Part A: Critical evaluation of a proteomics based research paper
A1. Define the term “Proteomics” 100 words
Ans. Proteomics is a branch of molecular biological science which deals with the study of structure and function of proteome at a larger-scale. The proteome refers to the complete set of proteins which a particular genome expresses at a particular time. The main aim of proteomics is to make an analysis of the different proteomes in an organism at different intervals of time. This is for highlighting difference between the proteomes at different times. There are three steps in proteomics, beginning from preparation of sample, acquisition of data by separating the proteins and in last the analysis of data by using methods like mass spectrometry.
A2. Explain the difference between Discovery and Targeted Proteomics 100 words
Ans. There are two types of researches in which the proteomics study is being divided for improving the scope and sensitivity of proteomic analysis. These two researches are discovery proteomics and targeted proteomics. The discovery proteomics works by optimising the identification of protein by putting more effort and time for each sample; thereby, reducing the total number of samples which are analysed. On the contrary, the strategies used in the targeted proteomics restrict the features that are to be monitored in the proceedings. After this, the chromatography is optimized, tuning of instrument and methods of acquisition is done so that highest throughput and sensitivity is achieved for large number of samples (hundreds or thousands).
A3. Describe the biological question addressed in the SILAC paper which you have been provided 100 words
Ans. The biological question which is being addressed in the given SILAC (Stable Isotope Labelling Amino Acid in Cell Culture) paper is “how the deletion of two major chaperone proteins, i.e. SSA1 and SSB1 proteins in yeast can disturb the chaperone interaction network in yeast?” SILAC-based approach has been employed in the analysis of the chaperone network and in examining the changes occur in abundance of local and global proteins. It has been highlighted in the paper, how systems modelling and quantitative proteomics can be utilised for rationalising of the emergent network properties. Apart from this, the changes that occur in HSP70 system for accommodating the loss of major proteins is also studied.
A4. i) Which species was studied?
Ans. Yeast (Saccharomyces cerevisiae)
ii) Which type of sample (for example, whole cells, cell lysate, an organelle, cell line)
Ans. In the study, the whole mutant cells (yeast gene deletion mutants) were used for analysis of the HSP70 system.
A5. Experimental Design
a) Which samples were compared?
Ans. Two mutants (SSB1 and SSA1) strains from HSP70 subfamily of cytosolic chaperones were selected and compared with the wild-type strain of yeast.
b) How many samples were compared? Help: were replicates included in the design, if yes provide details.
Ans. There had been a comparison of four samples of which three were the samples of mutant strains and one was the wild-type strain of yeast. The mutant strains included derivatives deleted for SSA1, SSB1 and CPR6 proteins.
A6. Explain the basic principles of SILAC. In your answer include the key reference for the methodology ie where the method was first described (list in full eg authors, title of manuscript, Journal, vol, page numbers). 300 words maximum
Ans. The SILAC is a simple approach used in MS (Mass spectrometry)-bases quantitative proteomics research wherein proteins are labelled in vivo. The basic principle on which this method is based upon relies on the direct addition of the stable isotopes of amino acids which are being selected for the research into the cell culture medium; thereby, enhancing the quality and accuracy of the quantitative analysis of cellular proteome as compared to other methods of labelling. In this procedure, a light or heavy amino acid form is selected first and then it is metabolically incorporated into the proteins.
SILAC labelling method was first developed at the Centre for Experimental Bioinformatics (CEBI) located at the University of Southern Denmark. ‘Stable isotope labelling in cell culture (SILAC) is a metabolic labelling approach first published in 2002 by the lab of Matthias Mann’ (Ong et al 2002). In this process, arginine and lysine are generally used in combination with trypsin digestion to ensure the labelling of all peptides (except c-terminal peptides) in the given sample. There are five major steps involved in the methodology of the SILAC. These include preparation of the SILAC labelling medium, cells adaptation to SILAX labelling media, SILAC cells being treated with the differential treatments, cell lysis and protein estimation and MS analysis and quantitation. SILAC has been used to label higher organisms, for instance flies (Sury et al. 2010) and mice (Kruger et al. 2008), by feeding them with labelled food.
A7. State the names of the softwares were used for i) protein identification, ii) relative quantification
i) Software used for Protein identification: MaxQuant Software
ii) Software used for relative quantification: MaxQuant Software
A8. How many proteins were identified and quantified in total?
Ans. The number of proteins that were identified and quantified was 1448.
A9. In general, changes in protein level, identified by Discovery based proteomic techniques such as SILAC, DIGE or iTRAQ can be validated using an antibody based methodology. Name two examples of these methodologies.
Ans. Two examples of antibody-based methodologies include enzyme-linked immunosorbent assay (ELISA) and western blot (WB).
