Surface Enhanced Laser Desorption Ionization Biology Essay

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Biological research in the last few decades has been characterises with introduction of the different analytical tools. The advent of Plasma desorption PD, Fast atomic bomb FAB, Matrix assisted laser desorption ionization MALDI and 2D-Gel electrophoresis 2D GE have made the analysis of protein in complex biological system using mass spectrometry MS possible.

However, most complex biological sample like serum, plasma, urine, saliva, cell lysate will require significant sample preparation and purification before being mass analyse. Commonly used purification method such as liquid chromatography, ion exchange chromatography, electrophoresis etc are often too laborious and consumed large amount of sample [30].

The foremost ionization technique used in the study of biomolecules was Plasma Desorption. It was introduced by Macfarlane and Torgerson in 1976 [16]. It involves the use of radioactive material; Californium 252Cf, an α emitter which gives two fission fragments [14, 16]. This ionization technique gives a fairly reasonable m/z measurement of molecules with molecular mass in the range of few kilodaltons. The use of radioactive element, low mass resolution and slow production of ions were its major disadvantages which limit its application to complex biological sample analysis [14, 16, 18].

The introduction of Fast Atomic Bombardment by Barber and group in early 1980s revolutionized the study of biomolecules through mass spectrometry [14]. However they suffer the disadvantages in that large peptides can not be study directly and chemical modifications of the peptides are sometimes requires [37].

MALDI was first developed by Tanaka et al. (1987,1988) and by Karas and Hillenkemp (1988) [16, 18, 38]. It is now among one of the most efficient ionization techniques for non volatile, high molecular weight compound especially proteins, biomolecules where it gives an improved sensitivity of high order of magnitude [18]. It works on similar principles like FAB.

As an ionization technique, MALDI offers a very high sensitivity where small sample as little as few hundred attomoles can analysed [14]. It is a high-thoroughput method that is also tolerant to the presence of contaminant like buffer and salts. Unlike in FAB, MALDI can be used in analysis of large carbohydrate without need for derivatization [13, 14].

Low mass resolution and inaccurate mass determination were major limitation of MALDI in biomolecules analysis [3]. Also the presence of high background noise due to high concentration of the matrix compound was another disadvantage [3].

The 2D PAGE technique which operates without the use of matrix was not an excellent alternative to MALDI. The technique is laborious and gives low resolution for proteins with extremes in molecular mass [12]. It also requires relatively large amount of sample [16].

With all these limitation associated to each techniques as they were being discovered, it now become imperative to develop an ionisation method that will be a high-thorough put, very sensitive and can selective fractionate before ionisation takes place.

The combination of MS analysis with biochemical or chemical modified surface will give a technique with significant high analytical power that will enhance the structural elucidation and identification of target protein [30].

This lead to the development of Surface Enhanced Laser Desorption Ionization SELDI in 1993. A technique that is now been widely used in the field of proteomics, disease biomarker and cancer detection [1, 12, 22, 24].

In this dissertation, we are going to look at the history and development of SELDI, the principles on which it operates its contribution to the development of analytical science and the possible limitations.

CHAPTER TWO

2.0 HISTORY OF DEVELOPMENT AND APPLICATION OF SELDI

The first work on SELDI was reported by Hutchens T. W. and Yip T. T. in June 1993 [1]. It is a modification of MALDI technique where the sample target; probe surface was redesigned to contain predetermined number of absorbing molecules. This selectively interacts with the sample depending on the chemistry introduced to the surface [1, 30].

This method of desorbing large biomolecules was originally designed based on two modes [1, 30]. These are Surface enhanced neat desorption SEND and Surface-enhanced affinity capture SEAC [1, 11, 14, 30].

SEND is a process by which analytes are desorbed and ionised without the need for matrix [30]. This direct method uses a chemically modified surface that contains some bound energy absorbing molecules which interact with sample molecules. It works on similar principle to MALDI [1]. This technique is still in germinal stage and has demonstrated limited application when compare to SEAC [30].

Most SELDI technology nowadays is based on SEAC. It is a method used in order to eliminate the suppression of molecular ionization in a complex mixture. It consists of a probe which used affinity capture devices and thus increases the detection of trace biomolecules in unfractionated samples [1, 14, 30].

