**3.1 Identification of somatropin (hGH)**

Recombinant hGH or somatropin consists of 191 amino acids with two disulfide bridges (Cys53-Cys165 and Cys182-Cys189) and promotes proteinogenesis as well as

**33**

*a*

*b*

*c*

*d*

*e*

*f*

**Table 1.**

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

**Peptide Mmass PMFa PSDb ISDc DICZEd NMR LC/MS**

GHRP-2 817.397 — X — — — — Gly-GHRP-2 874.419 — X — — — — GHRP-6 872.433 — X — — — — Gly-GHRP-6 929.455 — X — — — — Ipamorelin 711.385 — X — — — — MGF 2866.469 X X — — — —

Insulin Porcine 5772.766 X X — X X X Insulin Aspart 5821.611 X X — X X X DSIP 848.318 — X — — — — Thymosin-β<sup>4</sup> 4960.474 X X — — — —

Melanotan-II 1023.502 — X — — X — Bremelanotide 1024.510 — X — — X — Dermorphin 802.337 — X — — X X BPC-157 1418.692 — X — — X X Albumin bovinef > 66,000e X — — — — —

*Identification by peptide mass fingerprinting using enzymatic degradation as well as other modifications.*

*Identification and/or impurity profiling by double injection capillary zone electrophoresis.*

X — X X X X

X X — — — —

X X — X — —

3366.866 X X — — — —

1813.850 X X — — — —

fat mobilization and oxidation [51–53]. Recombinant hGH is used as a prescription drug to treat children's growth disorders and adult growth hormone deficiency. In the belief that the beneficial impact of somatropin on the growth can be extrapolated to healthy individuals, it is abused by bodybuilders and athletes [54]. However, many users are unaware of the correct dosage and how to prepare the solution for giving an injection. It has been demonstrated that supra-physiological dosages can have fatal consequences [55]. Apart from the undesired consequences following the abuse of somatropin, our investigations have shown that the illegally marketed products contained high levels of impurities such as endotoxins [50]. Endotoxins are associated with Gram-negative bacteria which can cause severe immune response and diseases in humans [56, 57]. Somatropin was identified through PMF and MALDI-ISD (see **Figure 2**)[48, 58, 59]. The availability of a compendial reference

*Illegally distributed peptides and proteins that have been analyzed by MALDI-ToF-MS and DICZE. The monoisotopic mass (Mmass) of the analytes and the employed analytical methodology is indicated.*

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

22128.68e

9111.576e

13,431e 23,114e 36,341e

De novo *sequencing by MALDI- post source decay.*

*Bovin albumin was detected in some of the samples.*

*Protein sequencing by MALDI- in source decay.*

Somatropin 22115.07

Long-R3-IGF 9105.385

hCG α - Chain β - Chain α + β

*Average molecular mass.*

Human Somatoliberin

AOD (Anti Obesity Drug) HGH fragment 177–191


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

*a Identification by peptide mass fingerprinting using enzymatic degradation as well as other modifications. b* De novo *sequencing by MALDI- post source decay.*

*c Protein sequencing by MALDI- in source decay.*

*d Identification and/or impurity profiling by double injection capillary zone electrophoresis. e Average molecular mass.*

*f Bovin albumin was detected in some of the samples.*

#### **Table 1.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

reflectron mode. Small peptides are, on the other hand, analyzed in reflectron mode and/or PSD mode directly. This strategy was applied to the identification of the

*The primary structure of the analyzed peptides. (A) Somatoliberin, (B) AOD, (C) GHRP-2, (D) glycine-GHRP-2, (E) GHRP-6, (F) glycine-GHRP-2, (G) Ipamorelin, (H) MGF, (I) long-R3-IGF (disulfide bridges: C6-C48; C47-C52 and C18-C61; asp at position 3 is replaced by Arg), (J) insulin Aspart, (K) insulin porcine, (L) DSIP, (M) Thymosine* β*4, (N) Melanotan II, (O) Bremelanotide, (P) Dermorphin and (Q ) BPC 157. For* 

*The sample to be identified is analyzed in both reflectron and linear modes in order to determine the molecular mass of the analyte. Depending on the size of the molecule it will be exposed to enzymatic digestion in order to be identified through PMF. Small peptides used to be identified by* de novo *sequencing in PSD mode.*

Recombinant hGH or somatropin consists of 191 amino acids with two disulfide bridges (Cys53-Cys165 and Cys182-Cys189) and promotes proteinogenesis as well as

following peptides and proteins (**Figure 4** and **Table 1**).