A10. Critically review the paper – did the methodology provide answers to the biological
question? (up to 300 words, include information on study design)
Ans. The methodology used in the SILAC paper has successfully provided the answer to the biological question. It has revealed the differences in the total content of the protein in the mutant strains and wild-type strains of the yeast. The differences had been assessed at both the local level, i.e. relative changes in the concentrations of the protein individually and at global level, i.e. total content of cellular protein. It has been revealed that the increase in the cell size did not cause change in the relative concentration of proteins. The network comparison between the mutant and wild-type strains revealed the impact on most of the topological parameters; thereby, suggesting that there is not much change in the networks. This is contrary result to what the native expectation from the perspective of networks which suggest that the major hub proteins deletion can be lethal in some cases as they are deleterious.
The study included the selection of four yeast gene deletion mutants for the quantification of SILAC-based proteome. LC-MS analysis was performed for which the cells were grown, harvested and processed. The further analysis in the study was performed on the three out four biological replicates of the proteins that were identified and quantified. After the studies, it was observed that the intracellular network of chaperone is resilient to deletions and general protein homeostasis is maintained by the network for adjusting to the removal of two HSP70 (SSA1 and SSB1) proteins. In silico graph theoretical approaches of folding workload was used in last for justifying the changes observed during the experiments for network theory. This approach has very well supported the observations made during the experiment that the network can compensate the deletion of major hub proteins by increasing the node workload.
Part B: Experimental design (B1), Bioinformatic analysis (B2)
B1. Describe how the SILAC experiment described in the manuscript provided (SILAC.pdf) could have been designed using an iTRAQ workflow.
To do this, provide:
- Explain the principles of the iTRAQ method.
Ans. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) is a MS-labelling method which is widely used in quantitative proteomics analysis. This method relies on the use of isobaric reagents for labelling the peptides and proteins (primary amines are labelled). The main principle on which this technique is based on is the covalent labelling of side chain amines and N-terminus amines of peptides from digestion of proteins with variable mass tags.
- A design of the experiment – this should be a figure with figure legend and the experimental design should include at least 2 biological replicates for each condition included in the analysis. (500 words)
Ans. The experiment for network analysis of SSA1 and SSB1 deletion mutants can be conducted by using iTRAQ labelling method. There are two types of reagents used in iTRAQ labelling method. These are 4 plex (in order to process up to 4 samples) and 8 plex (in processing up to 8 samples). In this case, we would employ the use of 4 plex reagent as we have four samples initially. The whole design of the experiment can be broken down into three main steps, i.e. preparation of samples, digestion of proteins, labelling, analysis and processing of data. The materials and methods required for designing the experiment are as follows:
- Preparation of Yeast Samples: Prior to making samples, there is a need to purchase the wild-type strain of yeast and the mutants deleted for SSA1, SSB1 and CPR6. The samples are prepared by growing yeast on a medium rich in nitrogen bases with a supplementation of amino acids including leucine, histidine, lysine and arginine.
- The protein samples are then separately (four sample preparation for WT-yeast, SSA1 mutant, SSB1 mutant and CPR6 mutant) dissolved in 0.5 M triethyl ammonium bicarbonate at pH 8.5. The next step in the experimentation is the reduction of the samples by adding a reducing agent.
- The samples are incubated for one hour at the temperature 60 degrees Celsius. This is followed by addition of a cysteine blocking agent for blocking the cysteine in the protein samples.
- After the cysteine is blocked, the protein digestion is done by adding trypsin in the samples and incubating the samples overnight at the temperature of 37 degrees Celsius. The samples are cleaned next day by using a zip-tip.
- After the protein is digested, the iTRAQ reagent is reconstituted in isopropanaol and then it is added to the sample of digested proteins. Here we have used 4-plex reagent, each of which would label the four sets of the peptides. After the proteins are labelled, all the samples are mixed in a single tube.
- The next step in this experiment is to facilitate the clean-up of the samples. This is done to purify the pooled samples on a column of strong cation exchange in order to remove the excess reagent which is not bound to the peptides.
- After the clean-up of samples, the samples need to be analysed using MS. This technique would enable a simultaneous quantitation and identification. The peptides which are labelled would react to the N-terminus; thus, helping in the selecting the peptide of interest.
In this experiment, we need to obtain global molecular view of the chaperone system and how it responds to the changes in yeast. For this purpose we quantified the proteome changes in the SSA1 and SSB1 mutants by utilising iTRAQ labelling approach. Two biological replicates out of four replicates in each sample of mutants were quantified and included in further analysis. MASCOT software was utilised for data processing as it supports the iTRAQ quantification data. It excluded the reporter ion masses from the processing and identified the spectra with fixed modification of iTRAQ. After the data processing, the network of chaperone in mutants would be constructed and further calculations would be done.
