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Protein Oxidation Biomarkers to Reveal the Age of Body Fluids Jolien M

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Protein Oxidation Biomarkers to Reveal the Age of Body Fluids Jolien M Protein oxidation biomarkers to reveal the age of body fluids Jolien M. Nienkemper 10458131 Date: 31-01-2018 Institute: University of Amsterdam Master Programme: Forensic Science Supervisor: Dr. Annemieke van Dam Co-Assessor: Prof. Dr. Ate Kloosterman List of contents: Introduction 2 1. What makes a good biomarker? 4 1.1 Biomarkers 4 2. The process of protein oxidation 5 2.1 Proteomics 5 2.2 Protein Oxidation 6 3. Oxidation products 7 3.1 Aliphatic residues 8 3.2 Aromatic residues 9 3.3 Sulfur-containing residues 10 4. Mass Spectrometry 12 4.1 Mass Spectrometry and biomarkers 12 4.2 From MS to immunoassays 14 Discussion 15 Conclusion 18 References 19 Appendix I: Table 1 23 Appendix II: Search Strategy 26 1 Abstract Knowing when a body fluid stain was left at a crime scene, can provide meaningful information on the order of events of a crime. A relatively recent development in the forensic field is to study the degradation pattern of proteins present in a body fluid, to determine the time of deposition. For this method to be successful, it is necessary to establish biomarkers which are capable of giving an indication of the age of a crime scene stain. The purpose of this article is to give an overview of the most common protein oxidation products on amino acid level, and to indicate which of these products have the potential of being used as a biomarker. It is discussed whether the biomarkers can be measured using Mass Spectrometry, and subsequently whether they can be detected through an immuno-based assay. For future research, it is advised to focus on the carbonyl groups, methionine sulfoxide, methionine sulfone, cysteines oxyacids, dityrosine and the different kynurenines for a more thorough investigation into their potential as a biomarker. Introduction With current techniques DNA can often be extracted from body fluids found at a crime scene, making identification possible. The next step is to use the information embedded in the body fluids to determine the time passed since deposition. Such information can be used to provide intelligence on the order of events of a crime, which can help to link a piece of evidence to the crime. For example, a fingermark found at a crime scene can be linked to a suspect with the help of a database. It is possible however, that the suspect claims that he had been at the location of the crime scene, but one week prior to the date of the crime. If no other evidence can be found against the suspect, he cannot be detained. In such a situation, it would have been helpful if the forensic examiners would have been able to determine at what time the fingermark was deposited. Currently, different study groups are looking into new ways to apply varying analytical techniques on certain types of body fluids to estimate the age of that particular body fluid. Most research in this area is still in its early stages however, and needs further investigation. Almost all body fluids contain proteins specific to that fluid, which can be used for identification of the biological material (Juusola & Ballantyne, 2003). Moreover, such proteins can function as biomarkers for age estimation. Over time proteins degrade into different products (Stadtman & Levine, 2000) which can be measured and used to determine the time since deposition. For example, it is known that the major component of blood, hemoglobin, converts into metHemoglobin once oxygen saturated. Inside of the body metHemoglobin is reduced back into hemoglobin, but outside of the body all hemoglobin will be oxygen saturated and change into hemichrome (Bremmer et al., 2011). Bremmer et al. (2011) studied the ratio of these three hemoglobin derivatives in dried blood stains with the use of diffuse reflectance spectroscopy. They found that each measurement, performed between 0 to 60 days after deposition, showed a unique combination of the three derivatives. With the help of this ratio, the age of a bloodstain could be estimated with, at most, a margin of uncertainty of 14 days. Bauer and colleagues (2003) took a different approach by looking into the degradation levels of RNA in blood, using RT-PCR to quantify the amounts of mRNA. Their results showed a significant correlation between the rate of RNA degradation and storage time. They do emphasize however, that storage conditions need to be known for 2 reliable age estimation, since, for example, heat and light could influence RNA fragmentation. p A similar approach was attempted for saliva samples, however, higher variability in overall protein concentrations within and between individuals and a high protein degradation rate made it hard to find a protein marker which was stable over time (Crosley et al., 2009; Ackerman et al., 2010). Saliva in general is found to be a very dynamic proteome, continuously being supplied with new proteins which are subsequently