**1. Introduction**

348 Myocarditis

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V & Torzewski, J. (2002) Complement and dilated cardiomyopathy: a role of sublytic terminal complement complex-induced tumor necrosis factor-alpha Myocarditis is an inflammatory disease of the cardiac muscle which might be related to viral (mainly parvovirus B19 and many others), protozoan (*Borrellia burgdoferi, Trypanosoma cruzi, Toxoplasma gondi*), bacterial (*Brucella, Corynebacterium diphtheriae, Gonococcus, Haemophilus influenzae, Actinomyces, Tropheryma whipplei, Vibrio cholerae, Borrelia burgdorferi, Leptospira, Rickettsia*), fungal (*Aspergillus*) and other non viral pathogens (Rezkalla SH et al., 2010, Blauwet LA et al., 2010, Cihakova D et al., 2010) infections; It has been reported, however, that this kind of inflammation might be caused by an hypersensitivity response to drugs (Kühl U et al., 2009). The final effect in each case is represented by myocardial infiltration of immunocompetent cells following any kind of cardiac injury.

Myocarditis presents with many symptoms, from chest pain that spontaneously resolves without treatment to cardiogenic shock and sudden death (Kühl U et al., 2010, Taylor CL et al 2010). The major long-term consequence is dilated cardiomyopathy with chronic heart failure(Lv H et al. 2011, Stensaeth KH et al 2011).

Nowadays, diagnostic tools available for this disease are mainly related to general investigations (such as electrocardiography) and analysis of the most abundant serum proteins, whose alteration is related to cardiac pathology, even if it's not specifically connected to myocarditis itself. Here we propose preliminary speculations on the serum proteins profiling (both in the expression level and in the characterization of post translational modifications) and on free peptides identification in myocardytis affected patients (compared to healthy individual), that could be helpful in finding specific markers for this pathology.

As many other inflammatory events this disease involves in fact different kinds of biological macromolecules, here we focus in particular on proteins. More than 50% of total protein content in a cell is post translationally modified. The pattern of Post Translational Modifications (PTMs) on proteins constitute a molecular code that dictates protein conformation, cellular location, macromolecular interactions and activities, depending on cell type, tissue and environmental conditions (Diernfellner AC et al., 2011, Savidge TC,

<sup>\*</sup> Equally contributed to the work.

A Proteomic Approach to Investigate Myocarditis 351

Guanidine, dithiothreitol (DTT), trypsin, α-cyano-4-hydroxycinnamic acid were purchased from Sigma. Iodoacetamide (IAM), tris(hydroxymethyl)aminomethane, calcium chloride and ammonium bicarbonate (AMBIC) were purchased from Fluka as well as the MALDI matrix 2,5-, α-cyano-4-hydroxycinnamic. Methanol, trifluoroacetic acid (TFA) and acetonitrile (ACN) are HPLC grade type from Carlo Erba, whereas the other solvents are from Baker. Gel filtration columns PD-10 are from Pharmacia, the HPLC ones from

Comassie Brilliant Blue was from Bio-Rad. PNGase F were purchased from Boheringer. Ion exchange resins Dowex H+ (50W-X8 50-100 mesh) was provided by BDH. Concanavalin A

Sera protein concentration was determined by Bradford assay method, using bovine serum albumin (BSA) as standard. Known amounts of BSA were diluted in 800 μL of H2O and then mixed to 200 μL of Comassie Brilliant Blue. 5 different BSA concentrations were determined by measuring absorbance at 595 nm and used to obtain a linear calibration curve. Three different sera dilutions were measured at 595 nm. Absorbance data were interpoled on the calibration curve, allowing the determination of protein concentration in

The depletion of the most abundant proteins from each serum sample was performed using Multiple Affinity Removal Spin Cartridges from Agilent. The procedure was performed at room temperature according to manufacturer's instructions and then immediately frozen to

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

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

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

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

Phenomenex, whereas the pre-packed columns Sep-pak C-18 are from Waters.

sepharose resin was purchased from Amersham Biosciences.

**2.2 Protein concentration determination** 

the different samples.

**2.3 Serum depletion** 

**2.4 Free peptides analysis** 

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

2% acetonitrile) from 7% to 60% in 50 min.

conversion in pyro-glutamic acid as variable modifications.


analyses.

MALDI-TOF/TOF

2011, Hao P et al.,2011). It is very well known that biological function of many proteins is strictly related to the presence of the appropriate set of PTMs. Most importantly, PTMs deregulation might be involved in the development of diseases , in fact, as a consequence of many pathologies, PTMs set might be altered (Dell A et al., 2001, Kim YJ. Et al 1997, Dube DH. Et al., 2005, Granovsky M. Et al 2000).

Proteomics investigations offer all useful tools to deeply investigate this kind of alterations. Two dimensional electrophoresis coupled with software mediated image analysis, affinity chromatographies and especially Mass Spectrometry (MS) present high levels of sensitivity, accuracy and reproducibility and these are all fundamental requirements that this kind of study needs.

As previously described by our group (Carpentieri A, Giangrande C, et al., 2010), serum glycoproteome characterization might be crucial in clinical investigation. In fact we showed that the *N*-glycan profiling of serum glycoproteins extracted from myocarditis affected donors compared to the ones of healthy people shows many peculiarities. We demonstrated that many of the extracted oligosaccharides are in fact incomplete or truncated structures whilst others show a high level of fucosylation, all these results fully matched previously published data about the glycosylation in proteins during chronic inflammation events (Dell A et al., 2001, Kim YJ. Et al 1997, Dube DH. Et al., 2005, Granovsky M. Et al 2000, Carpentieri A, Giangrande C, et al., 2010).

