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CBMS333 Functional Proteomics

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  • Course Code: CBMS333
  • University: Macquarie University
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  • Country: Australia

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Title: Report on Proteomics

Introduction

The biological term proteomics is used in describing the study of a full set of proteome or proteins which are expressed at a time provided in an organism, cell, organ or tissue. The current proteomics includes the integration of an inclusive of tools of protein-analytical information technologies and tools to reliably and quickly identify quantitative and qualitative variations in proteins. This will include recognition of changed protein expression related to the disease. This report also covers primarily for mass spectrometry, sample separations, separations, and protein fractionations for protein characterization and identification.

The structured program which is exploring facets of the data analysis and experimental design have been undertaken and also the technologies of sample-preparation which are practised exclusively for numerous types of samples such as mammalian fluids and tissue, plants, and micro-organisms. There is need of independent research of literature that is relevant to this topic. The practical section enables the understanding of the methodology and logic underpinning preparation of sample, protein separation, mass spectrometry, and fractionation of complex mixture.

Literature Review

In this report, the sample which was chosen was a grape skin which was analysed through the determination of its proteome. The two stages of ripening of the grape skin were analysed after a duration of two weeks apart. The samples were stored out of the refrigerator to enable a process that is natural to take place. The skin of the grapes were chosen since the tissues of the skin are present in the whole of the entire ripening and development of the grape and also the skin acts as a critical physical barrier for the protection of the grapes from threats which are external like external damages and pathogens (Akay, 2011, p. 156).

Proteins in Grape Skin

A protein is a chain of amino acids which are joined together by a band known as peptide among every amino acid. There are twenty different types of amino acids which form the major components of numerous proteins. Diverse amino acids have diverse functional groups on the chains located on their sides. The functional groups may be different chemically and co-operate with the functional groups on other different amino acids, such as in the similar molecule of proteins and other diverse molecules of proteins. The resultant compound normally determines the structure and shape of the protein molecules making up the skin of grapes which are used in the report (Daoud, 2011, p. 268).

Sample Preparation Methods

Grape skin disruption and initial solubility during the analysis of its proteome

The disruption of the of the skin tissues in the initial stage of proteomic fractionation strategy. This is done by homogenization of the tissues in the skin of the grapes by the use of cryogenic grinding or Doust homogeniser using the frozen grape skin in a pestle and motor. This requires being done swiftly in the presence of suitable inhibitors of protease to reduce degradation of photolytic of the grape skin. This alteration does not break the cells of the grape skin making the interactions and complexes of proteins to be preserved (Ferenc Darvas, 2016, p. 189).

In order to break and open the cells of the grape cells, mechanical strategies may be incorporated such as ultrasonic disruptions by the use of pressure devices or probe sonicators, cryogenic grinding by the use of bead mill, and thawing/freeze. The buffer selection utilized at this stage to solubilise the proteins is critical since if the protein cannot be solubilised, then analysis of the protein will not be possible. To maintain and retain the complexes of the protein and interactions which are normally easily and labile disrupted, PBS which is an example of physiological buffers in the presence of mild surfactants like dodecylmaltoside are very much recommended (Grandi, 2012, p. 159).  

Figure 1 above shows a flowchart of the proteome analysis strategy, fractionation, and solubilization of the grape skin (Hagen, 2010, p. 198)

If there is need of disruption of the cells of the grape skin, then the process should be done in the presence of surfactants such as SDS, C7BzO, and CHAPS or chaotropes such as guanidine, thiourea, and urea. The role of this cell disruption is to break all the bonds that are non-covalent and also disruption of tertiary and secondary structures of the grape skin. The table below shows the types of interactions between and within the proteins, method used during the process of disruption, and the energy needed during the disruption of the interaction of the proteins in the grape skin (Haroun N. Shah, 2011, p. 289).

Interaction nature

Disruption agent or method

Energy required

Electrostatic interactions

Chaotropes, charged surfactants, and  salts

2 to 5

Hydrophobic interaction

Surfactants, chaotropes

 

Hydrogen bond

chaotropic

3 to 8

Dipole-dipole

Van der Waals

Chaotropes and salts

0.3

Charge-dipole

Van der Waals

Chaotropes and salts

0.1

Disulphide bond

Reduction

40

Chaotropes

The method of chaotropes function through disruption of the hydrogen bonds of the water at the protein surface. When bonds between hydrogen and water join to the chaotropic instead of the proteins, the proteins will unfold leading to the exposure of interior protein. The chaotropic will then be removed from this solution since it is not needed through the use of buffer exchange (Hondermarck, 2016, p. 189).  

