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Introduction

Proteomics (study of proteome) is the large scale or systematic characterization of the proteome (proteins) of a cell, tissue or simple organism. Proteome is dynamic and can be defined as the set of proteins expressed in a specific cell under given physiological condition.

Proteins are macromolecule- long chains of amino acids. This amino acid chain is constructed when the cellular machinery of the ribosome translates RNA transcripts from DNA in the cell's nucleus. The transfer of information within cells commonly follows this path, from DNA to RNA to protein.

Proteins can be organized in four structural levels:

  1. Primary (1°): The amino acid sequence, containing members of a (usually) twenty-unit alphabet.
  2. Secondary (2°): Local folding of the amino acid sequence into a helices and ß sheets.
  3. Tertiary (3°): 3D conformation of the entire amino acid sequence.
  4. Quaternary (4°): Interaction between multiple small peptides or protein subunits to create a large unit

Each level of protein structure is essential to the finished molecule's function. The primary sequence of the amino acid chain determines where secondary structures will form, as well as the overall shape of the final 3D conformation. The 3D conformation of each small peptide or subunit determines the final structure and function of a protein conglomerate.

Proteomics can be subdivided into (a).Structural proteomics - in - depth analysis of protein structure, (b). Expression proteomics - analysis of expression and differential expression of proteins, (c). Interaction proteomics - analysis of interactions between proteins to characterize complexes and determine function.

In proteomics, the first step is sample preparation in which protein is extracted from samples such as blood, tissue, urine cells and cerebrospinal fluid. In the second step, we use methods such as 1D electrophoresis or 2D electrophoresis to separate different proteins. Then we try to cut proteins into peptides since peptides are easier to detect. In the forth step, we use mass spectrometry to detect peptides and peptides fragments based on their m/z. Finally, we can then determine the sequence of the protein using database search like MASCOT.

Sample Preparation for Electrophoresis (SDS-PAGE)

The purpose of sample preparation is to reduce the complexity of the sample prior to separation of the protein constituents and the type of sample preparation protocol used depends on factors such as solubility, size and isoelectric focus point of proteins. Sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) is used in visualizing protein expression bands (with commassie stain) by denaturing protein. It is necessary to denature all proteins in a sample in order to separate them solely on their mass in a gel matrix and not their electric charge. The disulphide bond contributing to tertiary structure is broken down using 2-mercaptoethanol or dithiothreitol (DTT) and this ensure further denaturing of the proteins.

The 12% resolving get mixture was prepared by adding together, 6.0ml protogel, 3.74ml resolving gel, 5.08ml doubly distilled water (ddH2O) and 150ul ammonium presulphate (10%) and the mixture was agitated to allow thorough mixing of the components. This was followed by the addition of 15ul TEMED. The mixture was thoroughly mixed together and loaded in between the plates of both cast immediately. Small volume of hydrated butanol added to the top of the gel to ensure a flat surface and exlude air interferring with the polymerization of the resolving gel. The gel was left for 20-30 minutes to allow it to set.

The 4% stacking gel was also prepared by adding together 1.04ml protogel, 2.0ml stacking gel, 4.88ml ddH2O. The tube was inverted to allow thorough mixing of the components. Then 40ul 10% ammonium presulphate was added followed by 8ul TEMED. The stacking gel was poured into the plates containing the resolving gel and was immediately combed using the well comb provided. The whole system was left to set for another 5-10mins. The comb was removed after setting and the cast was assembled in the gel running apparatus. This is the gel matrix for ID gel electrophoresis that is going to be used in separating the proteins in the plasma sample based on their molecular weight.

The running buffer was then poured into the tank of the running apparatus until full and the loading was position on top of the plates. This was followed by loading samples into the wells with 10ul protein markers into the first and second wells and the samples loaded into wells 3-10. The lid was placed on the tank and power set to 40mA and the system allowed to run for 45-50mins.

Protein Separation Using 1 Dimension Electrophoresis

Separation of proteins using 1D electrophoresis allows for quick global view of protein expressions and it can also be applied in targeted approach but further fractionation techniques may be required. ID gel electrophoresis separates proteins based on their molecular weight and travel time within the gel matrix. After completion of electrophoresis, the gel is removed from the running apparatus and a corner of it was cut off to mark as orientation point for reference and ready for coomassie brilliant blue staining.

The gel was transferred into a sandwish tub containing the staining solution (CBB, 50% methanol and 5% acetic acid) and was incubated for 20-30mins. After incubation, this was followed by coomassie destaining. The gel was rinsed in briefly in water to remove excess CBB stain and destaining solution (50% methanol and 5% acetic acid) was added. The destaining procedure was repeated to ensure the gel was fully destained.

