**3.9 Identification of human chorionic gonadotropin (hCG)**

Human chorionic gonadotropin (hCG) is a glycoprotein hormone consisting of α (92 amino acids) and β-subunits (145 amino acids) being noncovalently associated [86]. These subunits are, however, highly cross-linked internally through disulfide bridges, i.e., the α-subunit has five disulfide bridges [87], while the β-subunit has six [87, 88]. The protein is heavily glycosylated where oligosaccharides are attached to the protein backbone through asparagine and serine residues and constitute approximately 30% of the molecular mass [89]. The protein has been identified using MALDI-TOF-MS and DICZE [13, 50]. Approximately 40% of the amino acid sequence of hCG was confirmed upon PMF (**Table 5**) [13].


*b N.D. = Not detected.*

#### **Table 4.**

*MALDI peptide mass fingerprinting data from analysis of thymosin* β*4.*


#### **Table 5.**

*MALDI-PMF and MALDI-PSD analysis of human chorionic gonadotropin. The identified peptides from the* α *and* β *subunits are presented in the table below.*

The identification was confirmed by DICZE analysis of illegal samples together with the corresponding reference standard [13, 50].

#### **3.10 Identification of melanotan II (MII) and bremelanotide**

Melanotan, a melanocortin receptor agonist, is a cyclic-lactam bridge heptapeptide which induces melanogenesis (i.e., tanning of the skin), by activation of the MC1 receptor, being an analogue to alpha melanocyte hormone (α-MSH) [90]. The cyclic, lactam bridged structure of MII induces increased lipophilicity (**Figure 4**) [91].

Skin-tanning products that claim to contain MII are being advertised and sold on the illicit drug market. Injection of MII can result in systemic toxicity and rhabdomyolysis [90]. Bremelanotide (formerly PT-141) is an active metabolite of MII (**Table 1**).

These peptides were identified through the *top-down* approach by MALDI in PSD mode as illustrated in **Figure 9**.

#### **3.11 Identification of dermorphin**

Dermorphin is a μ-opioid receptor-binding peptide that causes both central and peripheral effects [92] (**Figure 4** and **Table 1**). This peptide, being originally isolated from the skin of the south American tree frog *Phyllomedusa sauvagii,* is classified as one of the strongest mammalian endogenous analgesic opioids [93, 94]. Dried frog skin containing dermorphin, has been used as a therapeutic agent by the Matses tribes of the upper Amazonian basin, to treat cuts during hunting expeditions [95]. The analgetic effects of dermorphin has been demonstrated in rat, horse, dog and white sea cod [92, 94]. It has been used illegally in horse racing as a pain killing agent, allowing horses to run even if injured.

This peptide, which was detected in several samples, was identified by MALDI in the PSD mode (**Figure 10**). The molecular structure was confirmed by NMR spectroscopy.

#### **3.12 Identification of body protecting compound 157 (BPC 157)**

BPC 157 being a partial sequence of body protecting compound (BPC) (Mmass = 40 kDa) is a synthetic peptide, which is composed of fifteen amino acids

**41**

**Figure 10.**

**Figure 9.**

*MALDI-PSD analysis of melanotan II.*

ful effects of this compound [97].

*MALDI-PSD analysis of dermorphin.*

*Identification of Peptides and Proteins in Illegally Distributed Products by MALDI-TOF-MS*

(**Figure 4** and **Table 1**). BPC was discovered and isolated from mouse gastric juice in response to stress stimuli in the gut mucosa [96]. BPC 157 is also known as Bepcin and PL. 14,736 or PL 10 [97]. This peptide fragment was speculated to be responsible for the BPC's physiological and protective effects [96]. However, it is unclear whether this peptide is endogenous to humans. BPC 157 is suggested to aid in tendon, ligament and muscle healing, and therefore its use as a quick injury healing in the sporting world is appealing. However, no proper clinical trials in human subjects have yet been performed to investigate the healing capability and the harm-

*DOI: http://dx.doi.org/10.5772/intechopen.95335*

*Identification of Peptides and Proteins in Illegally Distributed Products by MALDI-TOF-MS DOI: http://dx.doi.org/10.5772/intechopen.95335*