Surfactants

These are active surface agents which normally utilize compounds which are soluble in water which minimizes the liquid's surface tension or minimize the tension of the interface between a solid and a liquid or liquid and solid. The best solubilizing protein surfactant is the sodium dodecyl sulphate and anionic surfactant (Ian Humphery-Smith, 2013, p. 178).

Alkylation and Reduction of Cysteine

The final step of full protein disruption in the grape skin is the reduction of bridges of disulphide between alkylation and amino acids of the free thiol to inhibit the reformation of disulphides. The DTT is supplemented and permitted to react for thirty minutes or more followed by the addition of an agent of alkylation such as iodoacetamide (Joanna S. Albala, 2013, p. 248).

Fractionation Methods Involved in Proteomic of Grape Skin

Removal of Reagents and Contaminants

The techniques involved in a deduction of proteome complexity operate at their best when there is specifically protein and chosen reagents existing. These reagents are important in maintaining the solubility of the proteins during the technique of fractionation and precise to the fractionation technique.  Cells have a wide range of chemical compositions and not only proteins alone and this will affect the fractionation process. The contaminants should be eliminated or the technique of fractionation chosen initially which is not sensitive to the presence of the contaminants (Joanna S. Albala, 2013, p. 358).

Buffers and Salts

High salt concentration or specific physiological buffers may affect in numerous ways with chemical labelling, chromatography, and electrophoresis which involved SDC-PAGE and isoelectric. The removal of buffer and salt can be done through the following meter of conductivity: solid phase extraction, dialysis, precipitation, dilution, and buffer exchange by the use of gel chromatography filtration (Joanna S. Albala, 2013, p. 187).

Removal of lipids:   The surfactants which are present in the solution is enough to remove the lipids which are soluble hence preventing interference. Lipids can also be removed through precipitation or solvent extraction using acetone precipitation.  

Removal of nucleic acids: These components can be removed through ultra-centrifugation which huge nucleic acids may be are added to disrupt the interaction between nucleic acid and protein, endonuclease where an enzyme is engineered to work in denaturing solution or mechanical disruption where bead mills and ultrasonic probes are efficient at nucleic acids that are shear (Jozef Samaj, 2013, p. 179).

Desalting of Grape skin

Desalting though the centrifugal process is a faster method of carrying out the gel-filtration chromatography and also need a low-velocity centrifuge as compared to the system of chromatography. During the process of desalting using the centrifugal method, the volume of liquid is permitted to pass through the column which determines if the protein in the grape skin passes through and that the salt is retained in the column as shown in figure 2 below:

Quantification of Protein in Grape Skin

Before or during the fractionation process of grape skin, the amount and concentration of protein are the grape skin requires being measured. There are many methods for measuring the concentration of proteins and each present its own disadvantages and advantages.  These methodologies function by the complexity of the reagents with a given amino acids which produce visible fluorescence or colour which can be measured by the use of gel scanner or spectrophotometer. The signal from the grape skin is compared to a graph that is standard of protein concentration which is known (Liebler, 2014, p. 247).

The quantity of signal is related directly to the existence of amino acids in the grape skin hence the correctness depends on the correspondence of amino acids between the grape skin and the standard. This standard of measurement can be a sample of protein dried after being precipitated to a powder which can be weighed accurately in the laboratory before dissolution in a suitable buffer to a concentration that is known (Marko-Varga, 2013, p. 179).  

Analysis Methods

The method which can be used in the analysis of protein in the grape skin include:

MS analysis: In this method, there is the use of the technique of chromatography which is involved in the separation of peptide out before going through the mass spectrometer.

Comparative 2-DE: In this method, there is the separation of the proteins from the initial sample which is complex.

SDS-PAGE: In this analysis method, the electrophoresis is used and have been proved to be more effective because of the complexity of the skin. The SDS is used in making the protein to be more soluble. During the process of separating the protein out, it is done by molecular weight and not through points of isoelectric since the SDS wound affect it (Matthiesen, 2016, p. 278).