*molecular structures of somatropin and hCG see references [13, 50].*

**3.1 Identification of somatropin (hGH)**

**32**

**Figure 3.**

**Figure 4.**

*Illegally distributed peptides and proteins that have been analyzed by MALDI-ToF-MS and DICZE. The monoisotopic mass (Mmass) of the analytes and the employed analytical methodology is indicated.*

fat mobilization and oxidation [51–53]. Recombinant hGH is used as a prescription drug to treat children's growth disorders and adult growth hormone deficiency. In the belief that the beneficial impact of somatropin on the growth can be extrapolated to healthy individuals, it is abused by bodybuilders and athletes [54]. However, many users are unaware of the correct dosage and how to prepare the solution for giving an injection. It has been demonstrated that supra-physiological dosages can have fatal consequences [55]. Apart from the undesired consequences following the abuse of somatropin, our investigations have shown that the illegally marketed products contained high levels of impurities such as endotoxins [50]. Endotoxins are associated with Gram-negative bacteria which can cause severe immune response and diseases in humans [56, 57]. Somatropin was identified through PMF and MALDI-ISD (see **Figure 2**)[48, 58, 59]. The availability of a compendial reference

standard has made it possible to apply double injection capillary zone electrophoresis (DICZE) for both identification and impurity determination of somatropin products [50, 58, 59]. The DICZE-method provided complementary information on the native protein, providing a side by side comparison between the electrophoretic patterns of the reference standard and the analyte to be identified [50].

### **3.2 Identification of human somatoliberin**

Human somatoliberin, growth hormone-releasing hormone (GHRH), constitutes of 44 amino acids without any post-translational modification or disulfide bridge. Somatoliberin was first isolated from two pancreatic islet cell tumors, and subsequently from normal human hypothalamus [60–62]. The MALDI results from determination of the molecular mass, PMF and amino acid sequence revealed that the Asn8 (N), Gly15 (G) and Met27 (M) residues have, respectively, been replaced by Gln8 (Q ), Ala15 (A) and Leu27 (L) during the synthesis (see **Figures 4** and **5**). The peptide was successfully identified by PMF and *de-novo* sequencing of three of the tryptic peptides.

### **3.3 Identification of an anti-obesity drug (AOD)**

The AOD peptide is a fragment of the C-terminus of human growth hormone (fragment 177–191) where a tyrosine is added at the N-terminus. It is a cyclic peptide consisting of 16 amino acids with a disulfide bridge between cysteine residues at positions 7 and 14 in the peptide chain [63] (**Figure 4** and **Table 1**). The fragment is the minimum length of the hGH sequence that retains the lipolytic and antilipogenic properties of hGH [63–65]. The molecular peptide masses of its tryptic peptides complied with the peptide map of hGH fragment 177–191. The existence of the disulfide bridge between C7 and C14 was confirmed upon analysis of the non-reduced tryptic sample (**Figure 6**). This peptide has also been employed as a signature peptide for the identification of hGH [48, 50]. The amino acid sequences of three selected tryptic peptides were also confirmed.

### **3.4 Identification of growth hormone releasing peptides (GHRP)**

**35**

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

hormone secretagogue receptor [66, 67]. Ghrelin strongly stimulates food intake and GH release in humans [68–70]. These peptides were identified through *denovo* sequencing. The amino acid sequence of GHRP-6 differs slightly from that of GHRP-2, i.e., the amino acid residues dA and Naphthyl alanine (NalA) in GHRP2

Ipamorelin is a penta-peptide, being derived from GHRP-1 [71]. Ipamorelin like the other GHR-peptides, stimulates production of growth hormone [72]. Incorporation of aminoisobutyric acid (Aib) in the peptide chain increases the

**3.5 Identification of mechano growth factor (MGF) and long-R3 insulin-like** 

Insulin regulates the cellular uptake, utilization, and storage of glucose, amino acids, and fatty acids and inhibits the breakdown of glycogen, protein, and fat. Since more than one decade ago the illegal use of insulin has been noticed

MGF is a unique, spliced variant of IGF-1. MGF induces muscle cell proliferation in response to muscle stress and injury [74]. MGF and Long-R3-IGF1 were identified in several confiscated samples. Long-R3-IGF-1, an analogue of IGF-1, has 13 additional amino acids at its N-terminus (**Figure 4** and **Table 1**). IGF-1 mediates the anabolic and mitogenic activity of GH [75–77]. MGF and Long-R3-IGF1 were identified by sequence coverages of 100% and 43%, respectively

are replaced by H and dW in GHRP-6 (**Figure 4** and **Table 1**) [70].