Developments

Over the last decade, the major development on SELDI has been on the sample presenting surface (affinity probe) because of its role in the purification, modification, extraction, amplification, and/or desorption/ionization of the analytes [2]. Some of the criterion that were put into consideration in order to design a good affinity surface were given as; non-specific binding character, specificity of the bio-affinity process, surface capacity, mass spectrometric chemical noise and requirement for non-stringent immobilization condition [2].

2.1 Technical development

The early work on SELDI starts with sample-presenting surfaces; a solid phase on which analytes are deposited in a manner required for successful desorption [1]. The sample probe element surface is usually chemically modified to make the surface active. It was observed that the composition of the sample probe element, its surface and any additional structures on the surface used in absorbing energy or presenting samples for desorption greatly varied [1, 2, 30].

Two models were produced as examples of chemically modifying a surface and thus converting it from inert state to an active one which can be used to neatly desorb intact biomolecules [1].

2.1.1 SEND

The first work on SEND involves direct attachement of energy absorbing molecules the sample presenting surface thereby eliminating the need for matrix even for analysis of high molecular weight compound [1, 30]. The probe surface was modified by covalently linking α-cyano-4-hydrocinnamic acid to a sepharose bead surface [1, 30]. Ionization of biomolecules with this modified surface give mass spectrum that's has few matrix-analytes adduct, low number of multiples charge species, absent of matrix signal and enhanced analysis of low molecular weight compound [1, 30].

Further work on SEND revealed that the possibility of using self assembled monolayers of methy-N-(4-mercaptophenyl-carbamate) MMPC. This was covalently linked to a gold surface [1, 2, 30].

It was also revealed that molecules that perform poorly as matrix in convectional MALDI perfomed excellent well when use as immobilized energy absorbing molecules in SEND. This was confirmed using cinnamamide that were covalently bonded to sepharose 6B surface [1]. Cinnamamide was not an effective matrix when used in MALDI.

Recent reasarch has shown the ability of porous silicon to provide an excellent SEND surface [1, 39]. This was first described in desorption ionisation on silicon DIOS. The properties of silicon in term of thickness, porosity and resistivity can be harnessed with the proper choice of silicon wafer and etching condition [30, 39]. Modifying the surface will impart some chemistry onto it thus making it good substrate candidate for future SELDI techniques [30].

2.1.2 SEAC

The early work on SEAC was carried out by Hutchens and Yip. This involves analysis of infant urine on agarose beads surface modified with single stranded DNA[1]. The modified surface was able to capture a lactoferrin, an 80kDa glycoprotein that present in minute quantities in preterm infant urine [1, 30]. Apart from the ability to capture trace analyte in a sample, hutchen and his group were also able to demonstrate the ability of SEAC in allowing the removal of contaminants through washing thus enhancing the detection of analytes within a sample. [1, 30].

With the possibility of affinity-capture based on bead already established, the concept of Immobilized-metal affinity chromatography was accepted in analysis of biological sample. Many scientists have worked on this IMAC process [28, 29].

The IMAC techniques set the stage for the commercialization of SELDI [2]. It has lead to the production of many protein biochips which uses IMAC chemistry at the surface and now extensively used in the field of clinical and proteomics research [2, 11, 30].

Other surface probes developed over the years includes; antibody- and protein-affinity probes used in IgG and polypeptides capturing present in samples [31], lectin affinity capture used for detecting bacteria and virus in biological samples [32], hydrophobic interaction bio-probes and bio-molecular interaction probes [2].

2.2 Commercial Development and Application.

In the last late 1990s, SELDI was developed for commercial used by Ciphergen Biosystem based in California USA [30. 40]. It usually consists of ProteinChip which is the most important unit of the components. Since then it has be successfully used in medical, analytical and other basic research problems [9, 41].

What basically distinguished these probes are the chemistries incorporated into the surfaces to interact and retain specific compounds or those with similar properties.

These has made the techniques to be widely applicable in many fields such as cancer detection study [4,11], diseases biomarker discovery [4,10,15], toxicology [9,11], proteomics [2,8,9,10], neurology [11] and immunology [11].

CHAPTER THREE

3.0 PRINCIPLE OF SELDI

Based on the Hutchen and Yip experimental report, SELDI technique consist of three major components; the protein chip arrays (sample presenting surfaces), mass analyser and data analysis software [1, 12, 30,].

