2.3.5. Mass Spectrometry
The spots selected for identification were excised from silver-stained gels and destained with ferricyanide/thiosulfate on the same day as silver staining in order to improve the efficiency of the identification process [63
]. In gel digestion was performed with an automated protein digestion system, MassPrep Station (Waters, Milford, MA, USA). The gel plugs were washed twice with 50 µL of ammonium hydrogen carbonate (NH4
) 25 mM and 50 µL of acetonitrile. The cysteine residues were reduced by 50 µL of 10 mM dithiothreitol at 57 °C and alkylated by 50 µL of iodoacetamide 55 mM. After dehydration with acetonitrile, the proteins were cleaved in gel with 10 µL of 6.5 ng/µL of modified porcine trypsin (Promega, Madison, WI, USA) in NH4
25 mM. The digestion was performed overnight at room temperature. The generated peptides were extracted with 30 µL of 60% acetonitrile in 0.1% formic acid. Acetonitrile was evaporated under vacuum before nanoLC-MS/MS analysis.
NanoLC-MS/MS analysis was performed using a nanoACQUITY Ultra-Performance-LC (Waters Corporation, Milford, MA, USA) coupled to the SynaptTM High Definition Mass SpectrometerTM (Waters Corporation, Milford, MA, USA), or to the TripleTOF 5600 (Sciex,Framingham, MS, USA).
The nanoLC system was composed of ACQUITY UPLC® CSH130 C18 column (250 mm × 75 µm with a 1.7 µm particle size, Waters Corporation, Milford, MA, USA) and a Symmetry C18 precolumn (20 mm × 180 µm with a 5 µm particle size, Waters Corporation, Milford, MA, USA). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). 4 µL of sample were loaded into the enrichment column during 3 min at 5 µL/min with 99% of solvent A and 1% of solvent B. Elution of the peptides was performed at a flow rate of 300 nL/min with a 8–35% linear gradient of solvent B in 9 min.
The SynaptTM High Definition Mass SpectrometerTM (Waters Corporation, Milford, MA, USA) was equipped with a Z-spray ion source and a lock mass system. The system was fully controlled by MassLynx 4.1 SCN639 (Waters Corporation, Milford, MA, USA). The capillary voltage was set at 2.8 kV and the cone voltage at 35 V. Mass calibration of the TOF was achieved using fragment ions from Glu-fibrino-peptide B on the [50;2000] m/z range. Online correction of this calibration was performed with Glu-fibrino-peptide B as the lock-mass. The ion [M + 2H]2+ at m/z 785.8426 was used to calibrate MS data and the fragment ion [M + H]+ at m/z 684.3469 was used to calibrate MS/MS data during the analysis.
For tandem MS experiments, the system was operated with automatic switching between MS (0.5 s/scan on m/z range [150;1700]) and MS/MS modes (0.5 s/scan on m/z range [50;2000]). The two most abundant peptides (intensity threshold 20 counts/s), preferably doubly and triply charged ions, were selected on each MS spectrum for further isolation and CID fragmentation using collision energy profile. Fragmentation was performed using argon as the collision gas.
Mass data collected during analysis were processed and converted into pkl files using ProteinLynx Global Server 2.3 (Waters Corporation, Milford, MA, USA). Normal background subtraction type was used for both MS and MS/MS with 5% threshold and polynomial correction of order 5. Smoothing was performed on MS/MS spectra (Savitsky-Golay, 2 iterations, window of 3 channels). Deisotoping was applied for MS (medium deisotoping) and for MS/MS (fast deisotoping).
The TripleTOF 5600 was operated in positive mode, with the following settings: ionspray voltage floating (ISVF) 2300 V, curtain gas (CUR) 10, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 2, declustering potential (DP) 80 V. Information-dependent acquisition (IDA) mode was used with Top 10 MS/MS scans. The MS scan had an accumulation time of 250 ms on m/z [400;1250] range and the MS/MS scans 100 ms on m/z [150;1800] range in high sensitivity mode. Switching criteria were set to ions with charge state of 2–4 and an abundance threshold of more than 500 counts, exclusion time was set at 4 s. IDA rolling collision energy script was used for automatically adapting the CE. Mass calibration of the analyser was achieved using peptides from digested BSA. The complete system was fully controlled by AnalystTF 1.7 (Sciex) Raw data collected were processed and converted with MSDataConverter in mgf peak list format.
For protein identification, MS/MS data were interpreted using a local Mascot server with MASCOT 2.5.1 algorithm (Matrix Science, London, UK) against UniProtKB/SwissProt (version 2018_11, 558,898 sequences), without taxonomical restrictions. Spectra were searched with a mass tolerance of 15 ppm for MS and 0.05 Da for MS/MS data, allowing a maximum of one trypsin missed cleavage. Carbamidomethylation of cysteine residues and oxidation of methionine residues were specified as variable modifications. Protein identifications were validated with at least two peptides with Mascot ion score above 30.
Classical contaminants from human skin (keratins, filaggrin, desmoglein, involucrin) were removed from identifications. To cope with multiple identification issues from single 2D gel spots, univocal identifications were reported when the fist candidate was identified by at least twice more unique peptides than the next candidate or represented at least twice more spectra than the next candidate, and corresponded to the correct species (Mus musculus
). This allowed removal of contaminating serum proteins (Bos taurus
) from the identifications. In order to decrease the severity of the multiple identification problem, a graded identification strategy was used. A first identification of the spots of interest was attempted on analytical gels (loaded with 150µg protein, silver stained) when unsuccessful, more heavily loaded gels were used (250 µg protein, silver stained). If still unsuccessful, micropreparative gels (1 mg protein, colloidal Coomassie Blue-stained) were used. This strategy limited the problems associated with increased diffusion and streaking of proteins on 2D gels at high protein loads [64