**2.4 Free peptides analysis**

300 μL acetonitrile was added to 100 μL of serum, incubated at room temperature for 30 minutes and then centrifuged at 13000 rpm. Supernatants were collected and concentrated by vacuum centrifugation (SAVANT) and resuspended in 20 μL formic acid 0.1%. Samples were desalted by C18 Zip Tip (Millipore) and diluted 100 times prior mass spectrometry analyses.

### LC/MS-MS HPLC-Chip/Q-TOF 6520

Peptides were analyzed by a HPLC-Chip/Q-TOF 6520 (Agilent Technologies). The capillary column works at a flow of 4 μL/min, concentrating and washing the sample in a 40 nL enrichment column. The sample was then fractionated on a C18 reverse-phase capillary column (75 μm~43 mm in the Agilent Technologies chip) at flow rate of 400 nl/min, with a linear gradient of eluent B (0.2% formic acid in 95% acetonitrile) in A (0.2% formic acid in 2% acetonitrile) from 7% to 60% in 50 min.

Data were acquired through MassHunter software (Agilent Technologies). Proteins identification was achieved by using Mascot software (Matrix science), with a tolerance of 10 ppm on peptide mass, 0.6 Da on MS/MS, and choosing methionine oxidation and glutamine conversion in pyro-glutamic acid as variable modifications.

#### MALDI-TOF/TOF

Peptides were also analyzed by MALDI-TOF/TOF using a 4800 Plus MALDI-TOF/TOF (Applied Biosystems). Samples were mixed on MALDI plate to the matrix consisting of a

A Proteomic Approach to Investigate Myocarditis 353

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

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

Glycoproteins were puried using PBA-bound agarose (Sigma-Aldrich, Munich, Germany). 500 µl of sample previously diluted (1 : 1) with equilibration buffer (50 mM taurine/NaOH, pH 8.7, containing 3–10 mM MgCl 2 ) was incubated with 200 µl of pre-washed immobilized ligand resin for 1 h on ice and with gentle shaking. After transfer of the resin into 1,5 ml eppendhorf tubes, the non-binding fraction was collected by low speed centrifugation (10 s, 500 × g). The resin was then thoroughly rinsed with equilibration buffer (six washes of 150 µl each) and 1 N NaCl (three washes of 150 µl each). For nal elution of the bound fraction, a total of six washes (150 µl each) with taurine buffer containing 50 mM sorbitol were used.

Glycopeptides were lyophilized, resuspended in 10 mM AMBIC and incubated with PNGase F (5 U), for 12-16 h at 37°C. Deglycosylation was carried out also on unbound peptides, in order to release the glycans not recognized by Boronate affinity

The analyses to investigate the "peptidomic" both in healthy and in pathological samples were accomplished by using a gel-free approach. The peptide component of the eluted fraction was analyzed by tandem mass spectrometry. The stringency of scoring parameters of the MASCOT algorithm minimized the number of false positive identifications. Most MS/MS spectra giving positive hits were derived from doubly and triply charged precursor

Triplicate LC-MS/MS analysis of supernatants after ACN precipitation of serum proteins, showed the occurrence of many free peptides in both analyzed sera. As reported in Table 1, the total number of detected and identified peptides was 41. Among these, 9 peptides were unique in the pathologic sample. It should be noted that some peptides were identified in

Three successive fractions (150 µl each) were pooled before further analysis.

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

room temperature.

**2.9 Deglycosylation** 

chromatography.

both samples.

**3. Results and discussion 3.1 Free peptides analysis** 

ions that resulted predominantly in y-ion series.

Mass spectrometry and protein identification

pyro-glutamic acid, and asparagine deamidation.

**2.8 Boronate affinity chromatography** 

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 using air as collision gas. Spectra were manually interpreted.
