Analyse Biomolecules In Proteomics Biology Essay

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Mass spectrometry is a highly important analytical method used to analyse biomolecules in proteomics. Mass Spectrometry consists of three main parts; Ionization, Detection and Analysing. These are the three basic parts used in all Mass Spectrometers, however different types are used depending on the substances which are being analysed. The analysis of proteins begins with ionization of the molecules. There are many different types of Ionization sources. However, there are only two used when analysing biomolecules (http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm). The first is Matrix Assisted Laser Desorption Ionisation (MADLI) it is mainly used when analysing proteins which are thermolabile and non-volatile. The biomolecule is first co-crystallized in a Matrix by being attached to a target plate and being left to air dry (Gault, V. and McClenaghan, N. 2009 page). It is then bombarded with a high energy laser beam, which has a wavelength of 337nm and is a pulsed nitrogen laser. The excitation energy of the laser causes the biomolecule in the Matrix to ionize as it is rapidly vaporize into a gas. MALDI is used for samples of proteins which have small sample sizes, the sensitive detectors allow rapid analysis of the sample giving highly accurate results.

Electrospray Ionisation (ESI) is used to identify large and often polar proteins which range in molecular mass from 100 Da to over 1,000,000 Da. Electrospray Ionisation is used as an interface for Liquid Chromatography/ Mass Spectrometry (LC/MS); producing ions from fragments of unstable, thermally labile molecules (Watson J, Sparkman O. 2008. Page 503). The solute and a Nebulising gas are forced through a stainless steel capillary, this mixes them together. The tip of the capillary has an electric charge causing the sample to exit the capillary as highly charged droplets. The electric field is applied to the spray as the charged droplets pass through the counter electrode. The charged droplets first pass through a counter electrode which ionises the particles. When the solvent is in the presence of Nitrogen there is an endothermic loss of solvent by evaporation (Watson J, Sparkman O. 2008. Page 503). A Drying gas of Nitrogen is situated around the outside of the capillary to direct the charged droplets towards the mass spectrometer. The ions are then passed through a sampling code and then a skimmer which funnels the ions into a line. As the ions pass through the Electrospray Ionisation the pressure increases from normal atmospheric pressure to 1 Mbar (http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm). This causes ESI produces multiply charged ions.

Once the biomolecules have been ionized, they are then accelerated through a vacuum which has an electromagnet on either side which separates molecules. The molecules are separated depending on the mass or charge of the molecule. The unwanted ions are deflected by the electromagnet so only the required ions will reach the Detector and to be analysed. Grimm R and Beauchamp L have shown that the discharge and evaporation dynamics of the highly charged biomolecule droplets help to provide the optimization of the Electrospray Ionisation (Watson J, Sparkman O. 2008. Page 487).

The main analyser used in the Mass Spectrometry of Proteins and peptides is Time-Of-Flight (TOF) (Gault, V. and McClenaghan, N. 2009). This involves accelerating the ions down along drift tube. This is done to calculate the mass-to-charge ratio by measuring the time taken to reach the detector. TOF is mainly used in conjunction with MALDI and the most popular form of TOF is Quaduropole Time Of Flight (QTOF). (Igor V. Chernushevich, Alexander V. Loboda and Bruce A. Thomson YEAR). The lighter ions will go through the drift tube quicker than the larger ions. This allows them to be separated and give a Mass Spectrum graph which has a higher Mass Resolution. The results from the TOF analyser are then passed through an amplifier to a computer where the Mass Spectrum is shown on a computer. This allows the results to be compared with other results so the peaks can be identified easily.

Electrospray Ionisation Mass Spectrometry provides molecular weight information and when it is paired with Collisionally Activated Dissociation Mass Spectrometry/ Mass Spectrometry (CAD MS/MS) it can characterise a range of proteins (Bakhtiar, Hofstadler and Smith. 1996).this is done by gaining the ion spectrum by inducing the decomposition of the protein by inserting it into a collision cell. It then collides with molecules of Nitrogen, causing the initial molecules to be fragmented into separate product ions. Electrospray Ionisation Collisionally Activated Dissociation is also used in the mapping of peptides, including recognising where the phosphorylation sites are. The higher density of the multiply charged ions increases the effectiveness of revealing the sequence information of the proteins (Körner, Wilm, Morand. 1996).

