**2.7 Protein identification by mass spectrometry**

### *In situ* digestion

Protein spots were excised from the gel and destained by repetitive washes with 0.1 M NH4HCO3 pH 7.5 and acetonitrile. Enzymatic digestion was carried out with trypsin (12.5 ng/µl) in 10 mM ammonium bicarbonate buffer pH 7.8. Gel pieces were incubated at 4 °C for 2 h. Trypsin solution was then removed and a new aliquot of the same solution was added; samples were incubated for 18 h at 37 °C. A minimum reaction volume was used as to obtain the complete rehydratation of the gel. Peptides were then extracted by washing the

solution of 10 mg/mL α-cyano-4-hydroxycinnamic, whose preparation consisted in the resuspension of α-cyano-4-hydroxycinnamic in water and acetonitrile 10:1 (v/v). The instrument was calibrated using a mixture of standard peptides (Applied Biosystems). Spectra were register even in reflector positive. MS/MS spectra were performed with CID

IEF (first dimension) was carried out on non-linear wide-range immobilized pH gradients (pH 4-7; 7 cm long IPG strips; GE Healthcare, Uppsala, Sweden) and achieved using the EttanTM IPGphorTM system (GE Healthcare, Uppsala, Sweden). Analytical-run IPG-strips were rehydrated with 125μg� of total proteins in 125μl of rehydratation buffer (urea 8 M, CHAPS 2%, 0,5% (v/v) IPG Buffer, bromophenol blue 0,002%) for 12 h at 20°C. The strips were then focused according to the following electrical conditions at 20°C: 500 V for 30 min, from 1000 V for 30 min, 5000 V until a total of 15000 Vt was reached. After focusing IPG strips were equilibrated for 15 min in 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.05 M Tris-HCl, pH 6.8, 2% (wt/vol) DTT, and subsequently for 15 min in the same urea/SDS/Tris buffer solution but substituting the 2% (wt/vol) DTT with 2.5% (wt/vol) iodoacetamide. The second dimension was carried out on 12% polyacrylamide gels at 25 mA/gel constant current until the dye front reached the bottom of the gel. MS gel was

Gels images were acquired with an Epson expression 1680 PRO scanner. Computer-aided 2-D image analysis was carried out using the ImageMasterTM 2D Platinum software (GE Healthcare, Uppsala, Sweden). Differentially expressed spots were selected for MS

In order to find differentially expressed proteins, comassie stained gel image of serum proteins from healthy individual was matched with the one of myocarditis affected patient. The apparent isoelectric points and molecular masses of the proteins were calculated with ImageMaster 2D Platinum 6.0 using identified proteins with known parameters as

Relative spot volumes (%V) (V=integration of OD over the spot area; %V = V single spot/V total spot) were used for quantitative analysis in order to decrease experimental errors. The normalized intensity of spots on three replicate 2-D gels was averaged and standard

Protein spots were excised from the gel and destained by repetitive washes with 0.1 M NH4HCO3 pH 7.5 and acetonitrile. Enzymatic digestion was carried out with trypsin (12.5 ng/µl) in 10 mM ammonium bicarbonate buffer pH 7.8. Gel pieces were incubated at 4 °C for 2 h. Trypsin solution was then removed and a new aliquot of the same solution was added; samples were incubated for 18 h at 37 °C. A minimum reaction volume was used as to obtain the complete rehydratation of the gel. Peptides were then extracted by washing the

using air as collision gas. Spectra were manually interpreted.

**2.5 2D-gel electrophoresis** 

stained with colloidal comassie.

deviation was calculated for each condition.

**2.7 Protein identification by mass spectrometry** 

**2.6 Image analysis** 

analysis.

references.

*In situ* digestion

gel particles with 10mM ammonium bicarbonate and 1% formic acid in 50% acetonitrile at room temperature.

Mass spectrometry and protein identification

LC-MS/MS analyses were performed as previously described for free peptides.

Mass spectrometric obtained data were used for protein identification using the software MASCOT that compare peptide masses obtained by MS and MS/MS data of each tryptic digestion with the theoretical peptide masses from all the proteins accessible in the databases (Peptide Mass Fingerprinting, PMF). Database searches were performed in NBCI databank (National Center for Biotechnology Information), restricting the analysis to the pertinent taxonomies. The parameters used for the identification were: tolerance of 10 ppm on peptide mass, 0.6 Da on MS/MS, and cysteine carbamidomethylation as fixed modification. Variable modifications were methionine oxidation, glutamine conversion in pyro-glutamic acid, and asparagine deamidation.
