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.
\r\n\tParaffin waxes were used in different ways according to their characteristics such as chemical stability, non-poisonous, no phase separation with only a slight volume shift during phase transformation with a negligible degree of sub-cooling and complete thermal stability.
\r\n\r\n\tThe storage and management of thermal energy was seen as a prospective technology for efficient energy regulation and utilization. Phase change materials (PCMs), latent heat energy storage materials, can store and release significant quantities of waste heat energy during their phase transition; thus, they have enormous potential for efficient heat energy use.
\r\n\r\n\tBecause of their low costs, high latent heat and proper thermal characteristics such as little to no supercooling, low vapor pressure, self- behaviour, paraffin has been commonly used for energy storage applications. The type of shape-stabilized or structure-stable composites must be formed by injecting paraffin into porous materials as the supporting matrix in order to preserve the shape of paraffin and avoid leakage of the melted paraffin.
\r\n\r\n\t
\r\n\tThis book focuses on thermal energy storage. In particular, the commonly used materials at high temperatures, molten salts and concrete. Also the focus is on the most promising materials with paraffin such as; carbon nanotubes/ paraffin, Nano powder/ paraffin, by-products / paraffin and clay/paraffin in for thermal energy storage.
The challenge! How do you make this system?
It can be seen from Figures 1 and 2 that there are a number of significant differences. Perhaps, the most noticeable difference is that the multiplexing BOP control system depicted, in the previous figure, features dual redundancy hydraulic supplies and command paths (blue/yellow BOP control pods). These are not evident in the direct hydraulic BOP control system shown in Figure 1 [1, 2, 3].
The fundamental BOP control system for a surface BOP stack.
BOP multiplexing control system.
Both command and hydraulic pathways are extended very considerably in the subsea multiplexing version over the direct hydraulic surface BOP control system. Whether it has been considered by the reader at the point of reading the introduction, another very major and significant system design characteristic that is evident is the Class Society rules governing maximum closing times for BOP wellbore functions that represent the underlying design rationale in the development of the control system suited for the use in deep and ultra-deep water locations.
So, the starting baseline design is a system that is illustrated in Figure 1.
Essentially, this system is still in use today on land rigs, jack-ups, and tenders. Certainly, there are a number of design refinements on the basic system but the functionality and practical operability remain.
At this point, it should also be made clear that the core system requirements for the first land-based control systems and those encountered on sixth-/seventh-/eighth-generation ultra-deep water rigs are identical.
The BOP control system’s main purpose is:
To exercise efficient and reliable control over the blowout preventer stack in the event of a well influx when the primary well control barrier of the hydrostatic column of drilling fluids in the well has not contained the well influx in the hole. Hence, we can say here that primary well control has been lost.
Put another way, we can state that the blowout preventer is the very last mechanical barrier between the well and us and is known as “secondary well control.” All exploratory, appraisal, and development well barriers contain a secondary well control boundary.
In the most simplistic approach, we can now look at the immediate identified obstacles that reared up when the designers were contemplating the reality of making the current land-based system work subsea.
Let’s refer once again to Figure 1 in greater detail.
The normal hydraulic medium used in this closed system is 10 W (10 weight—density reference) mineral-based oil, and there are no environmental “leak” concerns because the system is shore-based or in the case of a bottom-supported drilling installation offshore (jack-up) surface application.
The following two figures further highlight the material requirements in a closed hydraulic control system where each end-user function (blowout preventer and valve hydraulic actuator) requires both a hydraulic supply and return line; this is in contrast to a subsea control system, which is an open hydraulic system (Figure 2).
The open hydraulic system, by definition, is one in which the displaced hydraulic fluid from the return/exhaust side of a hydraulic function is not routed via a dedicated return line back the accumulator unit reservoir but allowed to exhaust locally to the environment. In considering the application of BOP control subsea and in the marine environment, an open system must, essentially, employ a hydraulic medium which does not pollute or contaminate the environment in which displaced hydraulic fluid is being released into. Hence, a water-based hydraulic medium is utilized in all subsea BOP control systems (Figures 3 and 4).
