albedairy-mass sepctrometry first draft1.1

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Introduction:

Mass spectrometry is an analytical technique in which atoms or chemical molecules from

selected samples are positively ionised, then separated according to their mass-to-charge ratio

(m/z), and finally recorded using advanced technology and computers. Mass spectrometry is

relatively different from other similar biochemical analysis methods such as infrared,

ultraviolet rays and nuclear magnetic resonance spectrometric (Karas et al, 1987),

(Beckmann et al, 2004). All of these techniques are based on the principle of absorption of

electromagnetic radiation to transform the molecule from basic state of energy to an

electrically recognised condition (McLafferty, 1993). Not until recently, mass spectrometry

has become a potential diagnostic technology especially when both the Electrospray

Ionization (ESI) and Matrix-Assisted Laser Desorption Ionization (MALDI) instrumentations

were developed. These techniques combine both high sensitivity and efficacy, which makes

them perfect for commercial use in research and forensic laboratories (Fenn et al, 1989). The

new mass spectrometry technology together with the use of automated and semi-automated

samples introduction systems have provides the ability to perform high-throughput analysis

(Pacholski and Winograd, 1999). Analyzing proteins in their original composite forms and

performing this operation in high-throughput analysis that recognised the current mass

spectrometry technology as an important diagnostic tool for proteomics quantification(Seeley and Caprioli, 2008).

Mass spectrometry was first introduced in 1886 by Goldstein who discovered the

fundamental and activity of positive ions in a low-pressure electrical discharge tube. Later in

1922, Aston had characterised the isotopic abundances of around chemical elements.

However, the recognition of mass spectrometers and their use in the bio chemical science and

petroleum industry had presented only on the late 1940s. Nowadays, mass spectrometers are

widely used in a variety of disciplines. Mass spectrometry has progressed extremely rapidly

during the last decade, particularly between 1995 and 2009. This progress has led to the

introduction of exclusively new instruments. This has led to the development of new

applications. New high throughput mass spectrometry was recently developed to meet the

needs of the proteomic, metabolomic and other bimolecular applications (Seeley and

Caprioli, 2008). The combination of sensitivity, selectivity, speed and the capability of

obtaining structural information for proteins make the mass spectrometry a principle

technique for biochemical analysis of complex proteins (Mann and Kelleher, 2008). The

main essential components of a basic mass spectrometer include a sample inlet, ion source,



analyser, ion detector, and data system. The Mass spectrometry is capable of generating gas-

phase ions, then separating them according to their mass-to-charge ratio using electric fields

in an evacuated volume, and finally performs a count on the number of ions. A computer

system controls the operation in addition to storing and presenting the required data. Figure

(1) demonstrate some of the essential parts of mass spectrometer; where samples are

introduced to the instrument (inlets); how the ions are fragmented (analyser); how ions are

counted (ion detectors) and the type of output and how it is manipulated (data system) and

interpreted in the mass spectrum.

Figure (l). Schematic diagram of the main parts of a basic mass spectrometer.

The sample is introduced through an inlet to the ionisation source. The source generates

gas-phase ions, which are transferred to the mass analyser for separation according to their

mass-to-charge ratio. Sample introduction is important in mass spectrometry. In order to

perform mass analysis on a sample or specimen, which is initially at atmospheric pressure, it

must be introduced into the instrument in such a way that the vacuum inside the instrument

remains relatively unchanged. One of the sample introduction methods is by direct insertion

using an insertion probe and is a very effortless way to introduce a sample into device. The

sample is first placed onto a probe and then inserted into the ionization region of the mass

spectrometer, typically through a vacuum interlock. The sample is then subjected to any

number of desorption processes, such as laser desorption or direct heating, to facilitate the

ionization process. Direct Infusion is also by a duct column is used to introduce the sample in

a gas or liquid state. Direct infusion is also useful because it can efficiently introduce smallquantities of sample into a mass spectrometer without compromising the vacuum.



Ion Analyser:

Molecular and fragment ions are accelerated by direction of the charged particles through

the mass spectrometer. Uncharged molecules and fragments are pumped away. Ions travel

down the path based on their mass to charge ratio (m/z). With the introduction of ionization

sources that can vaporize and ionize variety of molecules, there is need to improve mass

analyser performance with respect to speed, accuracy, and resolution. Examples of ionization

include time-of-flight, time-of-flight reflection, and Ion Cyclotron Resonance (Beckmann et

al, 2004). Mass analysers have undergone many improvements over the past few years in

order to operate with MALDI and ESI and also accommodate other advancements in image

recognition (Bunch et al, 2004).

