Surface Enhanced Laser Desorption Ionization Biology Essay

Biological research in the last few decennaries has been characterises with debut of the different analytical tools. The coming of Plasma desorption PD, Fast atomic bomb FAB, Matrix assisted laser desorption ionisation MALDI and 2D-Gel cataphoresis 2D GE have made the analysis of protein in complex biological system utilizing mass spectroscopy MS possible.However, most complex biological sample like serum, plasma, piss, spit, cell lysate will necessitate important sample readying and purification before being mass analyse. Normally used purification method such as liquid chromatography, ion exchange chromatography, cataphoresis etc are frequently excessively arduous and consumed big sum of sample [ 30 ] .

The first ionisation technique used in the survey of biomolecules was Plasma Desorption. It was introduced by Macfarlane and Torgerson in 1976 [ 16 ] . It involves the usage of radioactive stuff ; Californium 252Cf, an ? emitter which gives two fission fragments [ 14, 16 ] .

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This ionisation technique gives a reasonably sensible m/z measuring of molecules with molecular mass in the scope of few kilodaltons. The usage of radioactive component, low mass declaration and slow production of ions were its major disadvantages which limit its application to complex biological sample analysis [ 14, 16, 18 ] .The debut of Fast Atomic Bombardment by Barber and group in early 1980s revolutionized the survey of biomolecules through mass spectroscopy [ 14 ] . However they suffer the disadvantages in that big peptides can non be study straight and chemical alterations of the peptides are sometimes requires [ 37 ] .MALDI was foremost 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 ionisation techniques for non volatile, high molecular weight compound particularly proteins, biomolecules where it gives an improved sensitiveness of high order of magnitude [ 18 ] . It works on similar rules like FAB.As an ionisation technique, MALDI offers a really high sensitiveness where little sample every bit small as few hundred attomoles can analysed [ 14 ] . It is a high-thoroughput method that is besides tolerant to the presence of contamination like buffer and salts. Unlike in FAB, MALDI can be used in analysis of big saccharide without demand for derivatization [ 13, 14 ] .Low mass declaration and inaccurate mass finding were major restriction of MALDI in biomolecules analysis [ 3 ] . Besides 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 usage of matrix was non an first-class option to MALDI.

The technique is arduous and gives low declaration for proteins with extremes in molecular mass [ 12 ] . It besides requires comparatively big sum of sample [ 16 ] .With all these restriction associated to each techniques as they were being discovered, it now become imperative to develop an ionization method that will be a high-thorough put, really sensitive and can selective fractionate before ionization takes topographic point.The combination of MS analysis with biochemical or chemical modified surface will give a technique with important high analytical power that will heighten the structural elucidation and designation of mark 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 malignant neoplastic disease sensing [ 1, 12, 22, 24 ] .In this thesis, we are traveling to look at the history and development of SELDI, the rules on which it operates its part to the development of analytical scientific discipline and the possible restrictions.

Chapter TWO


The first work on SELDI was reported by Hutchens T. W. and Yip T. T.

in June 1993 [ 1 ] . It is a alteration of MALDI technique where the sample mark ; probe surface was redesigned to incorporate preset figure of absorbing molecules. This selectively interacts with the sample depending on the chemical science introduced to the surface [ 1, 30 ] .

This method of desorbing big biomolecules was originally designed based on two manners [ 1, 30 ] . These are Surface enhanced orderly desorption SEND and Surface-enhanced affinity gaining control SEAC [ 1, 11, 14, 30 ] .SEND is a procedure by which analytes are desorbed and ionised without the demand for matrix [ 30 ] . This direct method uses a chemically modified surface that contains some edge energy absorbing molecules which interact with sample molecules. It works on similar rule to MALDI [ 1 ] . This technique is still in originative phase and has demonstrated limited application when comparison to SEAC [ 30 ] .Most SELDI engineering presents is based on SEAC.

It is a method used in order to extinguish the suppression of molecular ionisation in a complex mixture. It consists of a investigation which used affinity gaining control devices and therefore increases the sensing of hint biomolecules in unfractionated samples [ 1, 14, 30 ] .