Dimensional details for the land-based BOP control system.
Typical surface BOP stack, connected to BOP control system and hydraulic power unit via flexible hoses.
The fifth figure in this chapter (Figure 5) is a simple block diagram of the most simple of a subsea control system maintaining closed hydraulic flow paths, while Figure 6 shows the hydraulic flow path for one single BOP function, in this instance, a pair of ram type preventers. It should be appreciated that the single function hydraulic flow path depicted in Figure 6 must be repeated to provide control over all BOP functions. This multiplicity of hydraulic flexible hoses is the basis of the perceived problem.
Fundamental but not practical proposed subsea adaptation of the former surface BOP control system.
Direct hydraulic system with pneumatic control, one function.
Let us imagine that we are in the design team that were tasked back in the early 1950s to get this control system working subsea.
Armed with the system architecture described briefly in the previous three pages, a simple approach may have been along these lines.
Provide a frame for the surface stack1.
Install the hydraulic power unit on the rig topsides and run rigid pipe for the hydraulic supply and return lines to the moon pool area.
Install a hose reel to accommodate a hose bundle.
Interface the rigid pipework and arrange the hydraulic supply and return lines with flexible hoses to a removable hose stab plate to the hose reel end plate.
Spool sufficient hose bundle, containing the required number of supply and return hydraulic hoses, for the maximum operational water depth of the rig (let us say 750 feet).
On the BOP stack, connect the appropriately assigned hose (supply and return to each function) to that function.
Figure 7 here inserted as an A3 fold-out schematic is a labeled depiction of a typical surface stack hydraulic power unit (HPU) and its hydraulic manifold. This is for reference in the forthcoming explanations.
Skid Mounted Surface BOP Control HPU and Control Manifold.
So, assuming that we have suitably stabilized and secured the hose bundle through the water column, is this going to work [3]?
No, of course not! The reasons why not are many and varied and the following list attempts to capture all the impossible shortfalls. These are not listed in the order of importance and relevance necessarily that were facing the first design team as they struggled with all the obvious obstacles.
Let us assume that the surface BOP stack has now been submerged for service subsea. Let us use the stack shown in Figure 4 on page 77. This basic stack shown, using today’s nomenclature (API Standard 53) [4] is a Class 4 A1-R3, interpreted this means a total of four preventers, one of which is an annular and the remainder are ram type preventers. The hardware at the base of the stack is a NT2 adapter which nipples up to the riser down on a jack-up. The NT2 adapter is not a hydraulic function on a surface BOP stack and is manually operated by a circular array of mechanical locking dogs [5].
And let us add that the stack has two hydraulically actuated BOP mounted valves (one on the choke line and the other on the kill line) [6, 7].
Therefore to now sum the quantity of supply and return hydraulic hoses required to control the functions of this stack if it were underwater would be:
Annular preventer2 each 1 in. nominal diameter
Upper pipe rams2 each 1 in. nominal diameter
Shear blind rams2 each 1 in. nominal diameter
Lower pipe rams2 each 1 in. nominal diameter
Choke high closing ratio valve (HCR)2 each ½ in. nominal diameter
Kill HCR2 each ½ in. nominal diameter
Figure 8 overleaf shows a scaled cross section of a hose bundle that satisfies the requirements to provide the above functions with hydraulic power. We can see, with some spare hoses surplus to requirements, the OD of the entire bundle is only ~6 in.
Cross section, hose bundle for minimum outfitted. BOP stack. Hose # 1: annular preventer close; Hose # 2: annular preventer open; Hose # 3: upper pipe ram close; Hose # 4: upper pipe ram open; Hose # 5: shear/blind ram close; Hose # 6: shear/blind ram open; Hose # 7: lower pipe ram close; Hose # 8: lower pipe ram open; Hose # 9: choke HCR close; Hose # 10: choke HCR open; Hose # 11: kill HCR close; Hose # 12: kill HCR open.
