**1. Introduction**

In the beginning of last century, the first antimicrobial protein, lysozyme, was reported. A few years later, the best known antimicrobial compound, called penicillin, was discovered, which made research into natural antimicrobial proteins/peptides (AMPs) a very important research domain for therapeutic molecules that can be used against bacterial infections [1]. AMPs are naturally occurring small proteins (or peptides) in different organisms and are produced by many tissues and different cell types, acting as host defense molecules against bacteria, but with some also showing a fast antifungal, antiviral, antiparasitic and antitumor response [1–3].

The largest part of AMPs consists of antibacterial peptides with an inhibitory activity towards bacteria, both Gram-positive and Gram-negative [2]. Studies have revealed that AMPs exhibit an overall positive charge, allowing electrostatic interactions with negatively charged phospholipid groups in the bacterial membrane.

By this attribute, pores can be formed by AMPs to disrupt the membrane integrity. Some AMPs are able to cross the lipid bilayer, followed by disruption of intracellular functions such as blocking enzyme activity or inhibition of protein synthesis, both resulting in bacterial cell lysis [1]. For this reason, AMPs are often referred to 'natural antibiotics'.

AMP activity is not restricted to antimicrobial mechanisms, also AMP activity against parasites has been observed: a few AMPs are reported as antimalarial peptides and can possibly serve as new future drug targets against the malaria parasite. For example, cecropins have been shown to block the development of oocysts into sporozoites, while dermaseptins (and some derivatives) have been found to be able to permeabilize the host cell membrane [4].

Furthermore, a subset of AMPs have shown antifungal characteristics against some fungi commonly found in food and agriculture, but also against the common *Aspergillus* and *Candida albicans* infections [2]. These antifungal peptides can interact with fungal membranes to form pores, comparable to the AMP mechanism in bacteria, but they can also act by targeting the specific fungus cell wall or by acting as nucleic acid inhibitors through direct binding to nucleic acids [5]. A smaller part of AMPs also exhibit antiviral activity, by acting through different mechanisms. A first mechanism includes inhibition of virus attachment and cell membrane fusion. As an example, during the recent COVID-19 pandemic, the antiviral peptide (AVP) EK1C4 has been found to be very effective against S-mediated membrane fusion of the viral particles, thus inhibiting entry of the virus and thereby infection [6]. Another example of inhibition of virus attachment is demonstrated by dermaseptins, which possibly affect the lipid bilayer to alter the fusogenic properties of herpes simplex virus [7]. The virus for host cell infection can also be impeded by the direct action of certain AMPs, such as indolicidin, against enveloped virions, causing membrane instability by destruction of the virus envelope [8]. Combined, there is great potential for future therapeutic development of AVPs for both prevention as treatment of infection [6]. A small number of AMPs are believed to be active as anticancer peptides (ACP). It has been suggested that they specifically target the membrane of cancer cells through interaction with phospholipids, mainly phosphatidylserine, present at the outer leaflet of the cancer cell membrane in higher amounts compared to normal cells. Moreover, the ACP LTX-315 has demonstrated both cytolytic and immunogenic properties towards cancer cells, as LTX-315 induces tumor antigen and danger-associated molecular patterns (DAMPs) release, triggering an immune response towards the cancer cells [9].

AMPs are considered key components of the innate immune system, as shortly after an infection, these are promptly synthesized to neutralize a wide variety of pathogens, but through another mechanism compared to that of cytokines or phagocytes [10]. High concentrations of AMPs are usually required to exert an optimal pathogen killing activity, but *in vivo*, lower concentrations of AMPs are reported, in this case possibly acting as potent immune regulators, also leading to pathogen killing but rather through an indirect mechanism [11]. Besides permeabilizing lipid membranes and bacterial walls, their primary role as antimicrobicidal agents, other targets of AMPs are thus reported. Recently, some AMPs are found to also modulate immune responses in vertebrates, through chemotactic activity, attraction, activation and differentiation of leukocytes and monocytes, influencing Toll-like receptor (TLR) recognition and through secretion of proinflammatory cytokines and chemokines, although their underlying mechanisms have not been fully characterized yet [1].

