Lab 6 - MALDI-TOF Data Analysis

Question 1

What are the tasks of this lab?

Answer 1

• Data analysis
• Lab results/questions

Question 2

What should be recorded after receiving the printout of your mass spectrum?

Answer 2

The mass values of tryptic peptides that have a signal to noise ratio of approximately 10 to 1

Question 3

What website is used in this lab?

Answer 3

Protein Prospector

Question 4

What should the search parameters for this lab be?

Answer 4

• Database: SwissProt.2021.06.18
• Species: Homo Sapiens
• Digest: Trypsin
• Max # of missed cleavages: 1
• Constant modifications: None
• Molecular weight: 40000 Da to 60000 Da
• Max reported hits: 10
• Min # of peptides for match: 5
• Possible modifications: Oxidation of Methionine (M)
• Peptide masses are: Monoisotopic
• Mass tolerance: -/+1 D
• Instrument: MALDI-Q-TOF
• Data paste area: Mass of peptide

Question 5

Once the search has been conducted, what will you receive?

Answer 5

A Results Summary page that lists the top 10 hits ranked from the most probable to the least based on a score (MOWSE)

Question 6

Results: What protein was the top hit?

Question 7

Results: What was the sequence coverage of your peptide mass fingerprint?

Question 8

Results: For PTEN, how many mass fingerprints m/z matched PTEN theoretical tryptic peptides?

Question 9

Results: What properties (if any) indicate that it most likely is not the correct protein?

Question 10

What will unambiguously confirm your mass fingerprint work?

Answer 10

Obtaining partial amino acid sequence information from the MS/MS data

Question 11

What was done to two of the must abundant peaks from your mass fingerprint?

Answer 11

Were individually selected and fragmented at the peptide bond to produce various fragment ions

Question 12

How were the masses of the fragment ions recorded?

Answer 12

On a single spectrum

Question 13

What will the difference between pairs of masses (y and b ions) equal?

Answer 13

The mass of an amino acid allowing one to obtain some amino acid sequence

Question 14

What is the protocol for MS/MS analysis?

Answer 14

1) From list of detailed hits from the MS-FIT result, click on the hyperlink for the peptide mass that corresponds to the peptide mass from the MS/MS spectrum
2) Attempt to match the calculated fragment ion masses to the mass values present on your MS/MS spectrum
3) Label the y and b ion fragment ion series on the spectrum
4) Click on a hyperlink for the same m/z value for a peptide from a different protein listed on the MS-FIT putative identification list
5) Attempt to match the calculated fragment ion masses to the mass values present on your MS/MS spectrum
6) Make conclusions on which protein hit on the MS-FIT putative list is the correct protein identification