However, if we consider a BOP stack designed and built for subsea service (not a surface stack submerged!), the story is very different (Figure 8).
The subsea blowout preventer stack shown overleaf is an 18¾ in. nominal wellbore diameter, rated at 15000 psi maximum working pressure. This is denoted as “18¾—15 M.”
This particular blowout preventer stack is somewhat dated; “third generation” puts its age genre at around 15–20 years (Figure 9).
Third-generation BOP stack. Glomar Celtic Sea (Figure 10). 18¾ in.—15 M. Hose requirement—Mud boost valve: 2 × ½ in.; upper annular: 2 × 1½ in.; kill isolation valve: 2 × ½ in.; kill line connector: 2 × ½ in.; choke isolation valve: 2/½ in.; riser connector sec. unlock 1 × ½ in.; lower annular: 2 × 1½ in.; S/B rams: 2 × 1 in.; upper pipe rams: 2 × 1 in.; middle pipe rams: 2 × 1 in.; lower pipe rams: 2 × 1 in.; wellhead connector: 2 × ½ in.
Cross section, hose bundle for third-gen. BOP stack (scaled). Hose # 1: mud boost valve close; Hose # 2: mud boost valve open; Hose # 3: upper annular close; Hose # 4: upper annular open; Hose # 5: kill isolation valve close; Hose # 6: kill isolation valve open; Hose # 7: kill line connector extend; Hose # 8: kill line connector retract; Hose # 9: choke isolation valve: Close; Hose # 10: choke isolation valve open; Hose # 11: choke line connector extend; Hose # 12: choke line connector retract; Hose # 13: inner sweep valve close; Hose # 14: inner sweep valve open; Hose # 15: outer sweep valve close; Hose # 16: outer sweep valve open; Hose # 17: riser connector lock; Hose # 18: riser connector unlock; Hose # 19: riser connector sec. unlock; Hose # 20: upper outer choke close; Hose # 21: upper outer choke open; Hose # 44: lower outer kill close; Hose # 23: lower annular close, Hose # 24: lower annular open; Hose # 25: shear blind rams close; Hose # 26: shear blind rams open; Hose # 27: middle pipe rams close; Hose # 28: middle pipe rams open; Hose # 29: lower pipe rams close; Hose # 30: lower pipe rams open. Estimating size of hose reel required, rig operational water depth: 750 feet.
Given that the minimum outside diameter (OD) for the hose bundle is going to be around 7½ in. (with no spare lines in the bundled matrix) and the minimum critical bend radius (MBR) for this bundle, let us give the reel some arbitrary dimensions, as indicated in Figure 11.
Typical hose reel.
With these dimensions, the first wrap on this drum would store around 365 feet. Two wraps then would cover the water depth requirement of 750 feet. However, for the “storm loop” hose allowance in the moon pool to accommodate rig heave, another 250 feet would be required. This necessitates three wraps on this reel assembly.
The end plates’ diameter would be in the order of 22 feet diameter. The reel assembly, its prime mover, and brake assembly are large scale!
Typically, hose reel assemblies are installed at an intermediate elevation above the moon pool weather deck elevation on a mezzanine deck. Two such typical arrangements are shown in Figures 12 and 13.
Typical BOP hose reel: mezzanine-deck mounted.
Cameron hose reel installed on the Iran Alborz GVA 6000 semi-submersible drilling installation.
It has been shown that using bundled hoses of the required dimensions for the appropriate volumes demanded by the various BOP stack functions is impractical in terms of the physical challenges to build and install such hose reel topsides on a floating drilling installation. However, there are other issues with this concept, which can be summarized in the following list:
The hydraulic system is closed and therefore the friction losses encountered in the return hoses will effectively slow down the response times, which are clearly detailed and stated in the current specification of API 16D, Edition 4, 2004 [7].
