**3.5 Identification of mechano growth factor (MGF) and long-R3 insulin-like growth factor (IGF-1)**

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

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

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

#### *Mass Spectrometry in Life Sciences and Clinical Laboratory*


*a Glu-C cleaves at the C-terminus of either aspartic or glutamic acid residues. b The amino acid sequence of the peptide was determined.*

#### **Table 2.**

*MALDI peptide mass fingerprinting-data from analysis of mechano growth factor.*

#### **Figure 7.**

*MALDI analysis of intact long-R3-IGF and MALDI-PSD analysis of two tryptic peptides, i.e., m/z 1667.771 and m/z 1763.887.*

[78]. However, the misuse and wrong administration of insulin could cause the, so called, dead in the bed syndrome [79]. In bodybuilding, insulin works such as testosterone or hGH to consolidate muscle tissue. Insulin also prevents breakdown of muscles and vanishes rapidly from the body, since it has a very short half-time (*t*1/2) [80].

**37**

*a*

*b*

*c*

*d*

**Table 3.**

*Trypsinated B-chain.*

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

**Insulin Theoretical m/z**

Porcine (intact) 5774.635 5774.632 Aspart (intact) 5822.612 5822.618

[A-chain + Na]+ 2404.990 2404.758 [A-chain +1ME + Na]+a 2480.988 2480.769 [B-chain + H]+ 3398.682 3398.460 [B-chain +1ME + H]+a 3474.680 3474.486

[B-chain + H]+ 3446.667 3446.434 [B-chain + Na]+ 3468.648 3468.487 [B-chain +1ME + H]+a 3522.665 3522.422 [B-chain - (GFFYTDKT) + H]+c 2487.228 2487.030 [B-chain - (GFFYTDKT) + 1ME + H]+a, c 2563.226 2563.302

[GFFYTDK + H]+ 877.399 877.317 [GFFYTDKT + H]+ 978.457 978.446 [B-chain - (GFFYTDKT) + H]+c, d 2487.217 2487.030

[GFFYTPK + H]+ 859.425 859.345 [GFFYTPKA + H]+ 930.462 930.337 [B-chain - (GFFYTPKA) + H]+c 2487.228 2487.234 [B-chain-(GFFYTPKA) + 1ME + H]+ a, c 2563.226 2563.129

**[M + H]+**

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

Several illegal products containing insulin porcine or aspart have been analyzed. Insulin is composed of two peptide chains, i.e., A and B, which are joined by two inter-chain disulfide bonds. The A chain also contains an intra-chain disulfide bond (**Figure 4**). The results summarized in **Table 3**, demonstrate the applied strategy for the identification of porcine and insulin aspart. The insulin molecules were reduced using a potent reducing agent, i.e., 2-mercaptoethanol (ME). MS-analysis of the reduced samples resulted in a mass spectrum consisting of several signals from both reduced A and B chains. The A and B chains generated three and four signals, respectively, corresponding to the ME-modified peptide as described in **Table 3**. It is to be noted that the amino acid residues P and A at positions 28 and 30 in the B-chain, respectively, have been replaced by D and T in insulin aspart. Therefore, these insulin molecules are distinguished upon these differences. The tryptic digestion of the B chain yielded three peptide fragments of different sizes (**Figure 8** and **Table 3**). The molecular masses of these peptides were determined accurately, and the amino acid sequence of the tryptic peptides were determined in

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

PSD-mode.

Peptide chains from Insulin porcine:

Peptide chain from Insulin aspart:b

Tryptic peptides from Insulin aspart:

Tryptic peptides from Insulin porcine:

*Beta mercaptoethanol (ME) was used as reducing agent.*

*These peptides originate from insulin aspart, see Figure 8.*

*MALDI-TOF-MS analysis of insulin porcine and aspart.*

*The A-chains of insulin aspart and Insulin porcine are identical.*

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

Several illegal products containing insulin porcine or aspart have been analyzed. Insulin is composed of two peptide chains, i.e., A and B, which are joined by two inter-chain disulfide bonds. The A chain also contains an intra-chain disulfide bond (**Figure 4**). The results summarized in **Table 3**, demonstrate the applied strategy for the identification of porcine and insulin aspart. The insulin molecules were reduced using a potent reducing agent, i.e., 2-mercaptoethanol (ME). MS-analysis of the reduced samples resulted in a mass spectrum consisting of several signals from both reduced A and B chains. The A and B chains generated three and four signals, respectively, corresponding to the ME-modified peptide as described in **Table 3**. It is to be noted that the amino acid residues P and A at positions 28 and 30 in the B-chain, respectively, have been replaced by D and T in insulin aspart. Therefore, these insulin molecules are distinguished upon these differences. The tryptic digestion of the B chain yielded three peptide fragments of different sizes (**Figure 8** and **Table 3**). The molecular masses of these peptides were determined accurately, and the amino acid sequence of the tryptic peptides were determined in PSD-mode.


