**1. Introduction**

A broad range of proteins and peptides, for various purposes of enhancement, such as human growth hormone (hGH), i.e., somatropin, can be obtained from the illicit market. These products are mainly marketed as lyophilized formulations in small glass containers often without labelling. The customers are exposed to a range of potential harms, besides from the active components, including bacterial and fungal or viral infections which may arise from the fact that they are administered parenterally.

**Figure 1A** illustrates the total number of injection vials containing white lyophilized product cake being seized by the Swedish Customs during nine years in the past, i.e., 2010–2018. A large proportion of these samples, i.e., 64%, contained

#### **Figure 1.**

*Schematic illustration of the number of seized illicit products during 2010–2018 in Sweden (A), as well as the active peptides/proteins that have been identified in theses samples (B).*

human growth hormone or melanotan II. About a third of the seized vials, i.e., 27%, did not contain any active peptide or protein, while the remaining 9% of the vials contained other compounds (**Figure 1B**).

The concept of a proteolytic peptide pattern, i.e., protein peptide mapping (PPM), being characteristic of a protein was first demonstrated by SDS-PAGE [1]. In 1989, peptide sequencing by automated Edman degradation had a cycle-time of nearly one hour per amino acid residue. Samples of interest often contained complex mixtures of proteins, which usually required separation by SDS-PAGE followed by electroblotting onto a polyvinylidene fluoride (PVDF) membrane [2]. However, a more rapid approach to peptide sequencing is "peptide mass fingerprinting" (PMF). By PMF, proteins are enzymatically cleaved in a predictable manner and the sizes of the generated peptide fragments are specific for different proteins.

**29**

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

Subsequent analysis of the obtained peptides by mass spectrometry (MS) generates mass-to-charge ratio (*m/z*) values in the mass spectrum which in turn give rise to a characteristic "peptide mass fingerprint" of the protein [3, 4]. The fingerprint serves to identify the protein by comparison with *in silico* digests, i.e., search

engines attempt to match peptides from *in silico* digested proteins to those measured by the mass spectrometer [5–9]. Peptide mass fingerprinting with MS, which was first demonstrated with fast atom bombardment ionization in 1981, provides the possibility of identifying a protein at nanogram-level [5, 10–12]. Trypsin is a commonly used proteolytic enzyme for PMF, since it is relatively cheap, highly selective, and generates peptides with an average size of about 8–10 amino acids which are ideally suited for analysis by MS. It cleaves principally on the C-terminal side of arginine and lysine with the exception of Arg-Pro and Lys-Pro [2]. Limitations to protein identification by PMF include; I) The protein sequence must be present in a database for a successful protein identification. II) Proteins with extensive post-translational modifications may fail to yield good matches [13]. III) Different isoforms of a protein or alternatively spliced proteins may not be distinguished if the unique sequence regions are not observed in the peptide map. IV) Incomplete proteolytic digestion and differences in peptide ionization provide an incomplete mass fingerprint of the protein. Therefore, a complementary approach to PMF for protein identification is the use of tandem mass spectrometry (MS/MS), whereby tryptic peptide ions from the first stage of MS are dissociated along the backbone and then separated and detected in a second stage of MS to identify primary amino acid sequences [14–16]. Tandem mass spectrometry in conjunction with PMF provides even more specificity, thereby facilitating the identification [17, 18].

Since the innovation of sensitive commercial instrumentation based on MALDI-TOF MS in 1992, the technique has been widely used for protein identification due to its high sensitivity and mass accuracy, speed, extremely low material consumption, absence of multiple charge mass signals and relatively high tolerance toward additives and contaminants such as salts, matrix components and excipients [19–26]. Furthermore, MALDI is a micro-destructive analytical technique and the remaining material on the MALDI target plate can be archived for later analysis. The high sensitivity of MALDI implies that only a small aliquot of the digested protein is required for mass analysis, and the remainder can be used for alternative measurements. MALDI provides additional information regarding the primary structure of the protein by sequencing of selected tryptic peptide ions in post source decay (PSD) mode [27–34]. MALDI in-source decay (ISD) is another attractive method which generates partial sequence information of intact proteins with

The sequence information from MALDI-PSD or MALDI-ISD analyses can be used to validate protein identification. The singly charged ions generated by MALDI-TOF-MS are a mixture of b-, y- and a-ions accompanied by ions resulting

PMF-based protein identification is accomplished by searching a protein sequence database using different search engines such as ProFound [40], Mascot [41], or SEQUEST [15]. A value-based scoring system has been developed that facilitates the identification without accompanying amino acid data [42, 43]. Parameters which are considered to be important for the identification include; molecular mass, protein sequence coverage and the number of matching peptides [42]. However, presence of a signature peptide, being unique for a protein, facilitates the PMF-based identifications [44]. Prior reports suggest that a minimum of four matching peptides and a sequence coverage of at least 20% is necessary for positive PMF-based protein identification [45, 46]. The other alternative strategy

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

up to 20–50 amino acid residues [35] (**Figure 2**).

from neutral loss of ammonia or water [36–39].