The system offers zero redundancy and this is considered unacceptable for such a critical control system which must operate reliably and remotely in the “not-unlikely” event that the last mechanical barrier must be put in place immediately (shutting in the well). The prospect of building and installing an identical arrangement to the one illustrated is not in any way a practical solution whatsoever.
The hydraulic medium is environmentally unfriendly and illegal. The hydraulic medium used in surface BOP stack control systems cannot be used in subsea versions of the system (water-based, as described on page 75).
The system concept, as shown on previous pages, offers no hydraulic usable volume in storage on the subsea BOP stack, hence the drawdown effect on this system would be formidable and further exacerbate the response time issue for pipe and annular type preventers. Volumetric storage of hydraulic fluid subsea will be discussed in later sections.
What has not been discussed are the issues surrounding the realities of topsides and subsea terminations for hydraulic hoses, the minimum multiplicity exampled here is by no means the total number of stack functions now supplied to modern deep and ultra-deep water BOP stacks. The number of functions presented here for this illustrative exercise is 44, and to put that into today’s context, modern stacks boast in excess of 110 functions!
To this point in our design rational discussion on evolving control systems, the requirement for a BOP stack split disconnection has not been introduced. There is a myriad of situations in subsea drilling operations when we need to achieve a disconnection whereby the lower BOP is left latched on the subsea wellhead and the lower marine riser package is retrieved, either to surface or “positioned” in a stand-by location in the water column. The design architecture surrounding this design feature will be discussed in due course.
In light of the above, we can list the design features that are required for a reliable and fit-for-purpose subsea BOP control system [7]:
Provide redundancy
Comply with legislation
Practical installation
Install stored hydraulic fluid
Compliance: anti-pollution laws
Enhance functional multiplicity
Design allowance: disconnect
The ingenious design of the first BOP control system, which is now presented as an overview, was developed in the first half of the 1950s when the maximum rated water depth for drilling offshore off floaters was still under 1000 feet (305 m) [8].
The reasons for the water depth limitation are varied and not directly attributed to the restraints of the BOP control system. Some of these were marine, drilling plant topsides’ limitations and to a lesser extent, and capabilities of marine drilling riser (the mechanical connection between the subsea BOP stack and the drilling installation).
The immediate problems facing the pioneering design group:
How do we overcome the excessive dimensions of a simple closed hydraulic system deployed subsea?
How do we diminish the friction losses in the closed hydraulic system where the displaced fluid from the “other” side of the function slows the overall response times of the ram type and annular type preventers?
How do we build in redundancy to a point where the critical control system can satisfy the most stringent of regulators for reliability?
What can be done to minimize the risks of pollution to the marine environment?
Is there some way in which the volume/pressure drawdown effect can be reduced in the direct closed hydraulic system?
How should the topside equipment be arranged for optimum operation and account for rig motions?
What is required to configure the BOP stack to enable a disconnect while leaving the well secured in the coincidental event of a well influx?
Pictorially shown here are the basic principles that were proposed (Figure 14).
Concept and principles of the open BOP control system.
Pivotal to the success of the prototype design was the use of hydraulic relay valves which are installed in the newly conceived control pod(s) which are activated by a hydraulic pilot signal commanded from the surface. By the use of hydraulic relay valves and agreement that the displaced fluid volume from the “other” side of the function should exhaust directly through the “other side of the function” relay valve directly to the marine environment, it was immediately understood that the prior formidable size of the hose bundles could be greatly reduced, not least caused by using 3/16 in. pilot hoses in the bundle. The main hydraulic supply consequently consisted of one only nominal 1 in. diameter core hose within the bundle (see top right of the previous figure for details).
In order to have an open hydraulic system that exhausted hydraulic fluid directly to the marine environment, the hydraulic medium was changed from lightweight mineral oil to potable water dosed with additives in small percentages of dilution. This new hydraulic medium necessarily influenced careful material selection of both metal and rubber sealing components of the hydraulic valves, regulators, and other subsea control system components. Not only was the marine environment a factor in dispelling the consideration of the use of an oil-based hydraulic medium, but also differential pressure experienced across the thermoplastic wall of flexible hose at increasing hydrostatic pressure from the water column depth. This is discussed in the next sub-section.
