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Mass Spectrometry

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This assignment focuses on the methodology of peptide mass finger printing (PMF) and use of mass spectrum analysis software. The lecture notes provide a good foundation for this but you will have to carry out additional personal research to effectively answer the questions. The learning objective of the assignment is to give you an appreciation of the importance of bioinformatics and mass spectrometry in biomedical research technologies. Both mass spectrometry and bioinformatics are important skills in this area.

Please complete the following tasks:

The spectrum in the attached picture is a PMF profile obtained from a spot cut out of a 2 dimensional electrophoresis gel.

1) Identify the protein that produced the attached PMF mass spectrum, you may use any online resource to do this but I suggest the MASCOT server is a good place to start. The profile was produced using trypsin and is derived from a human cell lysate. List the amino acid sequence of each fragment.

2) The MASCOT server lists a number of chemical modifications for peptides. Which of these are likely to have occurred due to the electrophoresis used to isolate the protein? What effect will these have on the protein's mass?

3) Using dot points, outline the processes used to produce the PMF profile starting from protein lysate.

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Solution Summary

Mascot is a web-based tool for predicting protein sequences from PMF and MS/MS data. The
purpose of this lab is to familiarize you with the basic use of Mascot and the interpretation of
Mascot results.

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1) Identify the protein that produced the attached PMF mass spectrum, you may use any online resource to do this but I suggest the MASCOT server is a good place to start. The profile was produced using trypsin and is derived from a human cell lysate. List the amino acid sequence of each fragment.

The protein that produced the attached PMF mass spectrometry is enoyl-CoA hydratase , which has a top hit of 148.

The amino acid sequence of each fragment is : The matched peptides is in red:
1 MAALRVLLSC ARGPLRPPVR CPAWRPFASG ANFEYIIAEK RGKNNTVGLI
51 QLNRPKALNA LCDGLIDELN QALKIFEEDP AVGAIVLTGG DKAFAAGADI
101 KEMQNLSFQD CYSSKFLKHW DHLTQVKKPV IAAVNGYAFG GGCELAMMCD
151 IIYAGEKAQF AQPEILIGTI PGAGGTQRLT RAVGKSLAME MVLTGDRISA
201 QDAKQAGLVS KICPVETLVE EAIQCAEKIA SNSKIVVAMA KESVNAAFEM
251 TLTEGSKLEK KLFYSTFATD DRKEGMTAFV EKRKANFKDQ

2) The MASCOT server lists a number of chemical modifications for peptides. Which of these are likely to have occurred due to the electrophoresis used to isolate the protein? What effect will these have on the protein's mass? The proteins may have been treated with iodoacetamide at the N or C- termini. That is why Carbamidomethyl (C) is selected.

3) Using dot points, outline the processes used to produce the PMF profile starting from protein lysate.

1) The first thing you should do is follow the protocol for PMF.
a) Obtain the list of sharp peak intensity values from mass spectrometry data of the unknown sample.
b) Change the fixed modification into carbomidomethyl (H+ and OH- groups should be added).
c) Decoy set is invoked to delete the mismatch sequences during the prediction.
d) Click submit

Use the Mascot search:

User : TimT
Email : [email protected]
Search title : Prot-1
Database : NCBInr 20140323 (38032689 sequences; 13525028931 residues)
Timestamp : 5 May 2014 at 05:19:17 GMT
Warning : Specifying a protein mass disables query intensity in PMF-search
Warning : Specifying a protein mass disables mixture mode in PMF-search
Top Score : 148 for gi|1922287, enoyl-CoA hydratase [Homo sapiens]
Mascot Score Histogram
Protein score is -10*Log(P), where P is the probability that the observed match is a random event.
Protein scores greater than 88 are significant (p<0.05).

Concise Protein Summary Report

Help

Significance threshold p<
Max. number of hits

Preferred taxonomy

1. gi|1922287 Mass: 31807 Score: 148 Expect: 6e-08 Matches: 15

enoyl-CoA hydratase [Homo sapiens]
gi|14286220 Mass: 31835 Score: 148 Expect: 6e-08 Matches: 15

Enoyl Coenzyme A hydratase, short chain, 1, mitochondrial [Homo sapiens]
gi|30585161 Mass: 31948 Score: 148 Expect: 6e-08 Matches: 15

Homo sapiens enoyl Coenzyme A hydratase, short chain, 1, mitochondrial [synthetic construct]

gi|194097323 Mass: 31823 Score: 130 Expect: 3.8e-06 Matches: 14

enoyl-CoA hydratase, mitochondrial ...

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