Human dermal fibroblast secretomes (normal & Huntington's disease)
Description
Comparative analysis of the composition of the non-vesicular fraction of the secretomes of human dermal fibroblasts from a patient with Huntington's disease (HDDF line, sample #42) and a healthy person (DF262 line, sample #32).
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The protein samples were digested overnight by Trypsin Gold (Promega). Prior to reversed-phase fractionation, digested samples were resuspended in 50 μl of 1% (v/v) formic acid in water and filtered through 0.2 μm PVDF filter. Peptides were separated with a Chromolith CapRod RP-18e HR reversed-phase column (0.1 mm × 150 mm, Merck, Darmstadt, Germany) on a nano LC system (Eksigent NanoLC Ultra 2D+ system, SCIEX, Darmstadt, Germany). A total peptide amount of 600 ng was loaded and separated using a linear gradient of 0–50% B over 115 minutes followed by 50–100% B for 1 min and 100–100% B for 4 min at a flow rate of 400 nl/min. The mobile phases used were A, 5% acetonitrile with 0.2% (v/v) TFA in water and B, 60% (v/v) acetonitrile in water. The column was operated at a room temperature of 22-24 °C. The effluent from the column was mixed with matrix solution (CHCA 5 mg/ml, 0.2% (v/v) TFA in 95% methanol) containing two calibration standards bradykinin 2-9 (30 pM/ml) and ACTH 18-39 (60 pM/ml), at a flow rate of 1.4 μl/min. A micro-fraction collector was used to deposit 1 mm spots every 5 s, and a total of 704 fractions were collected in a 44x16 array for each nano LC run. The column was washed with a gradient (0–100–100% B for 5 min and 2 min respectively, at a flow rate of 800 nl/min) and equilibrated to 0% B for 3.5 minutes before subsequent injections. We analyzed the fractionated samples with a TOF/TOF 5800 System (SCIEX, Darmstadt, Germany) instrument operated in the positive ion mode. We set the MALDI stage to continuous motion mode. MS data was acquired at 3200 laser intensity with 1000 laser shots/spectrum (200 laser shots/sub-spectrum) and MS/MS data were acquired laser in-tensity at 3900 with a DynamicExit algorithm and a high spectral quality threshold or a maximum of 1000 laser shots/spectrum (250 laser shots/sub-spectrum). Up to 25 top precursors with S/N >40 in the mass range 750–4000 Da were selected from each spot for MS/MS analysis. We used the Protein Pilot 5.0 software (SCIEX, Darmstadt, Germany) with the Paragon algorithm 5.0 in thorough mode, for the MS/MS spectra search against the Uniprot human database. Carbamidomethyl cysteine was set as a fixed modification. False discovery rate (FDR) analysis was done by analysis of reversed sequences using the embedded PSEP tool. The MS/MS data was converted to mzidentml format for further analysis using Scaffold 4.0 software.