Cm10 proteinchip array

Second-voided urine samples were collected from each patient and, for ccRCC patients, before nephrectomy. The samples were processed as previously reported [20 ] and stored at -80°C until use. Prior to each analysis, the instrument performance was checked by the ProteinChip OQ kit (BIORAD). All the samples were analysed within a short time frame when the machine was working to standard. The SELDI analysis was carried out by loading 10 μg proteins from each patient in duplicate on CM10 ProteinChip array (BIORAD, cat. C57-30075) and following our previously described protocol [23 (link)]. After acquisition, the spectra were analysed by Protein chip DataManager™ 3.5 software (BIORAD, Hercules, CA, USA). The analysis was performed in a m/z range from 3,000 to 30,000 daltons, considering as real peaks those with S/N and a valley depth ratio greater than 4. All the spectra were normalized by means of total ion current. The reproducibility of the SELDI analysis, assessed as previously described [20 ], was comparable to that in our previous works [22 (link), 23 (link)]. Only mass peaks showing a statistically significant different expression (p<0.05) between cases and controls were considered for further analyses.

The cell culture supernatants from mouse TCs and BALBc/3T3 cells were analysed in triplicate on a weak cation-exchange (CM10) ProteinChip array (Bio-Rad Laboratories, Hercules, CA, USA). The chips were prepared and the samples were loaded according to the manufacturer’s instructions for low-stringency buffer conditions. The concentrations of all samples were equalized by adding binding buffer (0.1 M Sodium acetate, pH 4.0), and 1 μl of saturated energy absorbing molecule solution (sinapinic acid in 50% acetonitrile, 0.5% trifluoroacetic acid and 49.5% high-performance liquid chromatography grade water) was added twice and allowed to air dry.
The samples were analysed using time-of-flight mass spectrometry on a PBS II Protein Chip Reader (Bio-rad Laboratories, Hercules, CA, USA). The spectra were collected using the low-setting protocol: focus mass 5000 D, matrix attenuation 1000 D, sampling rate 800 MHz, energy 5000 nJ. The spectra were analysed using the ProteinChip Data Manager Software version 3.0.7 (Bio-rad Laboratories, Hercules, CA, USA). To improve the detection, the protein concentration was enhanced by freeze-drying the samples and reconstituting them in MilliQ ultrapure water (25% of the original volume).

Serum samples were collected and cryopreserved from each patient at baseline, 6 months, 12 months and 24 months. Samples were not available from all patients at each time-point; a total of 475 samples were available for SELDI-TOF mass spectrometry. Samples were randomized, and staff were blinded to the treatment arms. CM10 ProteinChip arrays (Bio-Rad Laboratories) were primed with binding buffer (50 mM ammonium acetate, 0.01% Triton X-100, pH 4·0) and incubated at room temperature (RT) for 5 min. A 1:10 dilution of serum in binding buffer was then applied to the array and incubated at RT for 1 hr. The arrays were washed twice with binding buffer and deionized water. Saturated sinapinic acid (0.7 µL) was applied twice to each spot on the arrays. Time-of-flight spectra were generated using a PCS-4000 mass spectrometer (Bio-Rad). Low-range spectra (mass/charge (m/z) ratio 0 – 20,000) were obtained at a laser energy of 3000 nJ, with a focus mass of 6000 and the matrix attenuated to 1000. High-range spectra (m/z 10,000 – 75,000) were obtained at a laser energy of 3900 nJ, with a focus mass of 30,000 and the matrix attenuated to 10,000. Mass accuracy was calibrated externally using All-in-One Peptide or Protein molecular mass standards (Bio-Rad).