Detector:

There are various types of detectors, however most work by producing an electronic

signal when bombarded by an ion. Timing mechanisms which combine those signals with the

scanning voltages permit the instrument to report which m/z strikes the detector. The mass

analyzer sorts the ions according to m/z and the detector records the load of each m/z. Regular

calibration of the m/z scale is necessary to maintain accuracy in the instrument. Calibration is

routinely performed by introducing a known protein or compound into the instrument andmodifying the presented data so that the compound's molecular ion and fragment ions are

stated accurately (Stoeckli et al, 2001). A detector records and counts the omitting ions. The

data system controls the various components of the mass spectrometer electronically, and

stores and manipulates the data used provided software and recording equipment. All mass

spectrometers have a vacuum system to maintain the low pressure (high vacuum) required for

operation. High vacuum minimises ion-molecule reactions as well as scattering and

neutralisation of the ions.

Occasionally, when performing a mass analysis there have been reports of a number of

problems which have to be observed. Small Peaks are some time observed to show a higher

molecular weight because of the natural isotopic abundance process of some molecules e.g.

C13

, and H2. Some other molecules like the labile proteins usually do not show their

molecular ions, so when performing a mass analysis the highest molecular weight peak

occurs at a less peak compared to the accurate molecular ions count. Typically, fragments

should be identified by the mass-to-charge ratio, and not by the mass results obtained in theanalysis.



Interpretation of mass spectrometry exercise:

Mass Molecular Contents583

483

392

374

198

165

137

91

57

43

M++

Boc = C4H9CO2

All molecules except C4H8CO2 + CH2C6H5

Loss of 209 from M+

C6H3 + CH3O + CO + NH + C4H

C6H3 + CH3O + CH3O + CO

C6H3 + CH3O + CH3O

C6H3 + CH2

C4H9 ( of the Boc)

CH3CO

A.

Molecular Weight Formula Molecular Ion

527 C25H33O6N5Si 528= M+●+H

Spectrum Ions

629= 1055= 529= 530= 531= 550=C4H5O3 2M

+●M

+●+2H M

+●+3H M

+●+4H M

+●+Na



B.

Molecular Weight Formula Molecular Ion257 C11H23O2N3Si 257= M

+●+H

Spectrum Ions

272= 515= 537= 259= 260= 261=

M+●

+CH3 2M+●

2M+●

+Na M+●

+2H M+●

+3H M+●

+4H

C.


397 C19H19O5N5 398= M+●

+H

Spectrum Ions

415= 399= 400= 401=

M+●

+NH3 M+●

+2H M+●

+3H M+●

+4H



D.


509 C33H39O2NSi 510= M+●

+H

Spectrum Ions

254= 511= 512= 513=

OTBDPS M+●

+2H M+●

+3H M+●

+4H

E.


253 C14H12O2N3 254= M+●

+H

Spectrum Ions

254= 255= 276= 288= 358= 391= 405= 507= 529=

M+●+H M+●+2H M+●+Na 276+C M+●+Bz 253+ 253+ M+●+H 2M+●+Na

4H+C7H5N2O 3H+C7H7N30



F.


352 C16H12O4N6 353= M+●

+H

Spectrum Ions

375= 354=

M+●

+23H M+●

+2H

G.


357 C20H27O3NSi 358= M+●

+H

Spectrum Ions

380= 280= 196= 715-718= 359= 360= 393=

M+●

+Na M+●

-Ph -162=(CH3)3CSiPh 2M+●

+H-4H M+●

+2H M+●

+3H M+●

+HCl



In summary, in mass spectrometry, a substance is bombarded with an electron beam

having sufficient energy to fragment the molecule. The ionised fragments which are produced

are speeded up in a vacuum through a magnetic field and are sorted on the basis of mass-to-

charge ratio. The MALDI mass spectrometer is illustrated in figure (2) where the device

noted to perform the following processes:

I. Produce ions from the sample in the ionization source.

2. Separate these ions according to their mass-to-charge ratio in the mass analyser.

3. Eventually, fragment the selected ions and analyse the fragments in the data analyser.

4. Detect the ions emerging from the data analyser and measure their abundance with the

detector that converts the ions into electrical signals.

5. Process the signals from the detector that are transmitted to the computer and control the

instrument through feedback.

