Question 1

Which amino acid contributes most strongly to the absorbance of proteins at 280 nm, and why is this property useful in protein quantification?

Accepted Answer

Tryptophan, because its indole ring absorbs strongly at 280 nm, allowing accurate spectrophotometric quantification of proteins.

Question 2

A protein has a pI of 5.0. At physiological pH (7.4), what is the net charge of this protein and how would it behave in ion-exchange chromatography using a cation-exchange resin?

Accepted Answer

Net negative charge; it will not bind to the cation-exchange resin and will elute quickly.

Question 3

Which of the following best describes the principle behind size-exclusion (gel filtration) chromatography?

Accepted Answer

Proteins are separated based on their molecular size, with larger proteins eluting first.

Question 4

During SDS-PAGE, what is the main factor that determines the migration rate of proteins through the gel?

Accepted Answer

The molecular weight of the protein, as SDS gives all proteins a uniform negative charge.

Question 5

Which method would you use to determine the isoelectric point (pI) of a protein, and what is the underlying principle?

Accepted Answer

Isoelectric focusing; proteins migrate in a pH gradient until they reach a pH equal to their pI, where their net charge is zero.

Question 6

A researcher adds ammonium sulfate to a protein solution and observes precipitation. Which phenomenon is being exploited, and what is the underlying mechanism?

Accepted Answer

Salting out; high salt concentration reduces available water for protein solvation, leading to precipitation.

Question 7

Which of the following statements about affinity chromatography is correct?

Accepted Answer

It separates proteins based on their specific binding to a ligand attached to the column matrix.

Question 8

In a two-dimensional gel electrophoresis experiment, what two properties of proteins are used for separation in the first and second dimensions, respectively?

Accepted Answer

First: isoelectric point (pI); Second: molecular weight

Question 9

A patient’s hemoglobin is analyzed and found to have a single amino acid substitution: Glu-6 to Val in the β-globin chain. What disease is this associated with, and what is the molecular consequence?

Accepted Answer

Sickle cell anemia; hydrophobic Val causes hemoglobin aggregation and fiber formation.

Question 10

Which of the following best describes the difference between bottom-up and top-down proteomics?

Accepted Answer

Bottom-up analyzes digested peptides, while top-down analyzes intact proteins.

Question 11

Which property of a solvent is most important in determining protein solubility, and how does the addition of ethanol or acetone affect this property?

Accepted Answer

Dielectric constant ($$\varepsilon$$); addition of ethanol or acetone decreases $$\varepsilon$$, reducing protein solubility.

Question 12

Which of the following is a key advantage of using mass spectrometry in proteomics compared to Edman degradation?

Accepted Answer

Mass spectrometry can analyze larger and more complex protein mixtures with higher sensitivity.

Question 13

In an ELISA assay for detecting antibodies against a virus, what is the role of the secondary antibody?

Accepted Answer

It binds to the primary antibody and is conjugated to an enzyme for detection.

Question 14

Which of the following statements about the Hoffmeister series is correct?

Accepted Answer

It ranks salts by their ability to precipitate proteins, with anions having the greatest effect.

Question 15

A protein sample is analyzed by SDS-PAGE and native PAGE. The SDS-PAGE shows a single band at 50 kDa, while the native PAGE shows a band at 100 kDa. What does this indicate about the protein’s structure?

Accepted Answer

The protein is a dimer of 50 kDa subunits, which dissociate in SDS-PAGE.

Question 16

Which of the following is NOT a typical application of proteomics?

Accepted Answer

Studying evolutionary relationships using DNA sequences.

Question 17

Which ionization technique in mass spectrometry is best suited for analyzing large biomolecules and why?

Accepted Answer

Matrix-assisted laser desorption ionization (MALDI); it produces singly charged ions and is tolerant to buffers.

Question 18

A protein mixture is separated by ion-exchange chromatography using a resin with negatively charged groups. Which proteins will elute first?

Accepted Answer

Proteins with the most negative charge.

Question 19

Which of the following best explains why ammonium sulfate is commonly used for protein precipitation?

Accepted Answer

It is high on the Hoffmeister series, has kosmotropic properties, and is effective at neutral pH.

Protein Analysis and Proteomics: Techniques and Applications

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