Proteins: Determination of Primary Structure

Question 1

General functions of proteins/peptides:

Answer 1

Enzymes:catalyze chemical rxns
Hormones:messengers regulating bodily functions
Storage:make essential substances readily available
Transport:carry substances thru body fluids
Structural:support / maintain cell shape
Protective:defend against foreign matter
Contractile:do mechanical work

Question 2

Conjugations:

a protein with a lipid is:

Answer 2

lipoprotein

Question 3

Conjugations:

a protein with a carbohydrate is:

Answer 3

glycoprotein

Question 4

Conjugations:

a protein with a phosphate is:

Answer 4

phosphoprotein

Question 5

Conjugations:

a protein with a heme is:

Answer 5

hemoprotein

Question 6

Conjugations:

a protein with flavin nucleotides is:

Answer 6

flavoprotein

Question 7

Conjugations:

a protein with a metal is:

Answer 7

metalloprotein

Question 8

a conjugation is…

Answer 8

a non-a.a. component of a protein

Question 9

techniques that separate protein by charge:

Answer 9

ion exchange chromatography
isoelectric focusing
electrophoresis

Question 10

techniques that separate protein by size:

Answer 10

dialysis and ultracentrifugation
gel electrophoresis
gel filtration (aka size exclusion chromatography)

Question 11

technique that separates protein by charge and size:

Answer 11

2D gel electrophoresis

Question 12

technique that separates protein by specificity:

Answer 12

affinity chromatography

Question 13

techniques that separate protein by polarity:

Answer 13

paper chromatography
reverse-phase chromatography
hydrophobic chromatography

Question 14

affinity chromatography…

Answer 14

separates biochemical mixtures based on highly specific interaction such as antigen-antibody, enzyme-substrate, or receptor-ligand

reversible, used for purification of one of interacting molecules

one component immobilized to create stationary phase while large is in mobile phase

Question 15

SDS-PAGE

Answer 15

sample put into gel, proteins migrate in electric field

SDS masks charge, so they move by weight

Question 16

Isoelectric focusing and 2D gel electrophoresis

Answer 16

first, separated by pI in gel with immobilized pH gradient (isoelectric focusing)

Then, put horizontally in an SDS-PAGE

final product has separated MW and pI on same gel

Question 17

Immunoblotting (Western Blot)

Answer 17

first, protein separated by electrophoresis, IEF, or 2D

then, put on membrane that blocks non-specific surfaces; incubate membrane with primary and labeled secondary antibodies

signal of antigen detected by chemiluminescence

determines protein expression in a biological sample

Question 18

Edman method for determining primary structures:

Answer 18

use multiple cleaving agents (to create overlapping fragments) on separate samples of polypeptide to sequence small fractions and then put back together

Edman’s reagent labels amino-terminal residues; successive rounds cleaves terminal residues over and over again to determine sequence

Question 19

Why has MS replaced chemical methods for determining primary structures?

Answer 19

no need for protein purification

sensitivity of technique allows for analysis of proteins in complex mixtures

high coverage within protein sequence

multiple proteins can be identified in a single analysis

can be used to determine post-translational modifications and their locations

Question 20

MS

Answer 20

peptide/protein is ionized

broken into peptides by proteases

MS further breaks into fragment ions & measures their mass

uses speed to separate based on weight

in all, measures mass/charge ratios of ions

set of masses used to determine primary sequences using protein databases

Question 21

proinsulin includes

Answer 21

the C peptide, which is cleaved from A and B chains in mature insulin