The best studied AMPs include the human defensins and cathelicidins and both have been shown to be chemotactic: β defensins (hBD) recruit (memory) T cells and immature dendritic cells through their chemotactic activity, suggesting

#### *Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human… DOI: http://dx.doi.org/10.5772/intechopen.99251*

they promote cellular immune responses via interactions with the G proteincoupled receptor CCR6 [11]. Another example of direct AMP chemotaxis includes cathelicidin LL-37, an AMP that has been proven to be chemotactic for neutrophils, monocytes and T cells, but not dendritic cells [11]. Additionally, an indirect chemotactic effect is possible by AMPs through inducing the release of pro-inflammatory cytokines and chemokines, to further refine and activate the innate, and eventually the adaptive, immune response [1, 11]. In synergy with particular immune mediators, LL-37 has been shown to enhance IL-6 and IL-10 cytokine production, even as the production of macrophage chemoattractant proteins (MCP-1 and MCP-3) chemokines, resulting in an strengthened (innate) immune response [12]. Toll-like receptors (TLR) are key players in innate immunity by recognizing microbe-associated molecular patterns (MAMPs). TLR activation leads to secretion of AMPs, but some AMPs, including cathelicidins, can modulate TLR-mediated inflammatory responses by strongly reducing LPS-induced TLR activation, mostly by inhibiting TLR4 [11, 13]. Lastly, AMPs, e.g. cathelicidins and β defensins, also exert a regenerative function by affecting wound healing, by stimulating migration, proliferation and tube formation of endothelial cells, through a cascade of activated pathways [11].

Overall, AMPs are important key players in host protection. Due to increasing antibiotic resistance, several AMPs have good potential therapeutic purposes, ranging from antimicrobial, anti-inflammatory and immunomodulatory properties. Also co-administration of AMPs with existing therapies can have good clinical outcomes [14]. Recently, the ACP LTX-315 which has been described earlier, demonstrated in phase I human clinical studies to be an effective drug, due to its immunostimulatory effect resulting in tumor necrosis [9]. Currently, a phase I clinical trial for transdermally accessible tumors is ongoing to evaluate the efficacy of LTX-315 monotherapy or in combination with immune checkpoint inhibitor immunotherapy [9]. Still, some limitations for the therapeutic use of AMPs need to be resolved: high proteolytic degradation of AMPs (i.e. susceptibility to proteases) is commonly observed, unpredicted toxicity is known to occur, chemical synthesis is costly and delivery of AMP targets to the site of infection can be very difficult [9, 14, 15]. As an example, LL-37 has proven to be very effective against Ebola virus infection, but its use as therapeutic molecule is limited as LL-37 is rapidly degraded and can lose its activity under certain conditions. These limitations were overcome with the design of an engineered LL-37 which prevents cell entry of the virus. The therapeutic outcome of these AMPs in animal models is ongoing, possibly combined with other small molecules that interfere with viral replication or together with virus-neutralizing antibodies [16].

AMPs mostly consist of 10 to 60 amino acids, including mainly basic and hydrophobic residues, resulting in positively charged molecules [1, 2]. They can be classified, based on their structure, into four categories; 1) linear extension structure, 2) α-helical AMPs, 3) AMPs consisting of β-strands stabilized by disulfide bonds and 4) both α-helical and β-sheet structures [2]. Due to their cationic properties, AMPs are easily detectable by mass spectrometric analysis. In addition, structural information of AMPs can be obtained by tandem mass spectrometry in which fragmentation spectra are obtained. Based upon this, the corresponding amino acid sequence can be determined and the precursor ion can be identified [17]. Even if the AMPs have a cyclic nature, due to their cysteine bond formation, mass spectrometry can be used as an identification tool, although a specific approach is needed [18–21]. The added benefit of using proteomic approaches to study AMPs is the fact that the majority of AMPs are post- or co-translational proteolytically processed from their large polyprotein precursor, resulting in the release of the active AMP. This is important as it allows for the identification of the active AMP in physiological

conditions. It overcomes the limitations of a genomics approach, as it can be complicated to predict the configuration of the exact active peptide from genomic sequences alone [22, 23]. In addition, due to their high positive charge, distribution of the AMP in a tissue can be analyzed using mass spectrometry imaging (MSI). MALDI MSI is a multiplexed analysis that allows the screening of hundreds of analytes simultaneously in a single tissue section without *a priori* knowledge of the present biomolecules [24–26]. Using MSI, a mass spectrum on every pixel of the tissue is recorded, representing all measured analytes by their mass-to-charge (m/z) ratio. The thousands of generated mass spectra can be combined. This thus provides not only structural information about the AMP, it also retains its spatial distribution and information on the relative abundance throughout the tissue [27]. This can overcome the limitation of antibody-related visualization with AMP-specific antibodies that are in some cases designed towards the non-active part of the AMP.

Overall, mass spectrometry (imaging) is an useful approach for the detection and identification of AMPs in their native and active form.