Figure (2): Structure diagram of the MALDI ionization instrumentation (Stoeckli et al, 2007)



Direct Analysis in Real Time (DART) Mass Spectrometry:

DART is a new technique has been lately revealed by major scientific and research

institutions, allowing the precise mass measurements in high resolution of solids, liquids and

gases chemical compounds (McDonnell and Heeren, 2007). DART is widely applied to

various biological and chemical applications using electronic impulse and the ionization of

polar and non-polar compounds present on metal, polymer, mineral and biological surfaces.

Standard construction of the DART system is illustrated in figure (3).

Figure (3): Simple schematic diagram of the DART ion source device.

Unlike other desorption ionization techniques, DART does not modify or change the

original samples. Scientific research had reported that DART tend to operator at a low

voltages ionizing radiation and therefore, reduces any risk of producing any radiations and

toxic solvents. The first DART device was constructed and tested in 2002, since then, DART

has been used increasingly in forensic applications. DART has been commercially available

for the past five years. Current research reviews have reported additional applications that can

be of interest to the forensic science that use advanced DART technology and also benefited

from the use of sophisticated computer systems (Seeley and Caprioli, 2008).

The resolution of a mass spectrometer represents its ability to separate ions of different

m/z. Mass resolution manifested in the sharpness of the peaks seen in the mass spectrum. An

instrument with high resolving power will be able to distinguish two peaks very close in mass

(McDonnell and Heeren, 2007). Calculating the resolution is done in one of two ways.



Magnetic instruments tend to give peaks which are essentially Gaussian in shape, and the

usual definition is R = m/Am, where m is the mass of an ion peak and Am is the distance to

the next overlapping peak.

Desorption Electrospray Ionization (DESI) Mass Spectrometry:

Desorption Electrospray Ionization (DESI) is a recent emerging technology in support to

the use of mass spectrometry. DESI present an exceptional advantage in contrast to the

conventional mass spectrometry techniques by the maintenance of biological samples under

optimum conditions outside the vacuum system and the rapid high-throughput analysis of

biochemical samples coupled with its ability of in situ detection (Wiseman et al, 2008).

Scientists have demonstrated the capacity of DESI systems for profiling of

phosphatidylcholine and many other biological materials using certain conditions in

biological tissues and the potential utilization of this approach for molecular imaging of

proteins and lipids (Takats et al, 2004), (Ho and Huang, 2002)

Figure (4): Detailed construction of the Desorption Electrospray Ionization (DESI)

(Wiseman, et al, 2006).

The development of the DESI instrumentation as an ion source for mass spectrometry

has emerged as an interesting alterative in cases where the analysis is unfortunately destroyed

by sample preparation process. The DESI ideally can be used as a direct screening test in

emergency toxicology, or in high-although put applications of forensic laboratories. It allows

for the rapid analysis of samples under ambient conditions and without any time consuming



preparation of samples (Ifa et al, 2007). The instrumentation mechanisms and applications of

DESI in forensics, chemistry and biology have been extensively reviewed and accredited.

Another application is presented by the analysis of commercial drugs and medications and

illegal drug substance. The DESI analysis is normally performed in minutes and only requires

minimal operation skills as the computer system control and operates all the analysis steps

following manufacturing operation procedures. These characteristics make DESI a suitable

technique for rapid, in situ analysis of samples in a variety of circumstances. For example,

DESI has been applied to the rapid analysis of chemical warfare agents and explosives

present on common surfaces e.g. paper, fabric, and human biological tissue. DESI has also

been used for the screening of cannabis samples, resulting in the rapid detection of the

unauthorised chemicals and drugs (Reyzer et al, 2003), (Wiseman et al, 2005).

Conclusion:

The main advantages of the mass spectrometry system are the high resolution, large

dynamic range, and highest sensitivity, which provide the maximum quality data presentation

for biological analyses especially in proteomics. Since this technology has been used, in

particular with the improvement of other closely related and supporting techniques, a

significant progress in providing sequencing and structural information about proteins and

other biochemical compounds have been recognized (Hsieh et al, 2007). The extensive

reputation is illustrated by the flexibility and eases of operation in combination with the use

high performance computers and well developed software. The technological challenge for

today's proteomics is to develop a system that will allow rapid fractionation of biological and

forensic samples and efficient identification of proteins and other chemicals. Currently, the

applications of mass spectrometry have covered most scientific aspects of biochemical

analysis and biological detection of molecules and chemical compounds. However, advanced

research and innovation of new instrumentations will enhance clinical research and improve

investigation techniques used in forensic science.



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