Possible exam questions (1 to 9 were explicitly mentioned by the professor, the rest are

questions of similar type based on the slides)

1. What is 2D PAGE, how does it work, what do you typically do after 2D PAGE?
2D PAGE is an electrophoresis technique to separate complex protein mixtures. It consists in
the use of two different physics feature: IEF (isoelectric focusing) and mass detection (SDS-
PAGE, PolyAcrylamide Gel Electrophoresis with Sodium Dodecyl Sulphate). After 2D PAGE we
have to adjust images obtained and detect the spots, where needed it is performed a
normalization of the spot intensities, and then compared to “master gel” to identify the spots
and do a database searching. It is so performed a western blot, n-terminal sequencing and
finally mass spectrometry

2. What is peptide mass fingerprinting?

Peptide Mass Fingerprinting (PMF) is an analytical technique for identifying unknown
protein. Proteins to be identified is first digested into peptides, whose absolute mass can be
accurately analyzed by a mass spectrometer.

3. Why it trypsin so commonly used for in-gel digestion? What is its specificity?
•Robust, stable enzyme
•Works over a range of pH values & temp.
•Quite specific and consistent in cleavage
•Cuts frequently to produce “ideal” MW peptides
•Inexpensive, easily available/purified
•Does produce “autolysis” peaks (which can be used in MS calibrations)

4. How does a typical MS spectrum look like? What are the axes? Is intensity always related to
the abundance?
A MS ANALYSIS GIVES THE MASS-TO-CHARGE RATIO (m/z) OF IONS...IN GAS PHASE
•Characterized by sharp, narrow peaks
•X-axis position indicates the m/z ratio of a given ion (for singly charged ions this
corresponds to the mass of the ion)
•Height of peak indicates the relative abundance of a given ion(not reliable for
quantitation)
•Peak intensity indicates the ion’s ability to desorb or “fly”

5. What is the difference between molecular ions and fragment ions?

Fragment ions are the result of passing unstable molecular ions in ionization chamber or in a
mass spectrometer.

6. What ionization sources are used in proteomics?

Electron Impact: The sample for analysis Is introduced into the ion source, either through a
solids inlet or through a gas chromatography column. Electron impact is an energetic
ionization technique and also produces fragmentations which are smaller parts of the original
molecule.
Chemical ionization: It is a low energy ionization process that avoids excessive fragmentation.
CI uses a reagent gas to gently transfer protons to the sample, usually producing (M + H) +
quasimolecular ions.
ESI: see electrospray
7. What are multiply charged ions? What is ESI? What is MALDI? Do they produce multiply
charged ions?
ESI=Electro Spray Ionization (soft ionization method) produce ions using an electrospray in
which a high voltage is applied to a liquid to create an aerosol. MULTIPLE CHARGED IONS
MALDI= Matrix Assisted Laser Desorption Ionization a soft ionization method that involves a
laser striking a matrix of small molecules to make the analyte molecules into the gas phase
without fragmenting or decomposing them. SINGLE POSITIVE CHARGE

8. What is the STRING database?

9. What is the difference between top-down, bottom-up and shotgun approaches in

proteomics? Slide p. 25 Proteins extracted from biological samples can be analyzed by
bottom-up or top-down methods. In bottom-up approach, proteins in complex mixtures
can be separated before enzymatic (or chemical) digestion followed by direct peptide
mass fingerprinting-based acquisition. In shotgun approach the protein mixture can be
directly digested into a collection of peptides then separated by multidimensional
chromatography on-line coupled to tandem mass spectrometric analysis . And in top-
down one, proteins in complex mixtures are fractionated and separated into pure
single proteins or less complex protein mixtures.

10. What is western blotting? Why do we use it?

The western blot (sometimes called the protein immunoblot) is a widely used analytical
technique used in molecular biology to detect specific proteins in a sample of tissue
homogenate or extract. The western blot is extensively used in biochemistry for the
qualitative detection of single proteins and protein-modifications (such as post-translational
modifications). It is used as a general method to specifically prove the existence of a single
protein in a mixture.

