Download Master course KEMM03 Principles of Mass Spectrometric Protein

Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
Exam
Master course KEMM03
Principles of Mass Spectrometric Protein Characterization
2010-10-29 kl 08.15-13.00
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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.
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1. MS basics and MS-instrumentation.
(10 p)
1982.2679
a. Explain why a laser is used in MALDI-MS, and explain why it is not needed in so
called electrospray instruments. 2 p
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?
2p
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? 1 p
d. What is the reflector used for in MALDI-TOF/TOF? 1 p
e. What is the time-gated ion selector used for in MALDI-TOF/TOF? 1 p
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
3205.5
1707.0641
4700 R ef lector Spec #1[B P = 1707.1, 6938]
100
1982.30590
Mass (m /z)
80
70
2565.6245
2689.6965
2514.4871
2641.5920
2308.4172
2365.4
2436.5339
2362.4436
2110.3767
1982.2679
KEMM03 101029_CE
2007.1792
1879.0590
1929.8
Mass (m /z)
1965.9457
1690.0374
1494.2
1826.1431
1676.0638
1735.0751
1555.8783
1473.8540
1605.8953
1422.8278
1285.7983
1195.7146
1024.6149
905.3419
943.3586
693.2526
862.4734
1058.6
2476.5254
0
623.0
732.2781
10
799.2935
30
982.3658
1001.5643
40
1157.6978
1046.6273
50
628.2285
% Intensity
60
20
1983.79561
6938.0
90
2801.0
1985.28532
1986.77503
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
2. Terms and expressions
(10 p)
a. Connect the terms 1-8 related to protein mass spectrometry with the correctly
corresponding terms a - h.
4p
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. 1672.92 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.
6p
.
A.
B.
C.
KEMM03 101029_CE
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
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?
2p
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? 1 p
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?
2p
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.
2p
f. The dynamic range in protein concentration in a human plasma sample is 106 - what does
that mean?
1p
KEMM03 101029_CE
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
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?
1p
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.
2p
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? 1 p
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).
1p
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?
1p
f. Assuming the protein identified based on the peptides above is 331 amino acids long, what
is the sequence coverage?
1p
KEMM03 101029_CE
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
g. A theoretical digest of the identified protein is shown below. What protease? Which is the
C-terminal amino acid of this protein?
2p
h. Explain why the isoelectric point is below three for peptide with mass 698.3 Da and above
12 for peptide with mass 917.6 Da.
1p
MH+
pI
KEMM03 101029_CE
Sequence
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
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? 2 p
A.
b. One of the peptides, with a peptide mass 1257.6345 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.
2p
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.
2.
3.
4.
What is the mass of the y1-ion?
1p
Is the peak with mass 559.35 a b-ion or a y-ion?
1p
Write the amino acid sequence of the b4 ion.
1p
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?
1p
5. One amino acid in the peptide has been blanked out. What amino acid is this? 2 p
KEMM03 101029_CE
4700 MS/MS Precursor 1257.64 Spec #1[BP = 175.1, 287]
175.1234
100
B.
287.3
90
559.3555
80
70
446.2587
% Intensity
60
1241.4260
688.4221
50
553.2192
682.3422
246.1545
40
1239.9277
719.4635
407.2292
30
588.2411
112.0890
20
230.1221
474.2959
208.1008
441.1875
570.2368
664.2266
1032.6442
818.4796
547.7806
1212.0283
795.3972
995.0720
702.8992
10
0
69.0
320.6
572.2
823.8
Mass (m/z)
1075.4
1327.0
Master course KEMM03 Principles of Mass Spectrometric Protein Characterization
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.
3p
b. Explain what ‘PTM’ means and state approximately how many PTMs are known.
2p
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?
1p
d. Explain how phosphopeptide peptides can be enriched if one wants to analyze
phosphoprotein by mass spectrometry.
1p
e. Is the mass deviation of a phosphorylated peptide 80 Da or 16 Da?
1p
f. The picture below illustrates that the plasma membrane contains many glycoylated proteins.
What does it mean that glycoproteins have “N-glycosylation sites”? 1 p
g. Give one example of how glycopeptides can be enriched for MS-analysis. 1 p
.
KEMM03 101029_CE