being removed by swallowing. Not only does is contain a great variety of proteins, it is also a very unstable fluid, susceptible to microorganisms and proteases (Helmerhorst & Oppenheim, 2007; Esser et al., 2008). In saliva, protein degradation starts from the moment a sample is deposited and is usually very rapid, making these proteins inadequate for long term age determination. However, a selection of degradation products has been found to increase in abundance over time and are therefore thought to be stable breakdown products of larger proteins, making them suitable biomarkers (Esser et al., 2008). Yet precautions should be taken towards the proteases in saliva and how they interact with these proteins. Seminal plasm proteins also show rapid degradation, but when stored under optimum conditions, relatively stable biochemical parameters can be found (Jimenez- Verdejo et al., 1994). An older research of Jimenez-Verdejo and colleagues (1994) studied the behaviour of a selection of semen specific enzymes and detected that, when all parameters were combined, the age of a semen stain could be calculated with a certain degree of precision. Nevertheless, none of the parameters was capable of giving a reliable age prediction on their own. A more recent study of Szykula (2016) found ‘new’ biomarkers in semen by analysing the length of proteins in aging stains with the help of mass spectrometry. Results showed dermcidin and semenogelin-2 to have the most significant change in length, making them interesting targets for validation studies. A promising study of van Dam et al. (2014) showed that with the help of fluorescence spectroscopy the age of the 55% of the male fingermarks could be determined. The method used is based on the principle of the oxidation of the proteins and lipids present in a fingermark, with tryptophan-containing proteins (Tryp) being the main contributor. Unsaturated lipids oxidize when exposed to air, which results in reactive oxidation products (LipOx). These products react with proteins and form fluorescent oxidation products (FOX) (Eq. (1)). [Eq. (1)]: LipOx + Tryp → FOX During the oxidation reaction tryptophan is degraded, causing the fluorescence intensity generated by tryptophan to decrease. FOX is being produced as a result of the reaction, which leads to an increase of FOX-induced fluorescence. From this fluorescence spectrum, a Tryp/FOX-ratio can be extracted, which can be used to determine the time of deposition. A downside to this method is the fact that fluorescence spectroscopy is not sensitive enough to measure the fluorescent reaction products of, for example, female donors, because of the lower excretion of skin components by women. Mass spectrometry is a technique which is far more sensitive and is capable of quantifying very minute amounts of proteins. This is particularly useful since there is still no method available to amplify proteins, as they do with PCR for DNA (Kussman et al., 2009). Since crime scene stains can have a low volume, it is important that the technique used to analyse these samples is sensitive enough to measure such small quantities. 3 This review will focus on the different forms of protein oxidation modifications and will try to answer the question ‘which biomarkers seem most promising in revealing the age of body fluids?’. Sub questions in need of an answer are: - What makes a good biomarker? - Which protein oxidation products are there? - Can MS be used to demonstrate the presence of oxidative modifications? - How do these biomarkers translate to practice? Proteins are chosen as the subject of this study since they are known to be the major component of almost every biological system, regardless of whether one considers tissue level, cellular level or body fluid level (Davies, 2005). Also, protein oxidation products are generally thought to be more stable compared to lipid oxidation products m. Moreover, proteins are found to be the main contributor to oxidation reactions with free radicals. Such knowledge, together with the finding that proteins have low turnover rates, and are therefore likely to accumulate higher amounts of oxidation products, makes that proteins are thought to be a suitable candidate for oxidation biomarker research (Davies et al., 1999). 1. What makes a good biomarker? 1.1 Biomarkers Before answering the main research question of ‘which biomarkers seem most promising in revealing the age of body fluids?’ it is discussed here what exactly a biomarker is, and what makes a good biomarker. Any biological molecule found in an organism that can be measured and quantified is a potential biomarker. In the medical field a biomarker is often a molecule that signals the presence of abnormal processes or diseases, but in the forensic context a biomarker can be used to indicate fairly common processes. One such example is using hemoglobin derivatives as biomarkers to measure the level of degradation of blood proteins. The body fluid from which biomarkers are aimed to be extracted should meet certain criteria.
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