11. What mass analyzers are used in mass spectrometry?

Quadrupole Mass Analyzer: four parallel metal rods with different charges
ORBITRAP: Ions are trapped in an electrostatic field between an inner and outer
electrode (Makarov, 2000).
TOF(Time of Flight):
Hybrid instruments /Tandem MS: combine two or more mass analyzers
A mass analyzer is the component of the mass spectrometer that takes ionized masses and
separates them based on charge to mass ratios

12. What is the difference between positive and negative ion modes in MALDI and ESI? How do
we decide which mode to use?
ESI: if sample has functional groups that accept H+ so positive detection is used; if functional
groups of the samples tent to lose a proton, then negative detection is used.
MALDI: positive modes generate M + H, negative ones M – H (single charged ions).

13. What is peptide de novo sequencing? What are the two main fragmentation methods used?

14. What is the aim of a quantitative proteomics experiment? Why do we perform quantitative
proteomics experiments?
 The expression of certain proteins changes upon cell stimulation or stress, or when
cells differentiate or turn into a disease state. Measuring the protein expression levels
as well as characterizing their post-translational modifications gives us information on
their cellular state. The goal of quantitative proteomics is to evaluate the relative
expression of proteins between two cellular samples being compared

15. What are the strategies for quantitative proteomics?

16. What are SILAC and ICAT? What does a typical spectrum of SILAC look like? What are the
pros and cons of these methods compared to each other?

17. How does label-free quantitative proteomics work? Which are the two types of
quantification? What are the pros and cons of this method?

18. What is MALDI imaging?

19. What is SELDI?

20. What are the experimental approaches to study protein-protein interactions in vivo and in
vitro?
21. What is the principle of yeast two-hybrid system?
22. What is the difference between yeast two-hybrid system and mammalian two-hybrid
system? How do we decide which one to use?
23. What is the principle of GST pull-down assays?
24. What is the principle of co-immunoprecipitation?
25. What are protein arrays? What are their advantages and disadvantages compared to other
experimental methods for studying protein-protein interactions?
26. What is the idea behind phage display?
27. What are resonance energy transfer-based techniques? What is the difference between FRET
and BRET?
28. What are protein complementation assays? What enzymes are used in this method?
29. Why is it useful to perform non-invasive in vivo imaging? What experimental methods are
used? What is BLI?
30. How do computational methods to predict protein interactions work? What are the
databases used?

Key abbreviations
2D-PAGE Two-dimensional polyacrylamide gel electrophoresis
BLI Bioluminescence imaging
BRET Bioluminescence resonance energy transfer
CID Collision-induced dissociation
DIGE Difference gel electrophoresis
ELISA Enzyme-linked immuno sorbent assay
Direct: an antigen coated to a multi-well plate is detected by an antibody that
has been directly conjugated to an enzyme.
Indirect: the antigen coated to a multi-well plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is
 applied. Next, an enzyme-labeled secondary antibody is bound to the first
antibody.
Sandwich: A first antibody (known as capture antibody) is coated to the wells.
The sample solution is then added to the well. A second antibody (known as
detection antibody) follows this step in order to measure the concentration of
the sample.
Competitive: The key event of competitive ELISA is the process of competitive
reaction between the sample antigen and antigen bound to the wells of a
microtiter plate with the primary antibody. High sensitivity
ESI Electrospray ionization
ETD Electron-transfer dissociation
FRET Fluorescence resonance energy transfer
GC Gel chromatography
HPLC High-performance liquid chromatography
ICAT Isotope-coded affinity tags
iTRAQ Isobaric tag for relative and absolute quantification
MALDI Matrix-assisted laser desorption/ionization
MS Mass spectrometry
MS/MS Tandem mass spectrometry
PTM Post-translational modification
PCA Protein-fragment complementation assay
QD-BRET Quantum dot/BRET (or Self-illuminating quantum dots)
Q-TOF Quadrupole time-of-flight mass spectrometry
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SELDI Surface-enhanced laser desorption/ionization
SILAC Stable isotope labeling with amino acids in cell culture
TOF Time-of-flight mass spectrometry
IEF Isoelectric focusing

Ionization methods pp77 (signed as slide 13)

MS pages 111(slide 1)- 134(slide 26)
Start from Pp 165