100% redundancy was provided by furnishing two identical systems which became color-coded blue and yellow. The system is arranged whereby one to the two identical sides of the system may be used at any given moment but never both. The redundancy satisfied both operators, oil companies, and more importantly, class societies and legislative bodies. Since the early systems, levels of redundancy have been revised, and standard operating procedures adopted formerly have been revised reflecting greater caution and conservatism. This will be discussed in due course.
By the introduction of gas pre-charged hydraulic accumulators, nominally 11 US gallons capacity each, the early problems of system drawdown effects were satisfactorily banished as the 1 in. hydraulic supply in either hose bundle maintained full system working pressure in the stack-mounted accumulator bottles.
This system quickly became field proven and a number of proprietary vendors produced their own systems, however it has to be said that all were based on the principles put forward originally by Paul Koomey and his design team.
The remainder of this sub-section concentrates on the general arrangement detail of the system and its operational characteristics and finally limitations identified for this system.
The hydraulic control system is always equipped with two control pods, designated as the blue or yellow pod. To maintain a fully redundant control system, both pods must be operational at all times.
Formerly, if a control pod becomes inoperable, drilling operations would be normally suspended and the BOP stack controlled with the working pod until repairs are completed and tested. This involved the retrieval to the surface of the defective pod, repair and test on surface before re-deploying subsea to latch back into its dedicated receptacle on the LMRP.
More recently with the advent of deep water drilling, the majority of oil companies will not allow continued drilling operations for the retrieval of one pod to surface for repair. If repairs are to be performed in the midst of a drilling program, drilling operations are suspended, and the well made safe and the entire LMRP retrieved to surface to repair the faulty control pod.
The active and selected control pod is normally alternated between the pods weekly or after a BOP stack test.
Koomey Shaffer introduced a 42 line retrievable pod which featured a double female receptacle design. The separate receptacles enable both the pod to be retrieved or else the entire LMRP (Figure 15).
The 42 line Koomey control pod.
Later, as the drilling contractors began to use BOP stacks with greater number of functions, Shaffer and others introduced a 64 line control pod, which, while featuring a different geometry (cubical rather than cylindrical) operated in the same manner and was intended for retrieval during drilling operations (Figure 16).
The Koomey Shaffer 64 line retrievable control pod.
Proprietary manufacturers of subsea hose bundle strive to provide a product which has a low volumetric expansion characteristic (VEC). This ensures that API closing times are not exceeded for ram type and annular type preventers. In the electro-hydraulic control system, the single greatest contributor to lengthening response times is the hydraulic pilot pressure build time and transport time.
The fluid parameters that govern the transmission time of a hydraulic signal through a thermoplastic tube are:
The density of the fluid
The viscosity of the fluid
The un-dissolved gas in the fluid
The bulk modulus of the fluid
The values may change, but this is usually associated with significant changes in the ambient operating temperatures. An extreme example is the difference in control fluid parameters in tropical climates. As opposed to climates in far northerly and southerly latitudes, there will be no monoethyleneglycol (MEG) added to the control fluid medium since the seawater temperature at the mudline is significantly above freezing point. (This is applicable for the relatively shallow water depths in which this type of BOP control system is used, and the previous statement is not true for ultra-deep water: >6000 feet.)
We can say that the density and viscosity of the fluid will remain close at their optimum values in this operational water depth.
One of the basic concerns in regard to using thermoplastic hose to transport hydraulic fluids to great depths is differential pressure across the tube wall. At great depths, the external pressure may be sufficient to collapse the hose. The pressure at which collapse takes place is dependent upon the hose construction and the nominal diameter of the hose [4, 7].