*a Beta mercaptoethanol (ME) was used as reducing agent.*

*c Trypsinated B-chain.*

*d These peptides originate from insulin aspart, see Figure 8.*

**Table 3.**

*MALDI-TOF-MS analysis of insulin porcine and aspart.*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

:

*Glu-C cleaves at the C-terminus of either aspartic or glutamic acid residues.*

*MALDI peptide mass fingerprinting-data from analysis of mechano growth factor.*

*The amino acid sequence of the peptide was determined.*

Glu-C digestion of the peptidea

Trypsin digestion of the peptide:

*a*

*b*

**Table 2.**

**Peptide fragments Theoretical m/z**

YQPPSTNKNTKSQRRKGSTFEERK 2869.169 2869.422

YQPPSTNKNTKSQRRKGSTFEE 2584.809 2584.816

GSTFEERKb 953.458 953.574 YQPPSTNKb 934.453 934.525 GSTFEERb 825.363 825.469 SQRb 390.199 390.186 NTKb 362.193 362.302

**[M + H]+**

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

**36**

**Figure 7.**

*and m/z 1763.887.*

half-time (*t*1/2) [80].

[78]. However, the misuse and wrong administration of insulin could cause the, so called, dead in the bed syndrome [79]. In bodybuilding, insulin works such as testosterone or hGH to consolidate muscle tissue. Insulin also prevents breakdown of muscles and vanishes rapidly from the body, since it has a very short

*MALDI analysis of intact long-R3-IGF and MALDI-PSD analysis of two tryptic peptides, i.e., m/z 1667.771* 

*b The A-chains of insulin aspart and Insulin porcine are identical.*

#### **Figure 8.**

*MALDI analysis of insulin aspart; analysis of reduced B-chain (A), MALDI-PSD analysis of tryptic B-chain (B), see Table 3.*

Double-injection capillary electrophoresis has also been applied for the identification of insulin molecules [81].

#### **3.7 Identification of delta sleep-inducing peptide (DSIP)**

The nonapeptide delta DSIP was first isolated from the cerebral venous blood of rabbits in an induced state of sleep during the mid-70s [82]. It was primarily believed to be involved in sleep regulation due to its apparent ability to induce slowwave sleep in rabbits. However, it has been demonstrated that short-term treatment of chronic insomnia with DSIP is not likely to be of major therapeutic benefit [83]. The peptide is marketed illegally presumably for the treatment of insomnia. The peptide was directly exposed to the PSD analysis in order to confirm its molecular mass and amino acid sequence (**Figure 4** and **Table 1**).

**39**

*a*

*b*

**Table 4.**

*N.D. = Not detected.*

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

Synthetic thymosin is a peptide consisting of 43 amino acids with artificial acetylation of the N-terminus (see **Figure 4** and **Table 1**). Thymosin has the potential of playing a significant role in tissue development, maintenance, repair, pathology and other important biological activities [84]. Some important biological activities of thymosin are related to the peptide sequence L17KKTET22 [85]. Illegally distributed thymosin products are claimed to promote a variety of beneficial biological functions, such as muscle building. The peptide was identified through

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

KTETQEKNPLPSKETIEQEKQAGES 2829.401 2829.219 Ac-SDKPDMAEIEKFDKSKLK 2151.090 2151.124 Ac-SDKPDMAEIEKFDKSK 1909.911 1909.698 Ac-SDKPDMAEIEKFDKa 1694.784 1694.765 NPLPSKETIEQEK 1512.780 1512.768 SKLKKTETQEK 1319.743 1319.729 Ac-SDKPDMAEIEK 1304.594 1304.498 ETIEQEK 876.421 876.356 TETQEKa 735.342 735.356 NPLPSKa 655.367 655.354 FDKSK 624.325 N.D.b QAGES 491.199 N.D.b SKLK 475.314 N.D.b FDKa 409.198 409.196 LKKa 388.282 388.286 LKa 260.187 260.168 SKa 234.135 234.151

**[M + H]+**

4961.484 4960.987

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

PMF and de-novo sequencing of the tryptic peptides (**Table 4**).

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

sequence of hCG was confirmed upon PMF (**Table 5**) [13].

*The amino acid sequence of the peptide was determined in the PSD mode.*

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

Ac*-*SDKPDMAEIEKFDKSKLKKTETQEKNPLPSK

ETI EQE-KQAGES

**Peptide fragments Theoretical m/z** 

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

**3.8 Identification of thymosin** β**<sup>4</sup>**

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