The Science Behind Maldi Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The acronym MALDI stands for Matrix Assisted Laser Desorption and Ionisation. MALDI is soft ionisation technique which according to Overberg et al 1992, Chapman 1996 and Caprioli et al 1996 is one of the most important techniques for ionising molecules that are non volatile and have high molecular weight. The technique has extremely wide applications which will be discussed though out the essay. These applications include; protein and peptide distribution, pathological markers, small molecules such as xenobiotics and finally lipids distribution (Zenobi and Knochenmuss 1999).

Firstly, the matrix protects the sample from the high energy of the laser by acting as a solvent for the analyte. The second important function of the matrix is to be able to absorb light energy and transfer this into excitation energy, this enables the analyte to become ionised by protonation or deprotonation. The matrix is usually an aromatic compound that has the ability to absorb UV light at a wavelength of 237nm, and it also has low volatility and will readily transfer protons to the sample molecules. (Towers et al 2010).

This soft ionisation technique works by an incident photon of laser light being fired at the sample. The matrix crystals in which the sample is embedded absorb the photons and rapid evaporation of the matrix occurs. This, in turn causes electrons and protons to be transferred resulting in protonated ions of the sample molecule emitted (Clench, 2010). The mechanism by which these ions are formed is still not properly understood but there are many ideas aimed at explaining the process.

MALDI initially produces primary ions which then stimulate the production of secondary ions. The simplest explanation found to how these ions are produced is that the ions are formed by multiphoton ionisation, in which the matrix absorbs the photons and direct photonisation occurs to give ionised species' (Clench, 2010). Although many people consider this technique to be true, for example Ehring et al in 1992 and Liao & Allison, 1995, the maths involved with energy potentials just don't add up (Zenobi and Knochenmuss 1999). Other models considered for MALDI ion formation include Excited State Proton Transfer (ESPT), which involves acid-base chemistry and it is said that if the matrix is excited by one photon it can then go on to donate one proton (Clench, 2010), but according to Knochenmuss (2006) due to ESPT being highly dependent on a charge stabilising environment

"this mechanism therefore appears to be rare, perhaps only active if matrix-analyte complexes are predisposed to proton transfer via strong asymmetric hydrogen bonds and stabilizing neighbour substituent's."

(Knochenmuss, 2006, p. 975)

Another possible model for the creation of ions is 'Energy Pooling' in which there isn't really ionisation going on at all. It seems due to the heat from the MALDI laser the ions that are already present in the sample just migrate out. As quoted by Knochenmuss (2006),

"strong interactions between closely packed chromophores in solids are common, and clusters are known to be produced in MALDI plumes"

(Zenobi & Knochenmuss, 1998, p. 345)

meaning that the theory of 'Energy Pooling' is a plausible theory.

Once MALDI has ionised the sample, the ions then need to be detected. To do this, the MALDI instrument is coupled to a mass spectrometer, for example a Time of Flight (TOF) instrument. TOF spectrometers are commonly used

"Owing to their widespread availability, high sensitivity, and unlimited m/z range"

(Cornett et al., 2007, p. 830)

The mass spectrometer will, using specialised software, generate a mass spectra of the sample. Mass spectra can be gained, either from a sample that has been spotted with matrix on a MALDI target plate, or directly from a tissue sample coated with matrix. When directly analysing an intact tissue section, (section = a thin, uniform, slice of tissue usually produced by a cryostat and typically around 10µm thick (Brown et al., 2004), different information can be gained. A low resolution spectra consisting of information from a specific area of the tissue is called "profiling" (Caldwell and Caprioli 2005). A profile spectra consists of mass to charge ratio information plotted against intensity. A high resolution spectra, can be gained from measuring the mass to charge ratio against intensity at many points all over the section. The xy coordinates of each spectra are remembered and so images can be produced for intensity of selected ion, hence the name "imaging" (Caldwell and Caprioli 2005).