The dominant property of differential pressure in this application arises from the differences between seawater and control fluid densities. Wherever in this type of system, there exists a degree of density difference across the hose tube wall, a chance of invoking hose collapse is possible. For instance, at a depth of 5000 feet, a thermoplastic hose containing a typical mineral oil as the hydraulic medium found in surface stack control systems will experience an overburden of around 220 psi, which is quite sufficient to collapse a hose. Pressures as little 30 psi can cause collapse of hoses with nominal diameters in the range of 3/8–½ in.
API specification 17E: specification for subsea production control umbilicals, states for collapse pressure [9]:
“The minimum value of external collapse pressure shall be 150% of the difference in the static head due to hydrostatic pressure at the maximum design depth less the static head at that depth due to the service fluid (hydraulic medium).”
Further unwanted differential pressure will be generated if the hydraulic lines are not 100% fluid filled. If any entrapped air is present in the tube length, the hydrostatic pressure will dominate and tube collapse will occur. This is easily eradicated by thorough purging and venting of all lines in the subsea umbilical hose bundle. The presence of air, however small, also dramatically increases response times due to the compressibility of gases [3].
The differential problem is overcome by choosing a hydraulic medium which has a specific gravity that is close to seawater.
Seawater has a gravity of ~1.03 and water is 1.00. Providing that the hydraulic medium is water-based with additives that only change the specific gravity to a new value remains close to that of the specific gravity of seawater then the possibility of hose collapse is virtually obviated.
Hoses, being composites with polymeric constituents are found to behave in a time dependent viscoelastic manner when applied load is a hydraulic charge as found within a pilot line hose.
The result of the viscoelasticity manifests itself in a pressure decay after initial pressurization. This is not detrimental for the pilot signals in this application since the hydraulic pilot-operated relay valves subsea “fire” and “vent” at pressures well below the nominal pilot pressure of 3000 psi. Figure 17 shows the pressure decay versus time. The typical time constraints are well beyond time of the hydraulic relay valves “firing” in this control system.
Pressure decay in thermoplastic hoses due to viscoelasticity.
Extensive laboratory testing has been performed to assess the changes in hose geometry subjected to a step positive change in internal pressure. The changes in geometry were measured using strain gauges, both axially and circumferentially affixed to the outer surface length of the hose under test.
The axial gauges measured any bending strains incurred and the circumferential gauges monitored hoop stresses. It was found that the hose length shortened with pressurization and this is explained by the layers of hose braiding attempting to establish a neutral lay angle during the buildup of pressure. The effect is almost instantaneous and remains constant, and hence the axial strain is not responsible for the viscoelastic effect.
Measured hoop strains correlate to observed pressure responses and shown typical viscoelastic behavior. In tandem with strain measurements, volume measurements have been recorded to estimate the variation in wall thickness. Such measurements have been quantified using two equations which account for the bulk modulus and pressure decay following initial viscoelastic expansion of the hose under test.
Results from these tests showed that both the internal and external diameters of the hose increased with pressurization although the OD significantly less than the ID of the hose.
Overall, this indicates that all hydraulic pilot hoses will “accept” more fluid when a pressure signal is initiated from the source and will duly expand in direct correlation with the VEC of the hose: dependent upon construction and materials. At pressure equilibrium (e.g., 3000 psi), the hydraulic pressure peak will transit the length of the hose at approximately the speed of sound.
All hoses that are constructed of material that use a composition of polymers and fibers may be classed as thermoplastic hoses. When subjected to pressure changes internally and externally, they exhibit a viscoelastic time-related response. After an initial pressurization, the pressure decays over a period of time as the hose dilates (see the previous figure) [3].
The extent of the dilation is dependent upon a number of factors such as hose material, construction, age, environment, and so on. A similar effect occurs when the hose is depressurized, this being a time-related contraction effect.
Against logical intuition, hoses bundled together exhibit greater volumetric expansion (VE) than identical hoses pressurized in isolation. There is a mathematical proof for these phenomena but suffice it to say that the reason is simply because there are effects from adjacent bundled hoses which remain pressurized against those vented to zero gauge.
It is known that aging in hoses reduces VE which acts in our favor (in drilling BOP controls) but is considered detrimental in production control systems.
