iTRAQ , SILAC

What is the main purpose of using iTRAQ in proteomics?

• To separate proteins based on size
  
  

• To label and quantify proteins from multiple samples

• To determine the secondary structure of proteins

• To identify post-translational modifications only

Answer explanation

iTRAQ is used for quantitative proteomics, allowing the simultaneous comparison of protein expression in multiple samples using isobaric chemical tags.

In iTRAQ labeling, which component ensures that all labeled peptides have the same total mass before MS/MS?

• Peptide reactive group

• Reporter ion group

• Balance group

• Fragmentation buffer

Answer explanation

The balance group offsets the mass of the reporter ion group, ensuring that all tagged peptides appear as identical masses in MS1. Differences are observed only in MS2.

Why do all iTRAQ-labeled peptides appear at the same m/z in MS1?

• They have different charges

• The balance group equalizes their total mass

• The peptides are fragmented before MS1

• The ionization source removes mass differences

Answer explanation

The balance group ensures that all iTRAQ-labeled peptides have the same total mass, making them indistinguishable in MS1. Only in MS2 do the reporter ions reveal their identity.

Which enzyme is commonly used to digest proteins before iTRAQ labeling?

• Trypsin

• DNA polymerase

• Lipase

• Kinase

Answer explanation

Trypsin is commonly used in proteomics because it cleaves proteins at lysine (K) and arginine (R) residues, generating peptides suitable for iTRAQ labeling and MS/MS analysis.

What is the main advantage of using iTRAQ over traditional 2D gel electrophoresis for protein quantification?

• iTRAQ allows multiplexing of samples

• 2D gels are more sensitive than iTRAQ

• iTRAQ can separate proteins based on charge and size

• iTRAQ is only qualitative, not quantitative

Answer explanation

iTRAQ enables the simultaneous analysis of up to 8 different samples, whereas 2D gel electrophoresis requires separate gels, making iTRAQ more efficient and reproducible.

What does SILAC stand for?

• Stable Isotope Labeling of Amino Acid Complex

• Stable Isotope Labeling by Amino Acids in Cell Culture

• Stable Ionized Labeling of Amino Carbon

• Sequential Isotope Labeling in Advanced Chemistry

Answer explanation

SILAC is a metabolic labeling technique used for quantitative proteomics where cells incorporate isotopically labeled amino acids, enabling mass spectrometry-based protein quantification.

What are the most commonly used labeled amino acids in SILAC?

• Lysine (K) and Arginine (R)

• Methionine (M) and Serine (S)

• Glutamine (Q) and Histidine (H)

• Aspartate (D) and Tyrosine (Y)

Answer explanation

These amino acids are chosen because trypsin, a proteolytic enzyme used in digestion, cleaves proteins at lysine and arginine residues, making their quantification easier in mass spectrometry.

What is the main purpose of using SILAC in proteomics?

• To measure RNA expression levels

• To quantify protein abundance changes in different conditions

• To visualize proteins under a microscope

• To create genetically modified cell lines

Answer explanation

SILAC is used for labeling proteins in living cells and tracking quantitative changes in the proteome during experimental conditions like stress, drug treatment, or disease models.

Why is SILAC considered better than other protein labeling methods?

• It provides high accuracy and reproducibility

• It does not require mass spectrometry

• It works for all types of cells, including dead cells

• It is faster than all other labeling techniques

Answer explanation

SILAC integrates heavy isotopes directly into proteins during synthesis, minimizing variability from sample processing. This allows for more accurate quantification compared to chemical labeling methods like iTRAQ or TMT.

Why is dialyzed serum used in SILAC experiments?

• To remove natural amino acids that may interfere with labeling

• To increase cell growth rate

• To increase the solubility of heavy amino acids

• To improve protein digestion by trypsin

Answer explanation

Dialyzed serum removes unlabeled amino acids, ensuring that cells only incorporate heavy isotopes.