The history of MALDI imaging

According to the Nobel Prize Committee it was a Japanese man named Koichi Tanaka from Shimadzu Corp who, in the mid eighties, developed the technique of soft ionisation using laser desorption to ionise proteins. In 2002 he shared a Nobel prize with John Fenn for his work (Griffiths 2008). There is however some controversy as to who actually developed the MALDI technique. At the University of Frankfurt in Germany, also in the mid eighties, Franz Hillenkamp and Michael Karas developed the MADI technique that is adopted today (BORMAN et al., 2003). The work that Hilenkamp and Karas published (Karas et al 1985) containing their developments coined the name MALDI. From the 1990s cheap lasers were developed and soon MALDI became a popular instrument within mass spec labs when teamed with mass spectrometers, for example, TOF to gain profiles of proteins and allowing the analysis of relatively complex mixtures from impure samples (Clench, 2010). In 1997 Richard M. Caprioli and colleagues at University of Texas managed for the first time to develop a MALDI-mass spectrometry (MALDI-MS) imaging system (Caprioli et al 1997). Fresh tissue taken from Rat spleanic pancreas' were successfully imaged. Matrix or C18 beads were used to coat the tissue samples, and images produced showed molecular ions and many peptides. The technique was extremely sensitive but not quantitative (Caprioli et al 1997). By 1999 Caprioli and his colleagues introduced direct protein analysis from imaging an intact tissue (Chaurand et al 1999). From 2000 to present, many scientific advances have been made. There have been examples of protein analysis on direct tissues for example, mouse brain by Stoeckli et al (2001), in addition lipids or phospholipids can be imaged, shown in figure 1, (Bunch 2010). Proteins have been identified that have potential to be used as cancer biomarkers (Zhenget al 2003). MALDI-MS can also be used to detect and image low Relative Molecular Mass (RMM) xenobiotics directly in tissue sections, for example, the detection of ketoconazole in the skin (Bunch et al 2004), and even in whole body tissue sections. Figure 2 shows the distribution of the anti cancer drug vinblastin throughout the thoracic and abdominal cavities of a rat (Trim et al 2008).

Figure 1: MALDI MS Image of a Phospholipid in Rat Brain (Bunch 2010)

Figure 2. MALDI-MS/MS images from the thoracic and abdominal cavities showing the distribution of the precursor and product ions of vinblastine (Trim et al 2008).

Applications of MALDI-MS imaging

MALDI-MS imaging has many applications when analysing biologics, especially when looking at proteins and peptides, and has proved to be a very versatile technique. Due to the fact that MALDI-MS has rapid analysis time and excellent sensitivity, it has found a place studying proteins, peptides, small molecules and xenobiotics in the research areas of neuroscience, cancer and the pharmaceutical industry. According to Cornet et al (2007)

"MALDI-IMS applications are typically classified into two broad categories according to the analyte: proteins and small molecules."

(Cornett et al., 2007, p. 803)

Proteins

Proteomics, the study of proteins, is a huge area of research and MALDI-MS imaging allows for proteins to be imaged and the located directly on a tissue sample. MALDI-MS imaging, when used to study proteins, offers complimentary information to gel electrophoresis and has the added advantage of being able to give spatial distribution of the protein (Berkelman et al., 2009). MALDI-MS imaging for proteomic studies can give an excellent insight to complex biological processes since the identity of the protein does not need to be known in advance (Stoeckli et al 2001), for example Stoeckli et al (2001) studied the protein and peptide distribution within brain tumors for proteins that are over or under expressed within brain tumors compared to normal brain tissue. This is a technique that could ultimately have the potential to be utilized, with an operating theatre, by a surgeon, to identify the margins of a tumor being removed. Proteomics is also important when looking at the physiology of an organ, for example Chaurand et al (2003) studied how spermatozoa mature as they pass along the epididymus, and the maturation depends on sequential interactions between proteins present in the epididymal fluid and the spermatozoa. MALDI-MS imaging was used to determine the protein composition within the epididymal fluid along the epididymus and confirm that there was a change in protein composition from the start of the spermatozoa journey to the end Chaurand et al 2003). This study has shown the potential of studying the epididymus for a better understanding of spermatozoa maturation and also that MALDI-MS imaging can be used to study any tissue, cell or organ.

Pathology

MALDI-MS imaging is becoming a key tool for pathology proteomic studies, and this is because of its ability to be performed on paraffin tissue sections and so can be used to hunt for biomarkers, for clinical diagnostic follow ups, and for closely monitoring protein products and metabolites of disease markers. In the research carried out by Chaurand et al (2003), detecting and identifying protein markers in colon cancer showed that;

"identification of unique protein modifications associated with cellular transformations, can provide new insights in to colon cancer and may provide tumour-specific markers useful in cancer diagnosis and treatment."

(Chaurand et al., 2001, p. 1321)

From looking at other literature available it seems that this is true for not only colon cancer but many other diseases such as Parkinson's disease. Skold, K. et al (2006) used MALDI-MS imaging to find biomarkers for Parkinson's disease and demonstrated that it is a

"powerful toolkit for sensitive and molecular specific detection of changes in proteins and peptides in experimental models of neurodegenerative disease."

(Sköld, et al., 2006, p. 268)

Small molecules

A unique feature of the MALDI MS imaging technique is its ability to not only study proteins, but to be able to analyse complex mixtures containing very small molecules as well. This technique is able to probe and map small molecules within tissues very quickly. However, as mentioned by Cornett et al (2007) when using MALDI MS imaging to look at molecules <1000 Da it seems there is a common problem with the matrix interference if the compound of interest has similar molecular weights to some matrix related ions.

Drug metabolism and Discovery

When talking about the small molecule applications of MALDI-MS imaging, the majority of the focus is on xenobiotics and their metabolites. This is because MALDI is an excellent tool for imaging a drug and/or metabolite(s) within an animal tissue and it eliminates the need for a labeling technique e.g. autoradiography (Solon et al., 2009). It was demonstrated by Bunch et al (2004) when studying the ketoconazole, and looking at its distribution within skin and imaging its permeation though the skins layers, that MALDI-MS imaging could produce excellent spatial data. This could prove to be useful when looking at optimizing pharmaceutical products that are applied topically.