In-gel Digestion (Coomassie Stained) with Trypsin

This step is carried out to cleave the protein into peptides as peptides are easy to analyze using mass spectrometry methods. The protein band made visible by the coomassie staining was excised from the gel by cutting out the gels part containing the protein of interest. The gel was cut into tiny bit of 1mm x 1mm and was transferred into 0.5ul microfuge tube. The gel pieces were washed with 80% acetonitrile and ammonium bicarbonate solution. The washing liquid was dispensed and acetonitrile was added to the gel. The gel pieces begin to shrink at this stage and the acetonitrile removed after 10mins. The gel was rehydrated using 7.6ul ammonium bicarbonate. This was followed by tryptic digestion of protein by adding 0.7ul trypsin and the components mixed together with vortex. The microfuge containing the gel pieces was placed in the incubator and left overnight to trypsinize.

ZipTip Cleaning of Peptides

ZiptipC18™ is a 10ul pipette tip containing a plug of C18 resin which is use for purifying and concentrating femtomolar to picomolar protein, peptides or oligonucleotide sample prior to mass spectrometry analysis.

The tryptic digestion was stopped by adding 2ul of 1% TFA to the digested protein and was vortex mixed. The ziptip was equilibrated before use by aspirating 8ul 80% acetonitrile 3 times and aspirating again in 0.1% TFA. The equilibrated ziptip was now used to aspirate the digested protein sample 15 times to enable the peptides to bind to the C18 resin. This was followed desalting and washing the bound peptide in 0.1% TFA before eluting the peptides with 8ul 80% acetonitrile. The sample was spotted on the MALDI TOF target plate with CHCA.

In 1D gel electrophoresis, proteins are separated based on their molecular weight after they have been denatured. The gel matrix acts as a sieve and further separate out protein by frictional effect. Tryptic digestion of protein is a process of cleaving proteins into peptides fragment at individual trysin site in the protein.

Other methods of fraction that could be used are:

  1. 2D SDS PAGE
  2. Affinity Chromatography
  3. Liquid chromatography

2. MASCOT DATABASE SEARCHING

  • In the peptide mass fingerprinting result, the protein with the highest score of 68 is 66KDa Human Albumin which correspond to 66KDa spot on the ID gel electrophoresis the protein was excised from. The peptide mass fingerprinting result reports that scores greater than 56 are significant p<(0.05) as shown in the MASCOT search result page. The protein match reported a score of 68 for the 66KDa human albumin protein indicating the score is significant and the match does not occur by chance. The search in this case is considered successful.
  • The expectation value or significant threshold is the number of matches with equal or better score that are expected to occur by chance. The expectation value for the default significant threshold p<0.05, is also 0.05. The score of 68 for human albumin in the peptide mass fingerprinting is above the protein score of 56 and the expectation value is 0.0036 which indicate that the score for the protein is significant since the lower the expectation value from the significant threshold, the more significant the score.

    In the histogram plot, the match for the protein 66KDa human albumin is well outside the green zone. Only match with no significance are inside the green zone of the histogram. This is another indication that the match for the protein is a real and highly significant.

  • When the mass tolerance of the peptide mass fingerprinting was set to 100ppm, the peptide mass fingerprinting search result reported no significant match and the protein score is lower than default score of 56 for which the hit becomes significant (MASCOT result page not attached). This is because there is no peptide mass in the 66KDa protein human albumin that fall within the mass tolerance. Though the reported match for the protein is the correct match since there was prior knowledge of the protein but the confidence level in the result is not significant. Increasing the mass tolerance to 150ppm, the PMF result reported a protein score of 56 which is exactly at the score limit of 56 set for which protein score greater than 56 becomes significant. This hit could be interpreted either as not significant or significant and in this case information about the protein is important. Reducing the mass discrimination increases the probability of false hits. When the mass tolerance was now reduced to 125ppm, the protein score for the 66KDa human albumin was 68 which indicated the result is more significant and there is a less than 1 in 20 chance of the hit been a random event (PMF result page attached).
  • The result of the MASCOT search from the LC-MS/MS reported protein score of 77 compared to 68 obtained from the tandem mass spectrometry which means that search result from LC-MS/MS is more significant than that obtained from MS/MS. This is because the protein bovin albumin was fractionated using liquid chromatography before MS/MS. Fractionation resolves proteins and reduces the complexity of the sample.

3. PROTEOMIC QUESTION

Proteomics is often considered after genomics in the study of biological systems. Proteomics is more complicated than genomics because while an organism's genome is more or less constant and stable under different physiological condition, the proteome differs from cell to cell and from time to time. Distinct genes are expressed in distinct cell type and this means that even the basic set of proteins which are produced in a cell needs to be determined. Proteomics confirms the presence of the protein and provides a direct measurement of the quantity present. Proteome is the entire complement of proteins expressed by the genome of a cell, tissue or organism. More specifically, it is the set of proteins expressed at a given time under defined physiological condition. Proteomics (study of proteome) is the large scale or systematic characterization of the proteome (proteins) of a cell, tissue or simple organism. Proteins are variable in different cell and tissue type in the same organism and in different growth and developmental stages of organism. Proteome is larger than genome, especially in eukaryotes in the sense that there are more proteins than genes which is due to alternative splicing of the genes and post-translational modifications like glycosylation and phosphorylation. The study of proteome enables quantitative and qualitative display of protein expressions patterns, assessment of global changes, comparative analysis of samples, identification of variant proteins and provides information from which biological hypothesis may be developed.

Genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the non-coding sequences. More precisely, the genome of an organism is a complete DNA sequence of one set of chromosomes; for example, one of the two sets that a diploid individual carries in every somatic cell. Functional genomics, which is an aspect of genomics deal with gene expression under various condition. Genomics encompass functional genomics which proteomics, metabolomics and transcriptomics are classed and structural genomics.

The challenges in proteomics research are:

  • Diversity in human cells-approximately 30,000 different sequence in human
  • Broad dynamic range, approximately 10-1,000,000 copies/per cell
  • Different forms of proteins as a result of post-translational modification
  • Size range
  • Quality challenges which include validation of experimental design, assessment of data quality, reproducibility and relating information generated by mass spectrometry and proteomics to biological question.

Targeted proteomics can be defined as an attempt to analyse or characterize a group of proteins present within a cell, tissue or organism. It seeks to ask more focus questions like protein groups, such as membrane proteins, phosphoproteins, glycoproteins, oxidative modification etc.

Experimental Design for the Detection of Cancer Specific Proteins in Plasma Using Cancer Tissue Targeted Approach

Cancer specific proteins produced from cancer cells or tissues can be used as a biomarker in determining cancer disease. These proteins are low abundant proteins which can be detected in plasma and the challenge is always focusing on a particular type of protein in the plasma that is expressed in the cancer cell or tissue.

Sample Preparation

  1. Collect 7ml of blood samples from both normal and cancer patient in two different centrifuge tubes containing heparin, EDTA, or citrate.
  2. Mix the content of the tube gently and centrifuge at 3000rpm for 10mins at 4 deg celsius.
  3. The plasma from the blood samples is separated out as a supernatant.
  4. The supernatant containing the plasma is collected for both the cancer and normal cell/tissue, divided into 4 x 250ul aliquot, stored at -80 deg celsius if transporting is required.
  5. The plasma samples are then denatured using urea and treated with sodium dodecyl sulphate (SDS)-ionic detergent.
  6. An aliquot of the plasma supernatant from the cancer and normal patient is silver stained before 2DE separation

2D Gel Electropheresis

2D gel electrophoresis separates in two dimensions. In the 1D separation, the plasma from the cancer and normal patient/cell/tissue is separated out into component proteins/peptides by their intrinsic charge (pKa) or isoelectric focusing points using IPG strip with pH gradient of 3 to 10. In the 2D separation, the proteins/peptides are further separated out on the basis of their molecular mass using SDS page. The proteins/peptides from cancer and normal cell or tissue are then quantitatively compared to determine the cancer specific proteins/peptides that are expressed in the cancer cell or tissue. At this stage further depletion of abundant proteins/peptides (albumin) could be done using chromatographic method (liquid chromatography). The gel containing the expressed cancer specific protein/peptide is excised, destained, trypsinized and ziptip cleaned for spotting on the MALDI-TOF target plate using dry droplet method for MS analysis.

Mass Spectrometry Analysis and Peptide Mass Fingerprinting

The cancer specific protein/peptide is spotted on MALDI-TOF target plate with CHCA and the spectra acquired. The spectra are then submitted for database searching using MASCOT to determine which protein/peptide has been modified in the cancer tissue or cell. This compares the charge to mass ratio (M/Z) of the tryptic peptides from the sample and matches to the database and generates the possible sequence for the proteins. Further mass spectrometry can also be carried out to resolve the most intense peak in the spectrum. This method is known as tandem mass spectrometry or MS/MS. The M/Z of the tryptic peptide precursor ion is selected, and subjected to collision induce dissociation (CID) to further fragment it. The spectrum acquired and submitted to database searching to predict the bny peptide/protein sequence and confirmation of protein identification.

ADVANTAGE

2D Gel electrophoresis

  1. Large numbers of proteins or poly peptides can be analyzed in a single run
  2. It can be used to study differential protein expression changes between cells
  3. It can be used to separate protein in it pure form and reduce complexity of a mixture

Mass Spectrometry

  1. Mass spectrometry can be used to analyze small and large protein molecules in biological samples like serum, plasma csf and tissue.
  2. It is easier to maintain and very flexible to use
  3. It is automated for high through-put analysis

DISADVANTAGE

2D Gel Electrophoresis

  1. Large amount of sampling handling and limited reproducibility.
  2. Difficult to separate low abundant proteins
  3. It is not automated for high through-put analysis

Mass Spectrometry

  1. Cannot analyse complex mixture on its own without coupling with online separation methods like HPLC
  2. Limited to the solid state
  3. Very Expensive instrumentation

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