Proteomics

Question 1

What is proteomics?

Answer 1

The large-scale study of proteins (functional molecules in cells)

Question 2

What is the proteosome?

Answer 2

The set of proteins expressed overall by a cell, tissue, or organism

Question 3

List 4 goals of proteomics.

Answer 3

• Identify and quantify proteins
• Determine where proteins are localized within systems
• Study modifications in different proteins
• Understand interactions, function, and activities of proteins

Question 4

What is 2D-GE?

Answer 4

• Two-dimensional gel electrophoresis
• Used to separate proteins based on two properties

Question 5

What are the 2 properties protein separation is based on?

Answer 5

1. Isoelectric point (pl) - pH at which a protein has no net electrical charge
2. Molecular weight - the size (mass) of the protein

Question 6

What is 2D-GE useful for?

Answer 6

• Analyzing complex protein mixtures
• Identifying differences in protein composition

Question 7

How do proteins appear once 2D-GE is completed?

Answer 7

• Spots on gel
• Each spot = unique protein based on MW and pl

Question 8

What is liquid chromatography (LC)?

Answer 8

• Used to separate proteins or peptides based on hydrophobicity

Question 9

What is hydrophobicity?

Answer 9

How much a molecule prefers water (polar environment) vs. an oily/non-polar environment

Question 10

What other technique is liquid chromatography often combined with?

Answer 10

Mass spectrometry (MS) to identify and quantify proteins

Question 11

List the 6 steps of LC-MS for protein analysis.

Answer 11

1. Protein extraction and digestion
2. LC: separating peptides by hydrophobicity
3. Ionization + MS detection
4. Mass spectra generation and peak analysis
5. Protein identification using databases
6. Protein quantification

Question 12

Describe protein extraction and digestion for LC-MS.

Answer 12

• Proteins extracted from a biological sample
• Extracted proteins digested into smaller peptides using enzymes
• Peptides prepped for LC separation

Question 13

Describe peptide separation for LC-MS?

Answer 13

• Peptide mixture loaded onto an LC column (stationary phase that interacts differently with water-loving and water-hating peptides)
• Gradient of solvents helps separate peptides based on hydrophobic properties

Question 14

Explain the differences in speed of hydrophobic vs. hydrophilic peptides through the LC column.

Answer 14

• Hydrophobic peptides take longer to travel through
• Hydrophilic peptides move by faster

Question 15

Describe the ionization and MS detection process of LC-MS.

Answer 15

• Separated peptides are ionized before placed in mass spectrometer
• MS instrument measures mass-to-charge ratio of each peptide

Question 16

Describe the generation of mass spectra and peak analysis for LC-MS.

Answer 16

• MS produces mass spectra (graphical representation of peptide masses)
• Each spectrum shows peak intensities

Question 17

What do stronger mass spectra peaks indicate about peptides?

Answer 17

Stronger peaks mean more abundant peptides

Question 18

Describe the protein identification process in LC-MS.

Answer 18

• Spectral data compared to a database to identify which peptides correspond to known proteins
• Software determines which proteins are most responsible for high-abundance peaks

Question 19

Describe protein quantification in LC-MS.

Answer 19

• Abundance levels of peptides are measured using peak intensities (height) and spectral counts (how often a peptide is detected)
• Tells us how much of a specific protein is present in a sample

Question 20

List 5 applications of proteomics.

Answer 20

1. Disease biomarker discovery
2. Drug target identification
3. Functional genomics
4. Systems biology
5. Personalized medicine