B2 : i) describe how you would use bioinformatics tools provided by EBI (e.g. provide a workflow or plan of analysis) to infer biological significance for the proteins.
Ans. EBI (European Bioinformatics institute) provides with bioinformatics tools to infer the biological significance of any protein of interest. One of the mostly usedbioinformatic tools at EBI is the InterProScan which is particularly used for the functional analysis of the proteins. By using this tool, one can analyze the functions of the protein of interest and can infer its biological significance.
ii) For protein with the accession number P10591, answer the following, briefly describing how you found out the information:
a) What is the protein name?
Ans. P10591 is the heat shock protein found in yeast. It can be determined by searching for the accession number in Interpro or Uniprot.
b) How many isoforms does it have?
Ans. There is no isoform of SSA1 protein according to the information available in the Uniprot.
c) What is the gene that encodes it?
Ans. The heat shock protein is encoded by SSA1 gene. This information is available online.
d) What is the amino acid sequence of the canonical form?
Ans. The amino acid sequence of the canonical form is obtained from the Uniprot database.
>sp|P10591|HSP71_YEAST Heat shock protein SSA1 OS=Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GN=SSA1 PE=1 SV=4
e) What are the primary interacting proteins (exclude information from text mining alone)
Ans. There are many interacting proteins of SSA1 as determined in the SILAC file. The primary interacting proteins for SSA1 include PAB1 (polyadenylate-binding protein), NAP1 (nucleosome assembly protein 1) and HSP70 which can determined by analyzing the Uniprot database.
iii) Describe the advantages of submitting proteomic data to PRIDE
Ans. PRIDE is proteomics identification database which is a public data repository of MS based data of proteomics. The main advantage of submitting proteomic data to PRIDE is that the original data of the researchers is stored without any control of the editor. This allows the large amount of original proteomics data across the globe to be shared publicly
Part C: Review and evaluation of proteomics technologies in bioprocess and biological engineering
C1. Describe the use of proteomics in biological engineering, include at three specific references in your discussion, of which one can be a review article. You can either discuss this in general terms or focus on a specific bioprocess related application. (500 words)
Ans. Proteomics is a fast growing and advancing discipline in scientific research which is used for wide range of investigations such as functions of proteins or genes in terms of their extracellular or intracellular environment or investigation that are at the level of global proteome. Proteomics research involves study of protein structure, its expression, interaction, localization and function in an organism. After reviewing the capacity of proteomics for characterization of differences in quantity and quality of proteins, the proteomics analysis has been widely used for various fields of biological engineering including biomedicine, discovering the potential biomarkers, characterizing the novel drug targets,etc., oncology, food microbiology and agriculture.
According to Gil, Ehsan, Joseph & Marco (n.d.):
Proteomics researchers aim to use experimental techniques that trade accuracy for volume in order to build up a complete picture of the function of large groups of proteins. Key research in the field focuses on the development of new high-throughput techniques and the computational machinery needed to analyze the data those techniques produce (p. 1).
The most important techniques used in the proteomics include 2-dimensional gels or liquid chromatography (LC) combined with protein microarrays and MS. The use of MS and 2D gels is common in the protein quantification and differential display of proteins. The main objectives of proteomics analysis include determination of differences in closely related species, difference in the growth rates of organisms, localization of proteins at subcellular level and influence of the environmental factors on the growth states.
There had been a rapid evolution in the sequencing technologies and tools of genome annotations after the sequencing of first complete genome of Haemophilus influenza in 1995. After this, there has been identification and sequencing of large number of genomes of different organisms. This study has further helped in developing vaccines for various diseases caused by different pathogens; thereby, proteomics analysis had played a vital role in biomedical science. The proteomics tools are particularly important in the manipulation of genome of organisms and designing of subunit vaccines. Rembert, Shih-Ting, Prashanth, Hamid, Robert, Carole, John, Quanshun, Saul & Scott (n.d.) suggested that ‘Identification of subunit vaccine candidates for bacteria causing infectious diseases has relied mostly on biochemical studies, identifying and characterizing protein and polysaccharide antigens from the bacterial cell surface’ (p. 1).
Apart from this, the proteomics research is being used for the discovery of biomarkers. This is one important potential area. The study of tumor cells (oncology) also relies on the proteomics research. According to Chandrasekhar, Dileep, Ester & Pramoda:
Analyse the protein expressions correlated to the metastatic process which help to understand the mechanism of metastasis and thus facilitate the development of strategies for the therapeutic interventions and clinical management of cancer. (2014, p. 6).
Proteomics research helps in characterization of expression of proteins and tumor cells function. The main role of proteomics in food technology is for controlling the quality of the final product along with its application in the standardisation and characterization of raw materials. It is now being used in agricultural fields for determining the interactions between the insects and plants; thereby, helping in the identification of genes in plants which are involved in the defensive response to insects.
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