In using the technique, few microlitres of the sample is deposited on the protein chip. After few periods of incubations, the analytes in the sample are capture on the surface due to adsorption, partition, electrostatic or hydrophobic interaction depending on their properties, the unbound components are removed by washing with appropriate solvent or buffer. The washed surface is allowed to dry before a matrix solution is added where it is allowed to crystalline with the capture analytes. This then follow by irradiating with laser beam where ionization takes place before being mass analyze in a mass spectrometer in a similar way to convectional MALDI-TOF-MS to give a mass spectrum which comprise of intensities against the m/z values of captured (bound) analytes [1, 2, 8, 12, 17, 24, 30]. This is the general procedure for SEAC. In case of SEND, the purified analytes is analysed directly as there is no need for matrix [2, 12, 30]. The schematic diagram for the various steps is given in Figure 1 and 2

. http://urology.jhu.edu/research/img1/proteomics13.jpg

Diagram reproduced from ref. 40

Figure : Schematic representation of SELDI-TOF-MS process:

Diagram reproduced from ref. 12

Figure : Schematic diagram of SELDI-TOF-MS main components

3.1 Sample presenting surfaces (chips).

As already pointed out earlier, the major difference between SELDI and MALDI technique is on the sample presenting surface (in SELDI) where some chemistry has been introduced through modification of the surface. These chips array are the central and most important unit of SELDI technique [12, 17]. They interact with analytes in the sample and thus introduced specificity into the process.

Many surface chips have been developed over the years and classified into two major group; Chemical surface and biochemical surface. These incorporate various types of surface properties ranging from broad binding features to the specific binding. [2, 12, 17, 30].

Both the chemical and biochemical probes are given in Figure 6.

Diagram reproduced from ref. 11

Figure : SELDI sample presenting surfaces (Chips); Upper chips represent chemical surface; bottom chips represent biochemical surface.

3.1.1 Chemical Surfaces

Chemical surfaces are usually made by modifying by classical chromatography media such as normal phase, reverse phase, ion exchange, and immobilised metal affinity chromatography IMAC. These surfaces give broad binding properties which are normally used in protein profiling and disease biomarker discovering. Binding of biomolecules to these surfaces is via hydrophobic, electrostatic, coordinate bonding or Lewis acid/base interaction [2, 12, 17, 30].

3.1.2 Biochemical surfaces

These are usually made by covalently linking any molecules of interest to the surface. These molecules include enzymes, antibodies, receptors, ligands, DNA etc depending on chemical nature of analytes of interest. These surfaces are very specific as they interact with biomolecules of similar properties only, thus increasing the enrichment of the capture analytes. The mode of binding interaction is similar to chemical surfaces [2, 30]. The advantage of these surfaces is that they can be used to determine specific molecular recognition mechanism in processes like enzyme-substrate, antibody-antigen, receptor-ligand and protein-DNA interactions by selectively capturing the target analytes in the complex mixtures of the sample [12].

3.2. Factors affecting SELDI technique.

Most of the parameters that affect SELDI respond usually occur at the sample presenting surface; being the heart of the technique. Some these factors include:

Non-specific binding: This occurs when analyte in the sample binds to other site on the probe surface apart from the area of interest. An ideal surface (chips) should have very low or no non-specific binding [2]. The non-specific binding could result due to poor surface chemistry on the probe. This leads to multi-mode retention of the analyte to the surface probe due to undesirable hydrogen bonding, hydrophobic, van der Waals or electrostatic interaction [2, 10]. Good surface chemistry on the probe will reduce this.

Type of surface (Affinity probes): This determines the specificity of SELDI process towards a particular analysis. These specificity can be designed to extend probe affinity to a wide range of analytes (biomolecules) or limit it to specific molecules depending on the assay and the surface types. This ensure that to get good SELDI response, appropriate surface probe should be used in which there is relationship in the chemistry of both the analyte and the probes [2, 16]. For example in the analysis of salivary peptides using five different types of chips; normal phase (NP20), weak cation exchanger (CM10), strong anion exchanger (Q10), reversed phase (H4) and immobilised metal affinity capture array with copper (IMAC-Cu), best result were achieved on CM10 chip at a given pH compare to other chips [10].