Over the last decennary, the major development on SELDI has been on the sample showing surface ( affinity investigation ) because of its function in the purification, alteration, extraction, elaboration, and/or desorption/ionization of the analytes [ 2 ] . Some of the standard that were put into consideration in order to plan a good affinity surface were given as ; non-specific binding character, specificity of the bio-affinity procedure, surface capacity, mass spectrometric chemical noise and demand for non-stringent immobilisation status [ 2 ] .


1 Technical development

The early work on SELDI starts with sample-presenting surfaces ; a solid stage on which analytes are deposited in a mode required for successful desorption [ 1 ] . The sample investigation element surface is normally chemically modified to do the surface active. It was observed that the composing of the sample investigation component, its surface and any extra constructions on the surface used in absorbing energy or presenting samples for desorption greatly varied [ 1, 2, 30 ] .

Two theoretical accounts were produced as illustrations of chemically modifying a surface and therefore change overing it from inert province to an active one which can be used to neatly desorb integral biomolecules [ 1 ] .

2.1.1 SEND

The first work on SEND involves direct attachement of energy absorbing molecules the sample showing surface thereby extinguishing the demand for matrix even for analysis of high molecular weight compound [ 1, 30 ] . The investigation surface was modified by covalently associating ?-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 figure 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 utilizing self assembled monolayers of methy-N- ( 4-mercaptophenyl-carbamate ) MMPC. This was covalently linked to a gilded surface [ 1, 2, 30 ] .

It was besides revealed that molecules that perform ill as matrix in convectional MALDI perfomed first-class good when usage as immobilized energy absorbing molecules in SEND. This was confirmed utilizing cinnamamide that were covalently bonded to sepharose 6B surface [ 1 ] . Cinnamamide was non an effectual matrix when used in MALDI.Recent reasarch has shown the ability of porous Si to supply an first-class SEND surface [ 1, 39 ] . This was first described in desorption ionization on Si DIOS.

The belongingss of Si in term of thickness, porousness and electric resistance can be harnessed with the proper pick of Si wafer and etching status [ 30, 39 ] . Modifying the surface will leave some chemical science onto it therefore doing it good substrate campaigner 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 piss on agarose beads surface modified with individual stranded DNA [ 1 ] . The modified surface was able to capture a lactoferrin, an 80kDa glycoprotein that nowadays in minute measures in preterm baby urine [ 1, 30 ] . Apart from the ability to capture hint analyte in a sample, hutchen and his group were besides able to show the ability of SEAC in leting the remotion of contaminations through rinsing therefore heightening the sensing of analytes within a sample.

[ 1, 30 ] .With the possibility of affinity-capture based on bead already established, the construct of Immobilized-metal affinity chromatography was accepted in analysis of biological sample. Many scientists have worked on this IMAC procedure [ 28, 29 ] .

The IMAC techniques set the phase for the commercialisation of SELDI [ 2 ] . It has lead to the production of many protein biochips which uses IMAC chemical science at the surface and now extensively used in the field of clinical and proteomics research [ 2, 11, 30 ] .Other surface investigations developed over the old ages includes ; antibody- and protein-affinity investigations used in IgG and polypeptides capturing nowadays in samples [ 31 ] , lectin affinity gaining control used for observing bacteriums and virus in biological samples [ 32 ] , hydrophobic interaction bio-probes and bio-molecular interaction investigations [ 2 ] .


2 Commercial Development and Application.

In the last late ninetiess, SELDI was developed for commercial used by Ciphergen Biosystem based in California USA [ 30. 40 ] . It normally consists of ProteinChip which is the most of import unit of the constituents. Since so it has be successfully used in medical, analytical and other basic research jobs [ 9, 41 ] .What fundamentally distinguished these investigations are the chemical sciences incorporated into the surfaces to interact and retain specific compounds or those with similar belongingss.These has made the techniques to be widely applicable in many Fieldss such as malignant neoplastic disease sensing survey [ 4,11 ] , diseases biomarker find [ 4,10,15 ] , toxicology [ 9,11 ] , proteomics [ 2,8,9,10 ] , neurology [ 11 ] and immunology [ 11 ] .

