Master course KEMM03 Principles of Mass Spectrometric Protein Characterization. Exam

Transcription

1 Exam Master course KEMM03 Principles of Mass Spectrometric Protein Characterization kl Use a new paper for answering each question! Write your name on each paper! Aids: Mini calculator, table with amino acids (one letter codes and residue masses). Maximum number of points is 60. Pass (Godkänd) corresponds to >50% (30) and Well pass (Väl godkänt) corresponds to >75% (45). Results are announced on course webpage and at Student expedition within one week. 1. MS basics and MS-instrumentation. (10 p) a. Explain why a laser is used in MALDI-MS, and explain why it is not needed in so called electrospray instruments. b. When a MALDI-TOF MS-spectrum is aquired the m/z-values are determined for the ions. Which is the physical parameter that is actually measured, and how is it converted into m/z? c. At atmospheric pressure the mean free path of a typical ion is around 52 nm. How come that the ions can travel several meters in the drift tube? d. What is the reflector used for in MALDI-TOF/TOF? e. What is the time-gated ion selector used for in MALDI-TOF/TOF? f. In a typical mass spectrum, each peak appears as a couple of peaks in magnification, why? And what is the monoisotopic and average mass of the peak below? 3 p Reflector Spec #1[BP = , 6938] Mass (m/z) % Intensity Mass (m/z)

2 2. Terms and expressions (10 p) a. Connect the terms 1-8 related to protein mass spectrometry with the correctly corresponding terms a - h. 4 p 1. Internal calibration 2. MH + 3. O-methylisourea 4. UniProt KB 5. PMF 6. Lectin column 7. MS/MS 8. Mascot a. Single charging b. MS c. Glycopeptides d. Search engine e. Lysine-terminated peptides f. mgf-file g Da h. Database b. Below are some structural formulas which illustrate various phenomena in protein mass spectrometry that you have worked with in this course. Give a short comment to each of A, B and C and explain what it shows. Your answers should include the following words: primary and tertiary amine, digestion, polypeptide backbone. 6 p. A. B. C.

3 3. Protein and peptide separation (10 p) Because the complexity of a proteome is large there is often a need to start a proteomic investigation by reducing the sample complexity. a. Which physical property of the proteins, or peptides, are the following separation techniques based on? 1. Gel filtration 2. Reversed phase chromatography 3. Isoelectric focusing 4. SDS-PAGE b. What charge does a protein have below and above its isoelectric point? c. In MALDI mass spectrometry the mass spectrum quality of a certain sample can often be improved by treatment with so called C18 microcolumns. Why? d. Describe a typical solvent suitable for loading the tryptic digest onto the microcolumn, and a typical solvent used for eluation. 2p e. Describe two types of peptides that could be lost from the sample in spite of the general improvement that is often obtained. f. The dynamic range in protein concentration in a human plasma sample is what does that mean?

4 4. Protein identification using Mascot (10 p) a. Mascot was used to search the Swiss-Prot database with a peak list derived from an MSspectrum obtained by tryptic digestion of a protein spot obtained from 2DE-separation of a soluble extract from E. coli with the output below. How can you tell whether a protein has been reliably identified, or not? b. In the Mascot output table below, two peptides contain missed cleavage sites. Which peptides and what cleavage sites? Answer with peptide masses, and indicate where in each peptide the missed cleavage site is. Use one-letter code and amino acid number in sequence. c. In the table above, calculate the mass difference between the values in the column named Observed and column named Mr (expt). What is this mass difference due to? d. For two of the peptides the mass error in ppm is now hidden. Calculate the mass error in ppm for these two peptides (show your calculation). e. If one would have used 25 ppm as the peptide mass tolerance setting when submitting the search, which peptides in the above table would not have been matched? f. Assuming the protein identified based on the peptides above is 331 amino acids long, what is the sequence coverage?

5 g. A theoretical digest of the identified protein is shown below. What protease? Which is the C-terminal amino acid of this protein? h. Explain why the isoelectric point is below three for peptide with mass Da and above 12 for peptide with mass Da. MH + pi Sequence

6 5. Evaluation of mass spectra (10 p) a. An MS-spectrum was recorded for the WrbA protein from E. coli (A). Which of the four most abundant peaks belong to this E. coli protein? What are the other peaks? A. b. One of the peptides, with a peptide mass was selected for further analysis by MS/MS. How is such selection of only one peptide mass possible to obtain? Exain how you do it practically when you are setting up a job to acquire MS and MS/MS data at the ABI 4700 MALDI TOF/TOF instrument, and what principle and what device in the instrument that makes this possible. c. On next page the MS/MS spectrum (B) for this peptide is shown, and the output obtained when the MS/MS-data were matched into Mascot. Answer the following questions: 1. What is the mass of the y1-ion? 2. Is the peak with mass a b-ion or a y-ion? 3. Write the amino acid sequence of the b4 ion. 4. Are mostly b-ions or y-ions detected in this MS/MS spectrum? This is commonly occuring in a MALDI-TOF/TOF instrument. What does it tell about the fate of the single charge extra protonupon precursor ion fragmentation? 5. One amino acid in the peptide has been blanked out. What amino acid is this?

7 4700 MS/MS Precursor Spec #1[BP = 175.1, 287] B % Intensity Mass (m/z)

8 6. Protein quantification and modification specific proteomics (10 p) a. isotope labbeling is commonly used to make relative quantification of how much there is of a certain protein in different samples.shortly state three different aways to incorporate isotopes into a protein to allaw quantification. 3 p b. Explain what PTM means and state approximately how many PTMs are known. c. The picture below illustrates how phosphoproteins can be resolved by 2DE. What is the explanation to why phosphorylated and non-phoshorylated forms of a protein can be separated by 2DE? d. Explain how phosphopeptide peptides can be enriched if one wants to analyze phosphoprotein by mass spectrometry. e. Is the mass deviation of a phosphorylated peptide 80 Da or 16 Da? f. The picture below illustrates that the plasma membrane contains many glycoylated proteins. What does it mean that glycoproteins have N-glycosylation sites? g. Give one example of how glycopeptides can be enriched for MS-analysis..