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EOM 1 - Test 1 > Lecture 3: Proteins I > Flashcards

Lecture 3: Proteins I Flashcards

Proteins

1
Q

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

A

30% total proteins

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2
Q

What is the difference between simple and conjugated proteins?

A

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

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3
Q

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

A

> 40

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4
Q

How many genes does the human contain?

How many are expressed at once?

A

~ 40K

10K - 15K expressed at once

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5
Q

What types of proteins are expressed at high levels?

What types are expressed at low levels?

A

Collagen, hemoglobin

hormones, signaling proteins

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6
Q

What are two sources of proteins for purification?

A

Natural or Recombinant

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7
Q

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

A
  1. Solubility
  2. Electrical Charge / Polarity
  3. Size and Shape
  4. Affinity
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8
Q

What techniques separate proteins on charge?

A
  1. Ion Exchange Chrom
  2. Isoelectric Focusing
  3. Electrophoresis
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9
Q

What techniques separate proteins on size?

A
  1. Dialysis and ultracentrifugation
  2. Gel electrophoresis
  3. Size exclusion chrom (gel filtration)
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10
Q

What techniques separate proteins on Charge and Size?

A

Two Dimensional (2D) Gel Electrophoresis

Hint: Two traits–so 2D

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11
Q

What techniques separate proteins on specificity?

A

Affinity Chromatography

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12
Q

What techniques separate proteins on polarity?

A
  1. Paper Chrom
  2. Reverse-phase chrom
  3. Hydrophic chrom
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13
Q

What does affinity chrom use to interact with proteins?

What are the phases?

Reversible?

When is Affinity Chrom BEST used?

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

Target in MOBILE
Ligand in STATIONARY

Typically reversible

Best for SPECIFIC molecules or groups

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14
Q

What trait does Gel Electrophoresis separate?

A

Molecular Weights and Relative Expression

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15
Q

How does SDS-PAGE work?

What does it separate on?

A

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

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16
Q

How are isoforms determined in clinical tests?

A

Isoelectric Focusing (IEF)

17
Q

What does isoelectric focusing (IEF) separate based on?

A

Isoelectric Points (pI)

18
Q

What are the two steps to 2D-gels?

What is final step?

A
  1. Subject to Isoelectric Focusing (IEF)
  2. SDS-PAGE
  3. Immunoblotting
19
Q

What does Western Blot Analysis (immunoblotting) determine?

A

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

Hint: Remember, PROTEIN

20
Q

What would be best test for a myeloma protein expression ?

A

Gel Electrophoresis

21
Q

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

What trait does this manipulate?

A

Antibodies/virus/toxins in bodily fluids

Affinity

Hint: Testing bodily fluids for SPECIFIC target

22
Q

What are two limitations of sequencing primary structures of proteins?

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

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

23
Q

What determines structure and function of protein?

A

Amino Acid sequence

24
Q

How is insulin synthesized?

A

Single peptide chain (preproinsulin)

25
Q

What is the mature form of insulin?

What connects them?

How long are mature forms?

A

2 Chains, due to cleavage of original

Disulfide bond

Chain A = 21 AAs
Chaine B = 30 AAs

26
Q

What are the steps to Edman Degradation?

A
  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
27
Q

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

A

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

28
Q

How are proteins ionized for MS analysis?

A
  1. Electrospray Ionization (ESI)
  2. Matrix-Assisted Laser Desorption (MALDI)

Small molecules can be ionized by heat

29
Q

What is cause of neonatal diabetes?

A

Mutations at the cleavage sites in conversion from preproinsulin to insulin

30
Q

What class of substance breaks proteins into peptides?

A

Proteases

e.g. trypsin

31
Q

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

A
  1. Break proteins with proteases
  2. Mass spec further fragments
  3. Measure based on M/z
  4. Analyze using protein database
32
Q

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

Two examples?

A

Post-Translational Modification

  1. Side chain modification
  2. Cleavage
33
Q

What are roles of post-translational modifications?

A

Regulation of protein activity

Dynamic jobs, some are reversible, such as phosphorylation and acetylation

34
Q

What are advantages of protein sequencing by MS?

A

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

35
Q

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

A

LC-MS

Hint: It’s quantitative and can process complex samples

EOM 1 - Test 1 (16 decks)

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