Minimizing the effects of VE promotes faster response times in hydraulically piloted BOP control systems since the pod-mounted relay valves will not “fire” until they have sufficient pressure in the hydraulic pilot signal: normally around 500–700 psi.
The following figures illustrate some of the effects of the volumetric expansion characteristic in thermoplastic hoses (Figures 18, 19, 20).
Time response curves from Hydril® empirical testing.
Typical response times for 1/4 in. and 1/2 in. Diameter thermoplastic hose.
VE curves for high pressure thermoplastic hose.
In this chapter, we have explored the evolving design technology that enabled a surface land-based BOP control system to be used reliably in the subsea marine environment. Further, design teams have provided the following system features to the subsea BOP control system:
100% inbuilt design redundancy: based on the high level of safety criticality of the subsea BOP control system (the last barrier). Given fundamental design principles of the subsea hydraulic control pod.
The use of hydraulic devices (such as relay valves and regulators) to satisfy legislative close times on BOP preventers and BOP-mounted valves (Choke and Kill).
An insight into the development of flexible hydraulic hose to maximize short response times by the limitation of the design VEC of elastomeric hose materials.
The principles employed to overcome hydraulic drawdown through extended lengths of hydraulic line/hose (accumulator 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 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.
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).
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. 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, 6, 7, 8, 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, 11, 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, 15, 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, 20, 21, 22, 23, 24, 25, 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, 28, 29, 30, 31, 32, 33, 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.
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).
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, 37, 38, 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 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].
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].
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 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:
2.5 μl 100 mM ME is added to 10 μl of the protein solution.
The protein is then incubated at 50°C for 15 minutes to reduce the S-S linkages.
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 darkness.
2.5 μl (10 μg/mL) trypsin is added to the mixture for the digestion. The reaction is performed at room temperature or at 37°C [13, 50].
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 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 following peptides and proteins, Figure 4 and Table 1.
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.
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 β4, (N) Melanotan II, (O) Bremelanotide, (P) Dermorphin and (Q) BPC 157. For molecular structures of somatropin and hCG see references [13] and [50].
Recombinant hGH or somatropin consists of 191 amino acids with two disulfide bridges (Cys53-Cys165 and Cys182-Cys189) and promotes proteinogenesis as well as fat mobilization and oxidation [51, 52, 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].
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, 61, 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.
MALDI-PMF (A) and MALDI-PSD (B) analysis of somatoliberin.
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, 64, 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.
MALDI-PSD analysis of AOD.
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 hormone secretagogue receptor [66, 67]. Ghrelin strongly stimulates food intake and GH release in humans [68, 69, 70]. These peptides were identified through de-novo 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].
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, 76, 77]. MGF and Long-R3-IGF1 were identified by sequence coverages of 100% and 43%, respectively, Table 2 and Figure 7.
Peptide | Mmass | PMFa | PSDb | ISDc | DICZEd | NMR | LC/MS |
---|---|---|---|---|---|---|---|
Somatropin | 22115.07 22128.68e | X | — | X | X | X | X |
Human Somatoliberin | 3366.866 | X | X | — | — | — | — |
AOD (Anti Obesity Drug) HGH fragment 177–191 | 1813.850 | X | X | — | — | — | — |
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 | — | — | — | — |
Long-R3-IGF | 9105.385 9111.576e | 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-β4 | 4960.474 | X | X | — | — | — | — |
hCG α - Chain β - Chain α + β | 13,431e 23,114e 36,341e | X | 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 | — | — | — | — | — |
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.
Identification by peptide mass fingerprinting using enzymatic degradation as well as other modifications.
De novo sequencing by MALDI- post source decay.
Protein sequencing by MALDI- in source decay.
Identification and/or impurity profiling by double injection capillary zone electrophoresis.
Average molecular mass.
Bovin albumin was detected in some of the samples.