**Figure 9.** *MALDI-PSD analysis of melanotan II.*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**Peptide position in the peptide chain**

AYPTPLR α-hCG; 36–42 817.446 817.482 TMLVQK α-hCG; 46–51 719.402 719.414 STNR α-hCG; 64–67 477.231 477.165 VTVMGGFK α-hCG; 68–75 838.439 838.471 SK β-hCG; 1–2 234.135 243.142 PR β-hCG; 7–8 272.161 271.996 EPLR β-hCG; 3–6 514.288 514.291 EPLRPR β-hCG; 3–8 767.442 767.469 DVR β-hCG; 61–63 389.204 389.228 FESIR β-hCG; 64–68 651.336 651.359

**Peptide fragments**

**Table 5.**

with the corresponding reference standard [13, 50].

killing agent, allowing horses to run even if injured.

**3.12 Identification of body protecting compound 157 (BPC 157)**

BPC 157 being a partial sequence of body protecting compound (BPC) (Mmass = 40 kDa) is a synthetic peptide, which is composed of fifteen amino acids

PSD mode as illustrated in **Figure 9**.

*and* β *subunits are presented in the table below.*

**3.11 Identification of dermorphin**

**3.10 Identification of melanotan II (MII) and bremelanotide**

The identification was confirmed by DICZE analysis of illegal samples together

*MALDI-PMF and MALDI-PSD analysis of human chorionic gonadotropin. The identified peptides from the* α

**Theoretical m/z [M + H]+**

**Determined m/z [M + H]+**

Melanotan, a melanocortin receptor agonist, is a cyclic-lactam bridge heptapeptide which induces melanogenesis (i.e., tanning of the skin), by activation of the MC1 receptor, being an analogue to alpha melanocyte hormone (α-MSH) [90]. The cyclic, lactam bridged structure of MII induces increased lipophilicity (**Figure 4**) [91].

Skin-tanning products that claim to contain MII are being advertised and sold on the illicit drug market. Injection of MII can result in systemic toxicity and rhabdomyolysis [90]. Bremelanotide (formerly PT-141) is an active metabolite of MII (**Table 1**). These peptides were identified through the *top-down* approach by MALDI in

Dermorphin is a μ-opioid receptor-binding peptide that causes both central and peripheral effects [92] (**Figure 4** and **Table 1**). This peptide, being originally isolated from the skin of the south American tree frog *Phyllomedusa sauvagii,* is classified as one of the strongest mammalian endogenous analgesic opioids [93, 94]. Dried frog skin containing dermorphin, has been used as a therapeutic agent by the Matses tribes of the upper Amazonian basin, to treat cuts during hunting expeditions [95]. The analgetic effects of dermorphin has been demonstrated in rat, horse, dog and white sea cod [92, 94]. It has been used illegally in horse racing as a pain

This peptide, which was detected in several samples, was identified by MALDI in the PSD mode (**Figure 10**). The molecular structure was confirmed by NMR

**40**

spectroscopy.

**Figure 10.**

*MALDI-PSD analysis of dermorphin.*

(**Figure 4** and **Table 1**). BPC was discovered and isolated from mouse gastric juice in response to stress stimuli in the gut mucosa [96]. BPC 157 is also known as Bepcin and PL. 14,736 or PL 10 [97]. This peptide fragment was speculated to be responsible for the BPC's physiological and protective effects [96]. However, it is unclear whether this peptide is endogenous to humans. BPC 157 is suggested to aid in tendon, ligament and muscle healing, and therefore its use as a quick injury healing in the sporting world is appealing. However, no proper clinical trials in human subjects have yet been performed to investigate the healing capability and the harmful effects of this compound [97].

**Figure 11.** *MALDI-PSD analysis of BPC 157.*

BPC 157 was recently identified in several confiscated vials for injection. The identification was carried out by MALDI in both PSD and reflectron modes (**Figure 11**). The amino acid sequence of the peptide was confirmed by NMR spectroscopy and LC-QTOF-MS.