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

Subsequent analysis of the obtained peptides by mass spectrometry (MS) generates mass-to-charge ratio (*m/z*) values in the mass spectrum which in turn give rise to a characteristic "peptide mass fingerprint" of the protein [3, 4]. The fingerprint serves to identify the protein by comparison with *in silico* digests, i.e., search engines attempt to match peptides from *in silico* digested proteins to those measured by the mass spectrometer [5–9]. Peptide mass fingerprinting with MS, which was first demonstrated with fast atom bombardment ionization in 1981, provides the possibility of identifying a protein at nanogram-level [5, 10–12]. Trypsin is a commonly used proteolytic enzyme for PMF, since it is relatively cheap, highly selective, and generates peptides with an average size of about 8–10 amino acids which are ideally suited for analysis by MS. It cleaves principally on the C-terminal side of arginine and lysine with the exception of Arg-Pro and Lys-Pro [2]. Limitations to protein identification by PMF include; I) The protein sequence must be present in a database for a successful protein identification. II) Proteins with extensive post-translational modifications may fail to yield good matches [13]. III) Different isoforms of a protein or alternatively spliced proteins may not be distinguished if the unique sequence regions are not observed in the peptide map. IV) Incomplete proteolytic digestion and differences in peptide ionization provide an incomplete mass fingerprint of the protein. Therefore, a complementary approach to PMF for protein identification is the use of tandem mass spectrometry (MS/MS), whereby tryptic peptide ions from the first stage of MS are dissociated along the backbone and then separated and detected in a second stage of MS to identify primary amino acid sequences [14–16]. Tandem mass spectrometry in conjunction with PMF provides even more specificity, thereby facilitating the identification [17, 18].

Since the innovation of sensitive commercial instrumentation based on MALDI-TOF MS in 1992, the technique has been widely used for protein identification due to its high sensitivity and mass accuracy, speed, extremely low material consumption, absence of multiple charge mass signals and relatively high tolerance toward additives and contaminants such as salts, matrix components and excipients [19–26]. Furthermore, MALDI is a micro-destructive analytical technique and the remaining material on the MALDI target plate can be archived for later analysis. The high sensitivity of MALDI implies that only a small aliquot of the digested protein is required for mass analysis, and the remainder can be used for alternative measurements. MALDI provides additional information regarding the primary structure of the protein by sequencing of selected tryptic peptide ions in post source decay (PSD) mode [27–34]. MALDI in-source decay (ISD) is another attractive method which generates partial sequence information of intact proteins with up to 20–50 amino acid residues [35] (**Figure 2**).

The sequence information from MALDI-PSD or MALDI-ISD analyses can be used to validate protein identification. The singly charged ions generated by MALDI-TOF-MS are a mixture of b-, y- and a-ions accompanied by ions resulting from neutral loss of ammonia or water [36–39].

PMF-based protein identification is accomplished by searching a protein sequence database using different search engines such as ProFound [40], Mascot [41], or SEQUEST [15]. A value-based scoring system has been developed that facilitates the identification without accompanying amino acid data [42, 43]. Parameters which are considered to be important for the identification include; molecular mass, protein sequence coverage and the number of matching peptides [42]. However, presence of a signature peptide, being unique for a protein, facilitates the PMF-based identifications [44]. Prior reports suggest that a minimum of four matching peptides and a sequence coverage of at least 20% is necessary for positive PMF-based protein identification [45, 46]. The other alternative strategy

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

human growth hormone or melanotan II. About a third of the seized vials, i.e., 27%, did not contain any active peptide or protein, while the remaining 9% of the vials

*Schematic illustration of the number of seized illicit products during 2010–2018 in Sweden (A), as well as the* 

The concept of a proteolytic peptide pattern, i.e., protein peptide mapping (PPM), being characteristic of a protein was first demonstrated by SDS-PAGE [1]. In 1989, peptide sequencing by automated Edman degradation had a cycle-time of nearly one hour per amino acid residue. Samples of interest often contained complex mixtures of proteins, which usually required separation by SDS-PAGE followed by electroblotting onto a polyvinylidene fluoride (PVDF) membrane [2]. However, a more rapid approach to peptide sequencing is "peptide mass fingerprinting" (PMF). By PMF, proteins are enzymatically cleaved in a predictable manner and the sizes of the generated peptide fragments are specific for different proteins.

contained other compounds (**Figure 1B**).

*active peptides/proteins that have been identified in theses samples (B).*

**28**

**Figure 1.**

#### **Figure 2.**

*MALDI in source decay analysis of a suspected illegal somatropin sample. The blue marked amino acid asp (D) is the deamidated form of Asn (N).*

for protein identification is the top down approach, where intact molecule ions are subjected to gas-phase fragmentation [47].

Proteins with posttranslational modifications, such as glycosylation, present additional challenges since the masses of the modified peptides are different and thus do not contribute to the identification. In such cases, the protein can be analyzed by capillary electrophoresis (CE), in order to explore the heterogeneity of the protein followed by comparison of its electropherogram with that of the corresponding reference standard [13, 48].