Peptide fragments | Theoretical m/z [M + H]+ | Determined m/z [M + H]+ |
---|---|---|
YQPPSTNKNTKSQRRKGSTFEERK | 2869.169 | 2869.422 |
Glu-C digestion of the peptidea: | ||
YQPPSTNKNTKSQRRKGSTFEE | 2584.809 | 2584.816 |
Trypsin digestion of the peptide: | ||
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 |
MALDI peptide mass fingerprinting-data from analysis of mechano growth factor.
Glu-C cleaves at the C-terminus of either aspartic or glutamic acid residues.
The amino acid sequence of the peptide was determined.
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.
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 [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 (t1/2) [80].
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.
Insulin | Theoretical m/z [M + H]+ | Determined m/z [M + H]+ |
---|---|---|
Porcine (intact) | 5774.635 | 5774.632 |
Aspart (intact) | 5822.612 | 5822.618 |
Peptide chains from Insulin porcine: | ||
[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 |
Peptide chain from Insulin aspart:b | ||
[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 |
Tryptic peptides from Insulin aspart: | ||
[GFFYTDK + H]+ | 877.399 | 877.317 |
[GFFYTDKT + H]+ | 978.457 | 978.446 |
[B-chain - (GFFYTDKT) + H]+c, d | 2487.217 | 2487.030 |
Tryptic peptides from Insulin porcine: | ||
[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 |
MALDI-TOF-MS analysis of insulin porcine and aspart.
Beta mercaptoethanol (ME) was used as reducing agent.
The A-chains of insulin aspart and Insulin porcine are identical.
Trypsinated B-chain.
These peptides originate from insulin aspart, see 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].
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 slow-wave 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.
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 PMF and de-novo sequencing of the tryptic peptides, Table 4.
Peptide fragments | Theoretical m/z [M + H]+ | Determined m/z [M + H]+ |
---|---|---|
Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETI EQE-KQAGES | 4961.484 | 4960.987 |
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 |
MALDI peptide mass fingerprinting data from analysis of thymosin β4.
The amino acid sequence of the peptide was determined in the PSD mode.
N.D. = Not detected.
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].
Peptide fragments | Peptide position in the peptide chain | Theoretical m/z [M + H]+ | Determined m/z [M + H]+ |
---|---|---|---|
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 |
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].
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.
MALDI-PSD analysis of melanotan II.
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.
MALDI-PSD analysis of dermorphin.
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, 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].
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.
MALDI-PSD analysis of BPC 157.
The proposed methods, based on PMF by MALDI-TOF-MS as well as analysis with DICZE, provided an efficient procedure for the identification of peptides and proteins in illegally distributed samples. The use of trypsin as a proteolytic enzyme generated peptide fragments which covered 40 to 80% of the amino acid sequences of the analyzed proteins. The presence of a signature peptide in the peptide map facilitated the analyte identification considerably. MALDI-TOF-MS was also applied in the PSD mode for the amino acid sequencing of selected tryptic peptides as well as small peptides, such as ipamorelin.
The double-injection CE method provided complementary information on the native protein in the presence of a reference standard. This provided the possibility of performing a comparison between the electrophoretic patterns of the reference standard and the analyte to be identified. In addition, the double-injection based identifications were carried out by comparing the corrected migration time of the analyte and the observed migration time of the reference standard.
ACHCA | α-cyano-4-hydroxycinnamic acid |
ACN | Acetonitrile |
Aib | Aminobutyric acid |
BPC | Body protecting compound |
DICZE | Double-injection capillary electrophoresis |
DSIP | Delta sleep-inducing peptide |
GH | Growth hormone |
GHRP | Growth hormone releasing peptide |
GHRH | Growth hormone releasing hormone (somatoliberin) |
hCG | Human chorionic gonadotropin |
hGH | Human growth hormone |
IGF-1 | Insulin like growth factor 1 |
ISD | In source decay |
Nle | Norleucine |
PMF | Protein mass fingerprinting |
PSD | Post source decay |
SA | Sinapinic acid |
TFA | Trifluoroacetic acid |
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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. 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