**3.6 Identification of insulin porcine and insulin aspart**

stability of the peptide (**Figure 4**) [73].

**growth factor (IGF-1)**

**Figure 6.**

*MALDI-PSD analysis of AOD.*

(**Table 2** and **Figure 7**).

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

GHRP, including GHRP-2, GHRP-6, Gly-GHRP-2, Gly-GHRP-6 and ipamorelin, as an agonist of the gut peptide ghrelin is an endogenous ligand for the growth

**Figure 5.** *MALDI-PMF (A) and MALDI-PSD (B) analysis of somatoliberin.*

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

**Figure 6.**

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**3.2 Identification of human somatoliberin**

**3.3 Identification of an anti-obesity drug (AOD)**

of three selected tryptic peptides were also confirmed.

*MALDI-PMF (A) and MALDI-PSD (B) analysis of somatoliberin.*

**3.4 Identification of growth hormone releasing peptides (GHRP)**

tryptic peptides.

standard has made it possible to apply double injection capillary zone electrophoresis (DICZE) for both identification and impurity determination of somatropin products [50, 58, 59]. The DICZE-method provided complementary information on the native protein, providing a side by side comparison between the electrophoretic

Human somatoliberin, growth hormone-releasing hormone (GHRH), constitutes of 44 amino acids without any post-translational modification or disulfide bridge. Somatoliberin was first isolated from two pancreatic islet cell tumors, and subsequently from normal human hypothalamus [60–62]. The MALDI results from determination of the molecular mass, PMF and amino acid sequence revealed that the Asn8 (N), Gly15 (G) and Met27 (M) residues have, respectively, been replaced by Gln8 (Q ), Ala15 (A) and Leu27 (L) during the synthesis (see **Figures 4** and **5**). The peptide was successfully identified by PMF and *de-novo* sequencing of three of the

The AOD peptide is a fragment of the C-terminus of human growth hormone

GHRP, including GHRP-2, GHRP-6, Gly-GHRP-2, Gly-GHRP-6 and ipamorelin,

as an agonist of the gut peptide ghrelin is an endogenous ligand for the growth

(fragment 177–191) where a tyrosine is added at the N-terminus. It is a cyclic peptide consisting of 16 amino acids with a disulfide bridge between cysteine residues at positions 7 and 14 in the peptide chain [63] (**Figure 4** and **Table 1**). The fragment is the minimum length of the hGH sequence that retains the lipolytic and antilipogenic properties of hGH [63–65]. The molecular peptide masses of its tryptic peptides complied with the peptide map of hGH fragment 177–191. The existence of the disulfide bridge between C7 and C14 was confirmed upon analysis of the non-reduced tryptic sample (**Figure 6**). This peptide has also been employed as a signature peptide for the identification of hGH [48, 50]. The amino acid sequences

patterns of the reference standard and the analyte to be identified [50].

**34**

**Figure 5.**

*MALDI-PSD analysis of AOD.*

hormone secretagogue receptor [66, 67]. Ghrelin strongly stimulates food intake and GH release in humans [68–70]. These peptides were identified through *denovo* sequencing. The amino acid sequence of GHRP-6 differs slightly from that of GHRP-2, i.e., the amino acid residues dA and Naphthyl alanine (NalA) in GHRP2 are replaced by H and dW in GHRP-6 (**Figure 4** and **Table 1**) [70].

Ipamorelin is a penta-peptide, being derived from GHRP-1 [71]. Ipamorelin like the other GHR-peptides, stimulates production of growth hormone [72]. Incorporation of aminoisobutyric acid (Aib) in the peptide chain increases the stability of the peptide (**Figure 4**) [73].
