How peptide mass fingerprinting and database searching

Reference no: EM133178618

Targeted Proteomics - workflow

You will be required to use a number of web-based databases and search tools to both identify and then quantify an unknown peptide.

Part 1: Peptide mass fingerprinting
You have been given a peak list generated from a MALDI MS experiment following extraction of a protein band resulting from SDS-PAGE. Trypsin was used to digest the target protein. The protein was isolated from a cell lysate from a human 3D cancer tumour model and is presented below.
• Outline the methodology of how peptide mass fingerprinting and database searching is utilized to help identify a protein from proteomic data sets?
(100 words)

By use of the MASCOT protein identification tool determine the potential ID of the target protein. Support in the use of this database will be given via the blackboard site located in the "Support Resources section".
From the data generated and a follow-up BLAST search construct a short response with details on;
• Protein putative identification with details on function?
• Probability score?
• What is the protein coverage?
• What masses went unidentified?
• What is the average mass accuracy of this mass spectrometer?
• Why do you think we missed finding some of the peptides?

(data analysis with explanation ~200 words equivalent)

Part 2: Protein identification from MS/MS data
In order to validate the putative ID from part 1, the most intence peak from the peptide fingerprinting experiment was investigated by MS/MS. You will find a text readable .mgf file containing relevant data on the blackboard site called C0001.mgf.
• Outline the minimumconsti tuents of this MASCO readable .mgf file?
By use of the MASCO protein ID tool perform a more detailed search on your target peptide. From the data generated construct a short response with details on
• Protein putative identifications?
• Probability scores?
• What was the sequences of the identified peptide?

(data analysis with explanation ~100 words equivalent)

Part 3: Relative Quantification
iTRAQ allows the relative quantification of peptides resulting from MS/MS data by recording the peak areas of reporter ions. You have each been given a unique data set to statistically analysis. Serum levels of the protein of interest have been proposed to be a marker of treatment efficacy for liver cancer patients following repeated administration of microwave therapy. Peak areas for the relevant reporter ions have been determined to study this effect with serum samples taken from a mouse containing a liver cancer xenograft subjected to increasing amounts of irradiation. Considering the data shown in Table 1 and taking the no treatment sample as the control, calculate the relative expression of the protein of interest in the test samples. Determine the significance the results and hence at which concentration at which the drug has a significant effect.

reporter ion	114	115	116	117	118	119
DrugmM	0	1	5	10	50	100
rep1	8773.6	9567.8	9477.6	9013.4	4351.6	2112
rep2	19737	22302.3	21032.4	21037.5	9460.5	4656.3
rep3	17300.8	16913.6	16860.8	18013.6	8861.6	4826.8
rep4	36593	33153	35010.6	34090.4	16021.8	8797.8
rep5	26047	24253.6	25333.3	21160.9	11364.3	6624.6

Part 4: Critical analysis of quantification methods

iTRAQ has been used in the example above to determine the relative abundance of the target peptide. In the following section critically compare alternative strategies for the quantification of peptide abundance in targeted and untargeted proteomics.
(500 words)

Reference no: EM133178618

Questions Cloud

Learning and professional development : In your opinion, what can be the action if the objective is to Train employees to handle different kinds of customers, Made adjustments on the menu that allows

Discuss the overall financial performance of your company : Using your financial analysis of one of the approved companies,Discuss the overall financial performance of your company in relation to the specific competitor

Write a great and exciting welcoming message : Consider yourself an HR manager of a Multinational Corporation, and you are required to deliver a presentation for all new employees.

Canadian occupational health and safety discussion : Jerry phoned today to say that he is going to be off work for a while. He is sending in the doctor's note but basically it looks like he is going to be off for

How peptide mass fingerprinting and database searching : Outline the methodology of how peptide mass fingerprinting and database searching is utilized to help identify a protein from proteomic data sets

Health and safety in the work area : Ensuring that the employee is safe and under conditions that do not affect their health is a task for the human resources department of any institution.

Estimated variable cost per unit of production : The following information is available for electricity costs for the first six month of the year: Estimated variable cost per unit of production

Several potential options for dealing with smith : Write a memo using the statement below. You should include several potential options for dealing with Smith, with an analysis of each option you identify in ter

Evaluate effectiveness of workforce strategic plan : For this task you will monitor and evaluate the effectiveness of the workforce strategic plan you have developed.

User Account

All Pages