The assays of proteins are sensitive to the reagents used in the preparation of the sample. The alternative methods that can be used instead of this method are densitometry and 1D PAGE in the determination of the concentration of the proteins. The methodology that has been chosen in this report on proteomics of grape skin for measuring the proteins in 1D PAGE due to some advantages which makes this method to be more effective and advantageous that other methodologies of proteomics. These advantages include:

  • In this method, the sample can be excised, subjected to MS, and in-gel digested.
  • 1D PAGE is companionable with numerous reagents used during the process of preparation of the samples.
  • The outcome of the assay is comparable directly and comparative quantitation may be done since 2D PAGE or 1D PAGE can be utilized during the process of fractionation.
  • The gel fixing after the process of electrophoresis eliminates substance that is contaminating which would affect the other assays(Michael Hamacher, 2014, p. 189).

SDS Page

The methodology that has been chosen in this report on proteomics of grape skin for measuring the proteins in 1D PAGE due to some advantages which makes this method to be more effective and advantageous that other methodologies as stated above. The mass spectrometer used in MS analysis has one great disadvantage which is that the ions being measured by this instrument can only be measured one type at a given time. Some of the provisions which the MS analysis have and other methodologies lack include the provision of pH which makes the process of separation of proteins with other compounds hence efficient separation (Misra, 2012, p. 179).

The commercially available mass spectrometers are also limited to certain ranges of masses that is determined by a quadrupole. The MS analysis is advantageous than the other two methodologies since it can be used to measure masses of both the proteins and peptides hence providing complementary information of the peptides present in the grape skin as opposed to the SDS PAGE which will only provide the mass of protein. The MS analysis also has nanoelectrospray which is normally used in the analysis of mixtures of molecules which are complex (Pedro Rodrigues, 2010, p. 278).  

The SDS-PAGE which is an abbreviation of Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis is the methodology suggested by this report of measuring the mass of proteins in the grape skin. For the separation of the proteins by their size, the molecules of the proteins should be coated with a compound which provides the specific molecules with a similar overall charge so that the intrinsic charge of the proteins at a given pH will not determine the process of separation.   

Preparation of the grape skin sample

The grape skin should be boiled during the preparation of the sample for the SDS-PAGE due to the urea presence since the proteins in the grape skin can denature at a temperature of 20 degrees. The SDS is added in the compound so as to coat the proteins and enabling their migration by the gel to be as a result of size alone. The digestion of proteomic into minute peptides is important for the fractionation of the protein and for sensitivity, detection, and measurement of the mass spectrometer (Sechi, 2011, p. 258).

There are a huge quantity of available enzymes of proteomic, however, a great number of projects involving proteomic is done by the use of trypsin due to the following properties: it is a minute protein hence can diffuse into gel, slight specificity which is defined, and cutting at arginine and lysine produces peptides possessing group of amine at each end of peptide. During the stage of isometric focusing by the use of pH gradient strip that is immobilised, the initial consideration is the determination of the load of protein that is to be used and the IPG gradient suitable for the grape skin being investigated which is shown in table 2 below:  

Type of stain

Concentration of protein in mg/ml

Silver staining

0.1 to 1.5

SYPRO ruby

0.1 to 1.5

Coomassie Blue

0.5 to 2.5

The pH gradient and the IPG length depend on the features of the grape skin sample. The trip of IPG requires to be rehydrated and there are options for which this method being either passive or actively rehydration. During the stage of 2nd dimension SDS after IEF, the major considerations in this stage are in relation to the choice of second gel dimension, placement of the IPG strip, and equilibrium of the IPG strips (Sechi, 2011, p. 169).

Analysis by Mass Spectrometer and Peptide Fractionation

The mass spectrometer measure mass of ions or molecules which as charged. The ions can be negatively or positively charged. The mass spectrometry is utilized in proteomics of grape skin as a way of identification of what proteins are present in the grape skin which may include gel band, liquid sample or gel spot. The proteins in the grape skin are initially digested with the enzyme to release peptides which are analysed easily as compared to the intact protein. Every mass spectrometers are composed of three sections which include a detector, mass filter, and an ion source.

The mass spectrometers may measure the masses of both proteins and peptides. When analysed, the mass spectrometer measures the charge or mass ratio of the proteins in the grape skin. The figure 2 below shows the mass spectrum of the average mass of peptide of the grape skin (Thongboonkerd, 2010, p. 187).  