### **2. Experimental**

#### **2.1 Sample preparation**

MALDI-TOF-MS is very tolerant to salts and sample matrices, hence it is seldom necessary to desalt the sample. However, sometimes it is necessary to use a C18 micro-column in order to fractionate a complex sample or enhance the target analyte concentration.

The sample to be analyzed is mixed with a matrix solution (1:1, v:v), e.g. sinapinic acid (SA) or alpha-cyano-4-hydroxycinnamic acid (ACHCA). One μl of the mixture is deposited on the MALDI target plate and allowed to air-dry (i.e., the dried-droplet method) before being placed in the mass spectrometer [19, 49].

#### **2.2 Proteolysis**

The analyte to be digested is dissolved in ammonium bicarbonate (50 mM, pH 7.9). The intact sample is directly analyzed by MALDI in order to determine the molecular mass of the analyte. Then, 200 μl of the solution is digested by addition of 2–10 μl trypsin (200 μg/ml in 10 mM HCl). The reaction is carried out at room temperature or at 37°C for 30 minutes up to 24 hours, depending on peptide or protein in question. It has been found that 30 minutes digestion of somatropin at room temperature generated enough tryptic fragments for the MALDI analyses [50]. For more complex proteins, such as human chorionic gonadotropin, the required time

**31**

**3. Results and discussion**

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

1. 2.5 μl 100 mM ME is added to 10 μl of the protein solution.

is performed at room temperature or at 37°C [13, 50].

period for proteolysis is found to be 24 hours at 37°C. Insulin porcine is digested at 37°C for 12 hours, while other peptides are digested at 37°C for 4 hours. In order to enable alkylation of the cysteine residues in a protein or peptide, it is reduced by using DTT or 2-mercaptoethanol (ME) followed by labelling of the free thiol groups with 2-iodoacetamide. The alkylation is carried out through the following

2.The protein is then incubated at 50°C for 15 minutes to reduce the S-S linkages.

4. 2.5 μl (10 μg/mL) trypsin is added to the mixture for the digestion. The reaction

MALDI-TOF analyses are performed using either an Autoflex or an Autoflex Max (Bruker Daltonics, Bremen, Germany) reflector type time-of-flight mass spectrometer, equipped with a pulsed nitrogen laser working at 337 nm and a smartbeam II laser working at 355 nm, respectively. The Autoflex instrument is operated in the positive ion mode with delayed extraction at an accelerating voltage of 20 kV and a variable voltage reflectron. The parameter settings are optimized to analyze peptides in reflectron mode. Before analysis, the instrument is externally calibrated with Bruker Daltonics standard peptide or protein mixtures. Peptide mass peaks occurring due to autolysis of trypsin (porcine) such as 842.51 and 2211.10 Da are also used for internal calibration. Mass spectra are obtained by averaging 250 laser shots (5× 50 shots) at different positions on the sample surface. All samples being used for post source decay (PSD) analysis are analyzed in the reflectron mode. The autoflex Max instrument TOF/TOF (2 kHz MS and 200 Hz MS/MS) operates in the positive ion mode. Metastable fragmentation is induced by laser (355 nm) without the further use of collision gas. The lyophilized samples are dissolved in 300 μL ammonium bicarbonate buffer (50 mM, pH 7.5). The liquid samples are diluted with same buffer. The wells of MALDI plate are spotted with 1 μl sample/matrix solution (1:1, v:v) and allowed to air dry before being placed in the mass spectrometer. ACHCA is used for analysis of peptides. About 20 mg of ACHCA is mixed in 1 ml of ethanol: acetonitrile (ACN) (1: 1 v/v) and 0.1% trifluoroacetic acid (TFA). SA is used for protein analysis. Two different solutions of SA in water and ethanol are made as follows: 1 - Saturated solution of SA in ethanol and 0.1% TFA; 2 - Saturated solution of SA in 50% acetonitrile (ACN) and 0.1% TFA. Solution 1 is first applied on the MALDI plate on which

the sample mixed with SA in 50% ACN and 0.1% TFA (1: 1) is then applied.

Illegally distributed lyophilized or liquid products being suspected to contain pharmacologically active peptides were seized by the Swedish customs. The analyte to be identified is analyzed in both reflectron and linear modes in order to determine its molecular mass (**Figure 3**). Large peptides and proteins are then exposed to trypsin digestion in order to obtain peptide-mass map upon MALDI analysis in

3. 2.5 μl 2-iodoacetamide (100 mM) is added into the mixture to interact with free sulfide groups of the cysteine residues at +4°C for 15 to 60 minutes in

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

**2.3 Apparatus and operating conditions**

procedure:

darkness.

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

period for proteolysis is found to be 24 hours at 37°C. Insulin porcine is digested at 37°C for 12 hours, while other peptides are digested at 37°C for 4 hours. In order to enable alkylation of the cysteine residues in a protein or peptide, it is reduced by using DTT or 2-mercaptoethanol (ME) followed by labelling of the free thiol groups with 2-iodoacetamide. The alkylation is carried out through the following procedure:

