChip Reader operated in a positive-ion mode, with an ion acceleration potential of 20 kV and a detector voltage of 2.8 kV. The molecular mass range investigated was from 1 kDa to 300 kDa. Time-lag focusing was optimized at either 5 kDa or 70 kDa for low and high mass ranges, respectively.

Processing of data included baseline subtraction and external calibration using a mixture of known peptide and protein calibrants. Peak detection (S/N>3) and peak clustering was performed automatically using ProteinChip® Software 3.2.

Biochips used throughout this study were CM10 (weak cation exchange), Q10 (strong anion exchange), H50 (hydrophobic surface) and IMAC30 (metal ion chelating surface) loaded with Cu⁺⁺ ions.

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4.2.6. PROTEIN IDENTIFICATION VIA MS/MS ANALYSIS

4.2.6.1. In-gel digestion

Spots were cut out from Colloidal Coomassie blue stained gels and subjected to in-gel trypsin digestion according to Shevchenko et al. [26] with minor modifications. The gel pieces were swollen in a digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/µL of trypsin (modified porcine trypsin, sequencing grade, Promega, Madison, WI) in an ice bath. After 30 min the supernatant was removed and discarded, 20 µL of 50 mM NH4HCO3 were added to the gel pieces and digestion was allowed to proceed at 37° C overnight. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Prior to mass spectrometric analysis, the peptide mixtures were redissolved in 10 µL of 5% formic acid (FA).

4.2.6.2. Protein identification by MS/MS

Peptide mixtures were separated by using a nanoflow-HPLC system (Ultimate;

Switchos; Famos; LC Packings, Amsterdam, The Netherlands). A sample volume of 10 µL was loaded onto a home-made 2 cm long fused silica pre-column (75 µm inner diameter, 375 µm outer diameter; Resprosil C18-AQ, 3 µm, Ammerbuch-Entringen, DE) at a flow rate of 2 µL/min. Sequential elution of peptides was accomplished using a flow rate of 200 nL/min and a linear gradient from solution A (2% acetonitrile, 0.1% FA) to 50% of solution B (98% acetonitrile, 0.1% FA) in 40 minutes over the pre-column in line with a homemade 10-15 cm resolving column (75 µm inner diameter; 375 µm outer diameter, Resprosil C18-AQ, 3 µm, Ammerbuch-Entringen, DE).

Peptides were eluted directly into a High Capacity ion Trap HCTplus (Bruker-Daltonik, Germany). Capillary voltage was 1.5-2 kV and a dry gas flow rate of 10 L/min was used at a temperature of 200°C. The scan range used was from 300 to 1800 m/z. Protein identification was performed by searching in the National Center for Biotechnology Information non-redundant database (NCBInr) using the Mascot program (http://www.matrixscience.com). The following parameters were adopted for database searches: complete carbamidomethylation of cysteines and partial oxidation of methionines, peptide Mass Tolerance ± 1.2 Da, fragment mass tolerance ± 0.9 Da, missed cleavages 2. For positive identification, the score had 124

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to be over the significance threshold level (P < 0.05). The MS analysis was carried out in the laboratory of Prof. Zolla (Laboratory of Proteomics, Department of Environmental Sciences, Tuscia University, Viterbo, Italy).