Figure 1: A mass spectrum of the protein Leucine Enkephalin. The results are from the University of Leeds. http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm > [Accessed: 16/01/13].

The protein Leucine Enkephalin is mainly tested by Mass Spectrometry because it is a quick and effective way to find out the molecular weight of the protein. Leucine Enkephalin is found in the brain of the human body which grows within a neurotransmitting peptide. This has the amino acid sequence Tyr-Gly-Gly-Phe-Leu. Analysis of this protein is required because it has similar properties to morphine (http://www.ncbi.nlm.nih.gov/mesh/68004743). The peptide has similar binding affinities to morphine (http://molpharm.aspetjournals.org/content/12/6/987.short), using mass spectrometry to analyse this peptide will allow the protein affinities to be tested and compared against the results which morphine has given. Currently Leucine -Enkephalin is being tested by Electrospray Ionisation Mass Spectrometry. Positive Ionisation conditions, adding a trace of formic acid, will be used to test the pentapeptide Leucine-Enkephalin to encourage protonation of the sample molecules. According to a known source (http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm) the theoretical molecular weight of Leucine-Enkephalin is 555.2962 Da. An accurate ESI mass spectrum should show a value of around 0.1Da difference to the theoretical value of the protein. Figure 1 has one MH+ peak of 555.1 Da, there is also a smaller peak at 557.2 Da, 558.3 Da and 578.1 Da. The three smaller peaks show that some of the naturally occurring carbon 12 atoms have been replaced with carbon 13 isotopes which are one Dalton higher. The peak at 557.2 shows the ion has one carbon 13 isotope present and the 558.3 Da peak has 2 carbon 13 isotopes present. The highest peak, 578.1 Da, is 23 Da higher than the theoretical value of Leucine-Enkephalin. Certain molecules in the sample have been ionised by a sodium cation (Na+) instead of the addition of a proton (H+) (http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm). When molecules are positively ionised, ions such as Na+, K+ and can ionise with molecules causing peaks which are a lot higher than the theoretical value to appear on the mass spectrum.

Future developments of Mass Spectrometry:

Using it in the development of protein therapeutics (http://www.ncbi.nlm.nih.gov/pubmed/21093608)

MALDI and ESI Mass Spectrometry are now being superseded by newer and more sophisticated software, e.g. FTCIR -MS - stands for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry - which utilizes the characterisation of in vitro cellular systems. (Understanding Bioanalytical chemistry book)

National Academy of Sciences http://phys.org/news/2012-09-mass-spectrometry-protein-assays-sensitivity.html Protein assays are being tested against antibodies to speed up drug discovery. This is done by using PRISM

Introduction to mass spectrometry book - MALDI Mass Spectrometry can be used to determine whether a certain peptide or protein is phosphorylated either before or after it has been treated with the enzyme phosphatase which cleaves the phosphate ester. The mass spectrum which is produced can be compared to a phosphorylated mass spectrum to see whether the protein is phosphorylated or not.

Introduction to mass spectrometry book - MALDI is being extensively used in the characterisation of proteins, including sequence information, charge derivatization. Combining MALDI with CID and Q-TOF has allowed the sequencing of proteins and peptides to extend to larger molecules and ones which contain metal ions. Using "Intensity fading" along side MALDI allows scientists to see the signals given off by low-molecular-mass binding of proteins.

MALDI TOF MS is being used to detect protein biomarkers in samples of cancer patients. This is allowing the direct analysis of proteins to identify which proteins are present in cancerous cells and tissues. INTRO TO MASS SPEC BOOK. Page 549 - 552

LC/MS is used alongside ESI in the research of neuropeptides Intro to mass spec page 674. Silberring J ans Ekman R. mass spectrometry and hyphenated techniques in neuropeptide research. John wiley & sons new York 2003 (http://onlinelibrary.wiley.com/doi/10.1002/jms.740/abstract;jsessionid=F5C5950050CD887737D1065F03DA9F64.d03t01?deniedAccessCustomisedMessage=&userIsAuthenticated=false) .

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