Thanks to the high sensitivity of the most modern analytical techniques, several research groups focus the research at level of peptides rather than at protein level (Taylor-Papadimitriou J. et al., 1994 Amado F et al., 2010, Menschaert G et al. 2010). In fact, these endogenous peptides are referred to as the peptidome. Initially, peptidomic analyses were conducted as a method to study neuropeptides and peptide hormones; these are signaling molecules that function in a variety of physiological processes (Ludwig M. 2011, Colgrave ML et al., 2011). Recent studies have found large numbers of cellular peptides with half-lives of several seconds, raising the possibility that they may be involved in biological functions (Gorman PM et al 2003).

Here we propose preliminary data to show molecular basis of myocarditis using a proteomic approach. The analyses were focused on the serum proteins profiling, namely the study of the expression level and the characterization of glycoproteins involved in the pathology. A classical two-dimensional gel electrophoresis procedure to obtain protein maps and a "gel-free" comparison of the glycoproteomes in healthy and myocarditis human sera by using advanced mass spectrometry were reported. Free peptides identification in myocardytis affected patients (compared to healthy individual), was achieved in order to provide possible specific markers for this pathology.

### **2. Materials and methods**

#### **2.1 Materials**

Human serum samples from 8 healthy donors (all Caucasian 4 males and 4 females aged between 60 and 80 years, all other clinical informations were covered by laws on privacy) and from 5 myocarditis affected patients (all Caucasian 3 males and 2 females aged between 60 and 85 years, all other clinical informations were covered by laws on privacy), respectively, have been obtained from the "Servizio Analisi" Policlinico, Napoli. Aliquots of serum samples from different donors were pooled in order to obtain an average overview of glycoforms distribution.

2011, Hao P et al.,2011). It is very well known that biological function of many proteins is strictly related to the presence of the appropriate set of PTMs. Most importantly, PTMs deregulation might be involved in the development of diseases , in fact, as a consequence of many pathologies, PTMs set might be altered (Dell A et al., 2001, Kim YJ. Et al 1997, Dube

Proteomics investigations offer all useful tools to deeply investigate this kind of alterations. Two dimensional electrophoresis coupled with software mediated image analysis, affinity chromatographies and especially Mass Spectrometry (MS) present high levels of sensitivity, accuracy and reproducibility and these are all fundamental requirements that this kind of

As previously described by our group (Carpentieri A, Giangrande C, et al., 2010), serum glycoproteome characterization might be crucial in clinical investigation. In fact we showed that the *N*-glycan profiling of serum glycoproteins extracted from myocarditis affected donors compared to the ones of healthy people shows many peculiarities. We demonstrated that many of the extracted oligosaccharides are in fact incomplete or truncated structures whilst others show a high level of fucosylation, all these results fully matched previously published data about the glycosylation in proteins during chronic inflammation events (Dell A et al., 2001, Kim YJ. Et al 1997, Dube DH. Et al., 2005, Granovsky M. Et al 2000,

Thanks to the high sensitivity of the most modern analytical techniques, several research groups focus the research at level of peptides rather than at protein level (Taylor-Papadimitriou J. et al., 1994 Amado F et al., 2010, Menschaert G et al. 2010). In fact, these endogenous peptides are referred to as the peptidome. Initially, peptidomic analyses were conducted as a method to study neuropeptides and peptide hormones; these are signaling molecules that function in a variety of physiological processes (Ludwig M. 2011, Colgrave ML et al., 2011). Recent studies have found large numbers of cellular peptides with half-lives of several seconds, raising the possibility that they may be involved in biological functions

Here we propose preliminary data to show molecular basis of myocarditis using a proteomic approach. The analyses were focused on the serum proteins profiling, namely the study of the expression level and the characterization of glycoproteins involved in the pathology. A classical two-dimensional gel electrophoresis procedure to obtain protein maps and a "gel-free" comparison of the glycoproteomes in healthy and myocarditis human sera by using advanced mass spectrometry were reported. Free peptides identification in myocardytis affected patients (compared to healthy individual), was achieved in order to

Human serum samples from 8 healthy donors (all Caucasian 4 males and 4 females aged between 60 and 80 years, all other clinical informations were covered by laws on privacy) and from 5 myocarditis affected patients (all Caucasian 3 males and 2 females aged between 60 and 85 years, all other clinical informations were covered by laws on privacy), respectively, have been obtained from the "Servizio Analisi" Policlinico, Napoli. Aliquots of serum samples from different donors were pooled in order to obtain an average overview of

DH. Et al., 2005, Granovsky M. Et al 2000).

Carpentieri A, Giangrande C, et al., 2010).

provide possible specific markers for this pathology.

(Gorman PM et al 2003).

**2. Materials and methods** 

glycoforms distribution.

**2.1 Materials** 

study needs.

Guanidine, dithiothreitol (DTT), trypsin, α-cyano-4-hydroxycinnamic acid were purchased from Sigma. Iodoacetamide (IAM), tris(hydroxymethyl)aminomethane, calcium chloride and ammonium bicarbonate (AMBIC) were purchased from Fluka as well as the MALDI matrix 2,5-, α-cyano-4-hydroxycinnamic. Methanol, trifluoroacetic acid (TFA) and acetonitrile (ACN) are HPLC grade type from Carlo Erba, whereas the other solvents are from Baker. Gel filtration columns PD-10 are from Pharmacia, the HPLC ones from Phenomenex, whereas the pre-packed columns Sep-pak C-18 are from Waters.

Comassie Brilliant Blue was from Bio-Rad. PNGase F were purchased from Boheringer. Ion exchange resins Dowex H+ (50W-X8 50-100 mesh) was provided by BDH. Concanavalin A sepharose resin was purchased from Amersham Biosciences.