Surface capacity: The capacity of a surface chip (array) can be defined as the total number of specific molecules that can explicitly bind to it [2]. This can be affected by the size of the analyte and its hydrodynamics radius depending on the assay types. The surface capacity is very important in determining the detection limit of given compound within complex sample [2].

Type of solid support: The nature solid support on which absorbing molecules are covalently linked also affect the SELDI responds. The ideal support should be resistant to desorption and/or ionization during the analysis process such that there is no array-mediated chemical noise which will reduced the quality of the resulting mass spectrum [2]. Most solid support materials are usually made from insulating material like nylon, polypropylene, polystyrene, glass etc [1]. Analysis of peptide mixtures on different solid support material were carried by Hutchen and Yip research group. The spectra in Figure 7 reveal the variation in signal to noise ratio among the different materials. Support materials made from glass was find to be ideal as it give a low signal to noise ratio [1].

Mass spectra reproduced from ref. 1

Figure : Mass spectra showing the effect of different types of solid support material on analysis of peptides mixtures

Non-stringent immobilization conditions: This describes various optimal biological conditions in which the analytes (most especially proteins) exist in its native environment. These native conditions must be maintained throughout the process of sample introduction, binding and washing. These conditions include pH, temperature, buffer composition and concentration. The effect of pH and temperature is particularly important in proteomics as various protein are denature at particular ranges of value [2]. For example, decreasing the pH of buffer from 8 to 6 in the analysis of saliva decreases the intensity of the peaks as proteins will be precipitated out of the solution [10].

3.3 Ionization mechanism

The mechanism of ionization of SELDI is similar to that of MALDI as it involves charge transfer [14]. The energy absorbing molecule (matrix) which was added to the capture analytes on the chip get ionised when irradiated with laser beam. The ionised matrix then undergoes charge transfer with the analyte to give a gas phase charge ion. Common ion formed include [M+H]+, [M-H]- or [M+nH]n+ [14].

CONTRIBUTION OF SELDI TO THE DEVELOPMENT OF ANALYTICAL SCIENCE

Since its introduction in 1993 and subsequent technical and commercial developments in the some years after, SELDI has found increasing use in the field of proteomics, disease biomarker, cancer studies etc.

One of the major and most important advantage of SELDI when compare to other techniques is its ability to analyse crude biological samples which thus eliminate the need for sample preparation [22, 24]. This has made it possible to analyse a whole cell lysate, biological fluid such as plasma, tear fluids and urine [22, 24]. Because of these unique advantages, the level protein profiling as greatly increased. An example of this is the recent publication in science journal on HIV research [27]. This involves Zhang and his group, with SELDI techniques, were able to discovered a small protein belonging to α-defensin family that were usually secreted following activation of CD8 T cells from long term non-progressors with HIV-1 infection.

The amount of sample require by SELDI is much lower than other profiling techniques e.g. 2Dgel electrophoresis [11, 22, 30]. Most clinical samples are always limited in term of sampled biofluids. The SELDI technique is very advantageous by ensuring optimal extraction of target compound from low amount of sample [11].

Since its commercial development of the protein biochips by Ciphergen Biosystem, it has made it possible to have several bio specific probes. This has accelerated the used of SELDI in several clinical proteomics research studies [2]. Most laboratories now used immobilised antibodies chips in the studies of Alzheimer's disease [36], cancer [4], neurological disorder and pathogenic organism [9].

Also its application as a diagnostic technology has contributed to the identification and characterisation of many cancer makers in many cell lines [22].

Its application has now increased in the characterisation of protein-protein interaction [33] and profiling of low molecular weight proteins secreted by various cell lines cultured in serum free medium [34]. Kwapiszewska and his group demonstrated the significance improvement of sample requirement in the protein profiling by their analyses of hemalaun-stained mouse lung cells with SELDI techniques. About 500-2000 cells from 30 interpulmonary vessels were only needed to generate profiles via SELDI-TOF with strong anion exchange SAX and weak cation exchange WCX chips [35]. This was a better development when compare with 2D-GE which will require about 50,000-250,000 cells thereby making the whole process more laborious.

It is now possible to generate protein profile from as few as 20-25 cells using SELDI technique. This may not be possible when 2D-GE or other similar techniques are to be used [22].