Chapter THREE



Based on the Hutchen and Yip experimental study, SELDI technique consist of three major constituents ; the protein bit arrays ( sample presenting surfaces ) , mass analyzer and informations analysis package [ 1, 12, 30, ] .In utilizing the technique, few microlitres of the sample is deposited on the protein bit. After few periods of incubations, the analytes in the sample are gaining control on the surface due to surface assimilation, divider, electrostatic or hydrophobic interaction depending on their belongingss, the unbound constituents are removed by rinsing with appropriate dissolver or buffer. The washed surface is allowed to dry before a matrix solution is added where it is allowed to crystalline with the gaining control analytes. This so follow by enlightening with optical maser beam where ionisation takes topographic point before being mass analyze in a mass spectrometer in a similar manner to convectional MALDI-TOF-MS to give a mass spectrum which comprise of strengths against the m/z values of captured ( edge ) analytes [ 1, 2, 8, 12, 17, 24, 30 ] . This is the general process for SEAC.

In instance of SEND, the purified analytes is analysed straight as there is no demand for matrix [ 2, 12, 30 ] . The conventional diagram for the assorted stairss is given in Figure 1 and 2. hypertext transfer protocol: //

jpgDiagram reproduced from ref. 40Figure: Conventional representation of SELDI-TOF-MS procedure:Diagram reproduced from ref. 12Figure: Conventional diagram of SELDI-TOF-MS chief constituents

3.1 Sample presenting surfaces ( french friess ) .

As already pointed out earlier, the major difference between SELDI and MALDI technique is on the sample showing surface ( in SELDI ) where some chemical science has been introduced through alteration of the surface. These french friess array are the cardinal and most of import unit of SELDI technique [ 12, 17 ] . They interact with analytes in the sample and therefore introduced specificity into the procedure.Many surface french friess have been developed over the old ages and classified into two major group ; Chemical surface and biochemical surface.

These incorporate assorted types of surface belongingss runing from wide binding characteristics to the specific binding. [ 2, 12, 17, 30 ] .Both the chemical and biochemical investigations are given in Figure 6.Diagram reproduced from ref. 11Figure: SELDI sample showing surfaces ( Chips ) ; Upper french friess represent chemical surface ; bottom french friess represent biochemical surface.

3.1.1 Chemical Surfaces

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


2 Biochemical surfaces

These are normally made by covalently associating any molecules of involvement to the surface. These molecules include enzymes, antibodies, receptors, ligands, DNA etc depending on chemical nature of analytes of involvement. These surfaces are really specific as they interact with biomolecules of similar belongingss merely, therefore increasing the enrichment of the gaining control analytes. The manner of adhering interaction is similar to chemical surfaces [ 2, 30 ] . The advantage of these surfaces is that they can be used to find specific molecular acknowledgment mechanism in procedures like enzyme-substrate, antibody-antigen, receptor-ligand and protein-DNA interactions by selectively capturing the mark analytes in the complex mixtures of the sample [ 12 ] .

3.2. Factors impacting SELDI technique.

Most of the parametric quantities that affect SELDI respond normally occur at the sample showing surface ; being the bosom of the technique. Some these factors include:Non-specific binding: This occurs when analyte in the sample binds to other site on the investigation surface apart from the country of involvement. An ideal surface ( french friess ) should hold really low or no non-specific binding [ 2 ] . The non-specific binding could ensue due to hapless surface chemical science on the investigation. This leads to multi-mode keeping of the analyte to the surface investigation due to unwanted H bonding, hydrophobic, new wave der Waals or electrostatic interaction [ 2, 10 ] . Good surface chemical science on the investigation will cut down this.Type of surface ( Affinity investigations ) : This determines the specificity of SELDI procedure towards a peculiar analysis.

These specificity can be designed to widen investigation affinity to a broad scope of analytes ( biomolecules ) or restrict it to specific molecules depending on the check and the surface types. This guarantee that to acquire good SELDI response, appropriate surface investigation should be used in which there is relationship in the chemical science of both the analyte and the investigations [ 2, 16 ] . For illustration in the analysis of salivary peptides utilizing five different types of french friess ; normal stage ( NP20 ) , weak cation money changer ( CM10 ) , strong anion money changer ( Q10 ) , reversed stage ( H4 ) and immobilised metal affinity gaining control array with Cu ( IMAC-Cu ) , best consequence were achieved on CM10 bit at a given pH comparison to other french friess [ 10 ] .Surface capacity: The capacity of a surface bit ( array ) can be defined as the entire figure of specific molecules that can explicitly adhere to it [ 2 ] . This can be affected by the size of the analyte and its hydrokineticss radius depending on the check types. The surface capacity is really of import in finding the sensing bound of given compound within complex sample [ 2 ] .Type of solid support: The nature solid support on which absorbing molecules are covalently linked besides affect the SELDI responds. The ideal support should be immune to desorption and/or ionisation during the analysis procedure such that there is no array-mediated chemical noise which will cut down the quality of the ensuing mass spectrum [ 2 ] .