For drug discovery it is especially useful when detecting both the parent drug and metabolite in the same experiment, even though they have different masses (Reyzer and Caprioli 2007). Reyzer and Caprioli (2007) compared clozapine distribution using MALDI-MS imaging with results from a tritium labeled clozapine autoradiography study, excellent correlation was observed. Meaning that, MALDI-MS imaging could be seen to be the better technique. This is because not only can distributional information be gained, but it can also identify parent and metabolite within the same animal, where as using a radiolabel cannot distinguish between the two. Other drug discovery studies using MALDI imaging that have taken place look at the distinguishing between precursor drugs and their metabolites, for example Khatib-Shahidi et al's (2006) study looking at olanzapine and its metabolites. This study managed to show the ability of MALDI-MS imaging to give simultaneous, unambiguous, localisation information on the different metabolites of the xenobiotic. This meant that there could be an evaluation of each compound within one animal, which could be extremely beneficial when looking at ways to cut down the amount of animals used within a pharmaceutical test. These examples show that MS imaging can be used in many ways for drug discovery

Phospholipids

Phospholipids are present throughout cells within the lipid membranes and have many functions including signaling and energy storage. They can play major roles within a disease due to defects in lipid metabolism. Due to the hydrophoicity of lipids they can be difficult to analyse and sometimes may require multiple stages of mass spectrometry to obtain good results (Cha and Yeung 2007). Recent developments in MALDI-MS have

"enabled direct detection of lipids as intact molecular species present

within cellular membranes". (Murphy et al 2007)

MALDI-MS imaging has been able to fill the gaps in lipid research and present the location of specific lipids within animal and plant tissue. Due to the amount of hydrogen bonds within a lipid, the ions that are created by MALDI have a significant mass defect, making it easier to separate between lipids and other biomolecules.

Figure 3: MALDI-MSI ion images from a section of mouse kidney. (Murphy et al 2007)

The images shown in figure 3b 3c and 3d indicate phospholipids present in abundance within a mouse kidney and figure 3a shows the cholesterol present within the kidney. The intensity shown doesn't just relate to concentration and so it cannot yet be used as a quantitative technique, showing quantitative analysis using MALDI-MS imaging is an area with possibility for improvement.

Limitations and advantages of MALDI-MS imaging compared to other MS imaging techniques

Currently there seems to be a handful of limitations with MALDI-MS imaging. For example, when analysing solutions using MALDI-MS, the signal from the sample can be difficult to identify, due to suppression from other things present in the tissue that have a higher signal(McCombie et al), an example of this is hemoglobin with in blood samples. Compared to electrospray ionisation MALDI-MS has the advantage that it has a higher tolerance to salts and buffers that could interfere with the signal, also sample preparation for the technique of MALDI is simpler (Zhang et al 2003).

The current method of discovering quantitative and spatial information for a pharmaceutical compound is the technique of Whole Body Autoradiography (WBA). A lot of information can be acquired from WBA and it is currently a mandatory procedure set by the FDA. A disadvantage of this technique is the time scale as it can take around three to four days to complete, whereas MALDI-MS imaging, depending on the size of the tissue being analysed, can have a run time as little as 10 minutes up to several hours (Solon et al., 2009). Also WBA cannot differentiate between parent drug and its metabolites that retain the radioactivity, which is obviously a big limitation, and something that can be achieved with MALDI-MS imaging. MALDI-MS imaging furthermore has the advantage that it doesn't require the analyte to be radio-labeled or tagged with fluorescence to be analysed, saving sample preparation time (Solon et al., 2009).

Conclusion

It seems there is a great deal of potential for MALDI-MS image based applications. When looking at the current applications of MALDI-MS imaging it is obvious that it already plays a big part in mass spectrometry research, but it seems that it is still in the development stages and there room for improvement. Improvements to quantification methods could lead to this technique being used regularly in drug metabolism testing, and become standard throughout the pharmaceutical industry. The time it takes to image a sample is continuously being reduced, and image resolution is also something that is continually improving. Recent advances of the technique focus on this need for technological improvements and the new matrices and more powerful lasers (Schwamborn & Caprioli, 2010), which will improve capability of the instruments by increasing their sensitivity giving better resolution and reduce the time needed for analysis.

As it stands this technique presently contributes to studying and understanding disease and with time will become a very powerful tool for disease investigation. Already healthy and unhealthy tissue samples can be compared to identify cancer biomarkers, and it is not inconceivable that this could one day assist in surgical procedures.

Although a little more research is needed for MALDI-MS imaging to become a quantitative method, with new developments occurring continuously, this could be sooner rather than later. The future is bright for this powerful analytical technique.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.