The technique can be automated through robotics system. This has the advantages of improving the reproducibility [11, 15]. This offer simplicity and high speed when compare with 2D-GE which is labour intensive and time consuming.

One of the major drawbacks in MALDI techniques that limit its uses in biological sample analysis is its inability to discriminate high salts content which could interferes with the signal. In SELDI however, this is not a problem as it is possible to clean up samples on the chip by the process of washing prior to analysis. This reduces technical variability on the chip and makes it possible for sample like cell lysates which has high salt concentration to be analyses immediately without need for being processed again [22].

The technique is a high throughput data generation one which can be used in analysis of low molecular weight protein (<25kDa) [15, 24]. This afford us the opportunity to analyses a significant large amount of sample in a relatively short period of time [11]. With this advantage, the chances of identifying a biomarker are increased. When compare with MALDI and 2D-GE, it has a very high sensitivity of quantification.

The availability of different variety of chips has made it possible for analysis of complex biological samples to be carried out into great extent. Numerous publication of analysis based on SELDI techniques in the area of toxicology [9, 11], immunology [11], neurology [11] have recently being published.

With SELDI, it is possible to carry out miniaturized on-chip pre fractionation of complex biological sample. This is necessary as the presence of higher-abundance protein can interfere with the process of identification and quantification of lower abundance one [11]. The efficient removal or separation of these higher abundance proteins thus improved the detection of the lower one. This also offer the chances of obtaining further specificity on the surface (chips) by combining various surface chemistry and/or washing condition like gradient elution in liquid chromatography [10 22]. This helps in determine the optimal conditions of binding for a target molecule (eg marker) and subsequent isolation of these targets [22].

Comparative overview of SELDI with MALDI and 2D-GE is given in Table 1 as follow.

Table : Comparison of important analytical features between SELDI, MALDI and 2D GE

Table reproduced from ref. 22

Recent Development in SELDI

Because of its presumed advantages in the area of protein profiling, disease biomarker discovery etc, SELDI was thought to be good in the genomic analysis of complex organism. While protein profiling mostly involves identification of a particular protein present in a given biological samples, genomic analysis involves looking at more than one protein in a given samples. It also involves looking at the sequence of the peptides in a given protein [30]. This means the multiple protein has to be digested to create an heterogenous pool of peptides which are then presented for desorption [30].

This has lead to the recent development of LDI-quadrupole-quadrupole-TOF (LDI-Qq-Qq-TOF) mass spectrometer. This instrument has the ability of performing collision induced dissociation CID. It has a protein chip interface which then makes it possible to carry out any protein chip based analysis. This hyphenated technique has an improved sensitivity in providing information about fmole level of peptides compare to pmole level usually observed in PSD [30].

Figure 8 gives the schematic diagram of LDI-Qq-Qq-TOF with protein chip interface.

Diagram reproduced from ref. 30

Figure : LDI-Qq-TOF MS with protein chip interface

This technique has been found to quickly reveal potential biomarker which are readily validated by analysis of dozens of relevant samples to obtain result which are statistically significant in term of expression and prevalence [30].

Future application

Because of its enhanced characteristics in term of sensitivity, high thorough put, lower sample requirement etc, it is expected the technology could successfully be apply to future drug discovery [11]. The believe is that in future, protein information may be use to obtain a unique fingerprint for every diseases. This would offer better diagnosis and prognosis of such diseases. With this, physicians will be well empower to prevent and/or treat diseases. SELDI-based protein chip technology is believed to be a key tool towards this dream [11].

Limitation

The major limitation is in the reproducibility of the techniques. Most protein chips are not readily available. The manufacturers prefer to sell the chips with the whole SELDI instrument. It has low mass resolution and sensitivity for higher molecular weight proteins [11, 24].

Conclusion

SELDI-TOF MS, a modification of MALDI TOF has increased precision and high thoroughput characteristics. It has wide application application in the proteomics and diseases biomarker discovery because of several advantages over other techniques. Its unique sensitivity (DL of fmole), speed, specificity, crude sample analysis and low sample requirements has been exploited in the field of proteomics and pathogenic notwithstanding its limitations. It is expected its contribution to analytical science in the few years to come will be far more than what is it is now.

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