Most solid support stuffs are normally made from insulating stuff like nylon, polypropene, polystyrene, glass etc [ 1 ] . Analysis of peptide mixtures on different solid support stuff were carried by Hutchen and Yip research group. The spectra in Figure 7 reveal the fluctuation in signal to resound ratio among the different stuffs. Support stuffs made from glass was find to be ideal as it give a low signal to resound ratio [ 1 ] .

Mass spectra reproduced from ref. 1Figure: Mass spectra demoing the consequence of different types of solid support stuff on analysis of peptides mixturesNon-stringent immobilisation conditions: This describes assorted optimum biological conditions in which the analytes ( most particularly proteins ) exist in its native environment. These native conditions must be maintained throughout the procedure of sample debut, binding and lavation. These conditions include pH, temperature, buffer composing and concentration.

The consequence of pH and temperature is peculiarly of import in proteomics as assorted protein are denature at peculiar scopes of value [ 2 ] . For illustration, diminishing the pH of buffer from 8 to 6 in the analysis of spit decreases the strength of the extremums as proteins will be precipitated out of the solution [ 10 ] .

3.3 Ionization mechanism

The mechanism of ionisation of SELDI is similar to that of MALDI as it involves charge transportation [ 14 ] .

The energy absorbing molecule ( matrix ) which was added to the gaining control analytes on the bit get ionised when irradiated with optical maser beam. The ionized matrix so undergoes charge transportation with the analyte to give a gas stage charge ion. Common ion formed include [ M+H ] + , [ M-H ] – or [ M+nH ] n+ [ 14 ] .


Since its debut in 1993 and subsequent proficient and commercial developments in the some old ages after, SELDI has found increasing usage in the field of proteomics, disease biomarker, malignant neoplastic disease surveies etc.One of the major and most of import advantage of SELDI when comparison to other techniques is its ability to analyze rough biological samples which therefore eliminate the demand for sample readying [ 22, 24 ] . This has made it possible to analyze a whole cell lysate, biological fluid such as plasma, tear fluids and urine [ 22, 24 ] . Because of these alone advantages, the degree protein profiling as greatly increased. An illustration of this is the recent publication in scientific discipline diary on HIV research [ 27 ] .

This involves Zhang and his group, with SELDI techniques, were able to detect a little protein belonging to ?-defensin household that were normally secreted following activation of CD8 T cells from long term non-progressors with HIV-1 infection.The sum of sample require by SELDI is much lower than other profiling techniques e.g. 2Dgel cataphoresis [ 11, 22, 30 ] . Most clinical samples are ever limited in term of sampled biofluids. The SELDI technique is really advantageous by guaranting optimum extraction of mark compound from low sum of sample [ 11 ] .Since its commercial development of the protein biochips by Ciphergen Biosystem, it has made it possible to hold several bio specific investigations.

This has accelerated the used of SELDI in several clinical proteomics research surveies [ 2 ] . Most research labs now used immobilised antibodies french friess in the surveies of Alzheimer ‘s disease [ 36 ] , malignant neoplastic disease [ 4 ] , neurological upset and infective being [ 9 ] .Besides its application as a diagnostic engineering has contributed to the designation and word picture of many malignant neoplastic disease shapers in many cell lines [ 22 ] .Its application has now increased in the word picture of protein-protein interaction [ 33 ] and profiling of low molecular weight proteins secreted by assorted cell lines cultured in serum free medium [ 34 ] .

