Lecture 3: Proteins I

Question 1

What percentage do structural proteins make up of the human body?

Answer 1

30% total proteins

Question 2

What is the difference between simple and conjugated proteins?

Answer 2

Conjugated include non-Amino Acid components, such as lipids, carbs, and heme.

Question 3

What is the minimum number of residues to fold into tertiary structure?

Answer 3

> 40

Question 4

How many genes does the human contain?

How many are expressed at once?

Answer 4

~ 40K

10K - 15K expressed at once

Question 5

What types of proteins are expressed at high levels?

What types are expressed at low levels?

Answer 5

Collagen, hemoglobin

hormones, signaling proteins

Question 6

What are two sources of proteins for purification?

Answer 6

Natural or Recombinant

Question 7

What are the 4 characteristics you can manipulate to separate proteins?

Answer 7

1. Solubility
2. Electrical Charge / Polarity
3. Size and Shape
4. Affinity

Question 8

What techniques separate proteins on charge?

Answer 8

1. Ion Exchange Chrom
2. Isoelectric Focusing
3. Electrophoresis

Question 9

What techniques separate proteins on size?

Answer 9

1. Dialysis and ultracentrifugation
2. Gel electrophoresis
3. Size exclusion chrom (gel filtration)

Question 10

What techniques separate proteins on Charge and Size?

Answer 10

Two Dimensional (2D) Gel Electrophoresis

Hint: Two traits–so 2D

Question 11

What techniques separate proteins on specificity?

Answer 11

Affinity Chromatography

Question 12

What techniques separate proteins on polarity?

Answer 12

1. Paper Chrom
2. Reverse-phase chrom
3. Hydrophic chrom

Question 13

What does affinity chrom use to interact with proteins?

What are the phases?

Reversible?

When is Affinity Chrom BEST used?

Answer 13

1. Antigen-Antibody
2. Enzyme-Substrate
3. Receptor-Ligand

Target in MOBILE
Ligand in STATIONARY

Typically reversible

Best for SPECIFIC molecules or groups

Question 14

What trait does Gel Electrophoresis separate?

Answer 14

Molecular Weights and Relative Expression

Question 15

How does SDS-PAGE work?

What does it separate on?

Answer 15

Sulfate groups ( negative ) mask ( positive ) protein charge, making net negative–which allows to interact with field according to MOLECULAR WEIGHT and EXPRESSIONHo

Question 16

How are isoforms determined in clinical tests?

Answer 16

Isoelectric Focusing (IEF)

Question 17

What does isoelectric focusing (IEF) separate based on?

Answer 17

Isoelectric Points (pI)

Question 18

What are the two steps to 2D-gels?

What is final step?

Answer 18

1. Subject to Isoelectric Focusing (IEF)
2. SDS-PAGE
3. Immunoblotting

Question 19

What does Western Blot Analysis (immunoblotting) determine?

Answer 19

Relative protein expression in biological sample, and specific amino acid sequence.

Hint: Remember, PROTEIN

Question 20

What would be best test for a myeloma protein expression ?

Answer 20

Gel Electrophoresis

Question 21

What would ELISA (Enzyme Linked Immunosorbent Assay) be used for?

What trait does this manipulate?

Answer 21

Antibodies/virus/toxins in bodily fluids

Affinity

Hint: Testing bodily fluids for SPECIFIC target

Question 22

What are two limitations of sequencing primary structures of proteins?

Answer 22

1. Can’t predict positions of disulfide (cystine) bonds

2. Can’t identify amino acids post-translationally modified

Question 23

What determines structure and function of protein?

Answer 23

Amino Acid sequence

Question 24

How is insulin synthesized?

Answer 24

Single peptide chain (preproinsulin)

Question 25

What is the mature form of insulin? What connects them? How long are mature forms?

Answer 25

2 Chains, due to cleavage of original Disulfide bond Chain A = 21 AAs Chaine B = 30 AAs

Question 26

What are the steps to Edman Degradation?

Answer 26

1. Edman's Reagent (phenylisothiocyanate) labels amino-terminal 2. PTH derivative can be released under mild conditions to generate new amino-terminal residue 3. Multiple rounds can be used to sequence

Question 27

Does Edman Degradation work for large proteins (>100)? How so?

Answer 27

Yes, you must use multiple cleaving agents and analyze overlapping fragments

Question 28

How are proteins ionized for MS analysis?

Answer 28

1. Electrospray Ionization (ESI) 2. Matrix-Assisted Laser Desorption (MALDI) Small molecules can be ionized by heat

Question 29

What is cause of neonatal diabetes?

Answer 29

Mutations at the cleavage sites in conversion from preproinsulin to insulin

Question 30

What class of substance breaks proteins into peptides?

Answer 30

Proteases e.g. trypsin

Question 31

What are the steps to analyze protein by Liquid Chrom Mass Spec?

Answer 31

1. Break proteins with proteases 2. Mass spec further fragments 3. Measure based on M/z 4. Analyze using protein database

Question 32

What allows vast number of proteins to be synthesized from relatively small number of genes? Two examples?

Answer 32

Post-Translational Modification 1. Side chain modification 2. Cleavage

Question 33

What are roles of post-translational modifications?

Answer 33

Regulation of protein activity Dynamic jobs, some are reversible, such as phosphorylation and acetylation

Question 34

What are advantages of protein sequencing by MS?

Answer 34

High specificity Sensitivity--quantitative analysis of protein in complex mixtures High coverage Multiple proteins can be ID'ed in single run Post-translational modifications can be determined

Question 35

What would be used to screen bodily fluids for concentrations of amino acids, fatty acids, or other metabolites?

Answer 35

LC-MS Hint: It's quantitative and can process complex samples