The mass resolution refers to the ability of the mass spectrometer to resolve or differentiate masses that are closely related which has been illustrated in the figure above.

Bioinformatics Method of Analysis

In bioinformatics method of analysis, peptide sequence is subjected to a wide range of methods of analysis to understand its evolution, structure, and function. The methodologies used involves biological sequence and sequence alignment. After the completion of the MS analysis, the resulting file of data has the multiply charged peptide perceived, the masses fragment produced by precursor’s CID and the duration at which the ion was fragmented and discovered which is known as the time of retention (Visith Thongboonkerd, 2013, p. 179).

The table 4 below shows the output of the peptide in the grape skin:

Conclusion

This report also covers primarily for mass spectrometry, sample separations, separations, and protein fractionations for protein characterization and identification. In this report, the sample which was chosen was a grape skin which was analysed through the determination of its proteome. The two stages of ripening of the grape skin were analysed after a duration of two weeks apart. The samples were stored out of the refrigerator to enable a process that is natural to take place. The practical section enables the understanding of the methodology and logic underpinning preparation of sample, protein separation, mass spectrometry, and fractionation of complex mixture.

Bibliography

Akay, M., 2011. Genomics and Proteomics Engineering in Medicine and Biology. Paris: John Wiley & Sons.

Daoud, S. S., 2011. Cancer Proteomics: From Bench to Bedside. New York: Springer Science & Business Media.

Ferenc Darvas, A. G. G. D., 2016. Chemical Genomics and Proteomics, Second Edition. Michigan: CRC Press.

Grandi, G., 2012. Genomics, Proteomics and Vaccines. Michigan: John Wiley & Sons.

Hagen, J. v., 2010. Proteomics Sample Preparation. Toledo: John Wiley & Sons.

Haroun N. Shah, S. E. G., 2011. Mass Spectrometry for Microbial Proteomics. Colorado: John Wiley & Sons.

Hondermarck, H., 2013. Proteomics: Biomedical and Pharmaceutical Applications. Paris: Springer Science & Business Media.

Hondermarck, H., 2016. Proteomics: Biomedical and Pharmaceutical Applications. Chicago: Springer Science & Business Media.

Ian Humphery-Smith, M. H., 2013. Microbial Proteomics: Functional Biology of Whole Organisms. Michigan: John Wiley & Sons.

Joanna S. Albala, I. H.-S., 2013. Protein Arrays, Biochips and Proteomics: The Next Phase of Genomic Discovery. New York: CRC Press.

Jozef Samaj, J. J. T., 2013. Plant Proteomics. Paris: Springer Science & Business Media.

Liebler, D., 2014. Introduction to Proteomics: Tools for the New Biology. Colorado: Springer Science & Business Media.

Liebler, D. C., 2014. Proteomics in Cancer Research. London: Wiley.

Marko-Varga, G. A., 2013. Proteomics and Peptidomics: New Technology Platforms Elucidating Biology, Volume 46. London: Elsevier.

Matthiesen, R., 2016. Mass Spectrometry Data Analysis in Proteomics. Brazil: Springer Science & Business Media.

Michael Hamacher, K. M. K. S. A. v. H. B. W. H. E. M., 2014. Proteomics in Drug Research. London: John Wiley & Sons.

Misra, A., 2012. Challenges in Delivery of Therapeutic Genomics and Proteomics. China: Elsevier.

Pedro Rodrigues, D. E. A. d. A., 2010. Farm Animal Proteomics: Proceedings of the 3rd Managing Committee Meeting and 2nd Meeting of Working Groups 1, 2 & 3 of COST Action FA1002, Vilamoura, Algarve, Portugal, 12-13 April 2010. Michigan: Wageningen Academic Pub,

Sechi, S., 2011. Quantitative Proteomics by Mass Spectrometry. Chicago: Springer Science & Business Media.

Thongboonkerd, V., 2010. Renal and Urinary Proteomics: Methods and Protocols. Paris: John Wiley & Sons.

Visith Thongboonkerd, J. B. K., 2013. Proteomics in Nephrology. Toledo: Karger Medical and Scientific Publishers.

Vito G. DelVecchio, V. K., 2013. Applications of Genomics and Proteomics for Analysis of Bacterial Biological Warfare Agents. Paris: IOS Press.

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