Kwapiszewska and his group demonstrated the significance betterment of sample demand in the protein profiling by their analyses of hemalaun-stained mouse lung cells with SELDI techniques. About 500-2000 cells from 30 interpulmonary vass were merely needed to bring forth profiles via SELDI-TOF with strong anion exchange SAX and weak cation exchange WCX french friess [ 35 ] . This was a better development when comparison with 2D-GE which will necessitate about 50,000-250,000 cells thereby doing the whole procedure more arduous.It is now possible to bring forth protein profile from every bit few as 20-25 cells utilizing SELDI technique. This may non 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 bettering the duplicability [ 11, 15 ] . This offer simpleness and high velocity when comparison with 2D-GE which is labour intensive and clip consuming.One of the major drawbacks in MALDI techniques that limit its utilizations in biological sample analysis is its inability to know apart high salts content which could interferes with the signal. In SELDI nevertheless, this is non a job as it is possible to clean up samples on the bit by the procedure of rinsing anterior to analysis. This reduces proficient variableness on the bit and makes it possible for sample like cell lysates which has high salt concentration to be analyses instantly without demand for being processed once more [ 22 ] .

The technique is a high throughput informations coevals one which can be used in analysis of low molecular weight protein ( & A ; lt ; 25kDa ) [ 15, 24 ] . This afford us the chance to analyze a important big sum of sample in a comparatively short period of clip [ 11 ] . With this advantage, the opportunities of placing a biomarker are increased. When comparison with MALDI and 2D-GE, it has a really high sensitiveness of quantification.The handiness of different assortment of french friess 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 country of toxicology [ 9, 11 ] , immunology [ 11 ] , neurology [ 11 ] have late being published.With SELDI, it is possible to transport out miniaturized on-chip pre fractional process of complex biological sample.

This is necessary as the presence of higher-abundance protein can interfere with the procedure of designation and quantification of lower copiousness one [ 11 ] . The efficient remotion or separation of these higher copiousness proteins therefore improved the sensing of the lower 1. This besides offer the opportunities of obtaining farther specificity on the surface ( french friess ) by uniting assorted surface chemical science and/or rinsing status like gradient elution in liquid chromatography [ 10 22 ] . This helps in determine the optimum conditions of adhering for a mark molecule ( eg marker ) and subsequent isolation of these marks [ 22 ] .

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

Table: Comparison of of import analytical characteristics between SELDI, MALDI and 2D GE

Table reproduced from ref. 22

Recent Development in SELDI

Because of its presumed advantages in the country of protein profiling, disease biomarker find etc, SELDI was thought to be good in the genomic analysis of complex being. While protein profiling largely involves designation of a peculiar protein nowadays in a given biological samples, genomic analysis involves looking at more than one protein in a given samples. It besides involves looking at the sequence of the peptides in a given protein [ 30 ] . This means the multiple protein has to be digested to make an heterogeneous pool of peptides which are so 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 executing hit induced dissociation CID. It has a protein bit interface which so makes it possible to transport out any protein bit based analysis. This hyphenated technique has an improved sensitiveness in supplying information about fmole degree of peptides compare to pmole degree normally observed in PSD [ 30 ] .

Figure 8 gives the conventional diagram of LDI-Qq-Qq-TOF with protein bit interface.Diagram reproduced from ref. 30Figure: LDI-Qq-TOF MS with protein bit interfaceThis technique has been found to rapidly uncover possible biomarker which are readily validated by analysis of tonss of relevant samples to obtain consequence which are statistically important in term of look and prevalence [ 30 ] .

Future application

Because of its enhanced features in term of sensitiveness, high thorough put, lower sample demand etc, it is expected the engineering could successfully be apply to future drug find [ 11 ] . The believe is that in future, protein information may be usage to obtain a alone fingerprint for every diseases. This would offer better diagnosing and forecast of such diseases.

With this, doctors will be good empower to forestall and/or dainty diseases. SELDI-based protein bit engineering is believed to be a cardinal tool towards this dream [ 11 ] .


The major restriction is in the duplicability of the techniques. Most protein french friess are non readily available. The makers prefer to sell the french friess with the whole SELDI instrument. It has low mass declaration and sensitiveness for higher molecular weight proteins [ 11, 24 ] .


SELDI-TOF MS, a alteration of MALDI TOF has increased preciseness and high thoroughput features.

It has broad application application in the proteomics and diseases biomarker find because of several advantages over other techniques. Its alone sensitiveness ( DL of fmole ) , velocity, specificity, crude sample analysis and low sample demands has been exploited in the field of proteomics and infective notwithstanding its restrictions. It is expected its part to analytical scientific discipline in the few old ages to come will be far more than what is it is now.


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