Biochemistry Exam #2 (Module 3)

In this mass spectrum, click on the peak that represents lactoglobulin in a state whose m/z value is approximately one half of its molecular weight.

(L+ 2 H) ^2+

A protein that is a tetramer with four identical subunits of 10 kD each would show major bands on a properly run SDS-PAGE gel at

10 kD

(Exam question 9) A mixture of five proteins are analyzed by MALDI-TOF. Their molecular weigths are 3 kD, 22 kD, 100 kD, 225 kD and 515 kD. A peak appears in the mass spectrum at 33.3 kD. Which protein most likely gave this peak?

100 kD

In the 2D gel image below, the red spot represents and unmodified version of a protein with a molecular weight of 150 kD. Which grey spot is appropriately placed to represent a version of that protein that has been phosphorylated at a serine reside (a phosphate group has been covalently attached to the serine hydroxyl group)? The SDS-PAGE portion of the gel spans from 10 kD to 2000 kD.

4

(Exam question 16) You start a protein purification with 15 g of a crude mixture of mitochondrial protein and measure the enzyme activity and get 150,000 units of activity. You then do several rounds of chromatography, and end up at 150 mg of protein that assays to give 100,000 units of activity. The percent yield of the enzyme you are trying to purify is

67%

(Exam question 14) A tetrapeptide contains alanine, lysine, glutamate, and methionine. If cyanogen bromide cleaves the tetrapeptide into a tripeptide and free glutamate, and trypsin cleaves the tetrapeptide into two dipeptides, what is the sequence of the tetrapeptide?

AKME

(Exam question 31) A small protein was completely hydrolyzed and found the contain Arg + Cys + 2 Lys + Met + Met + Thr + Val. Based on the observations below, reconstruct the protrin sequence. a. Sanger's reagent (FDNB) treatment and hydrolysis resulted in DNP- Arg being released. b. Carboxypeptidase A treatment gave: i. Val ii. All the rest c. Cyanogen bromide trearment gave: i. Lys-Cys-Met-Arg (in some order) ii. Thr- Val-Lys (in some order) d. Trypsin treatment gave: i. Arg ii. Lys iii. Cys-Thr-Lys- Met (in some order) iv. Val

Arg-Lys-Cys-Met-Thr-Lys-Val

(Exam question 17) A sample of blood proteins is separated on a 2D-gel. If the patient is young enough to express significant amounts of both fetal and adult hemoglobin, and fetal hemoglobin has less positive charges than adult hemoglobin but the sizes are nearly identical, where would they be found on the gel relative to one another?

At the same position in the SDS-PAGE dimension, but fetal hemoglobin will be found at lower pH in the isolectric focusing dimension

In ion-exchange chromatography, molecules are separated based on

Charge

(Exam question 13) A reagent that selectively cleaves after methionine residues is

Cyanogen bromide

In ion exhcange chromatography using diethylaminoethyl (DEAE) cellulose, which of the following amino acids would elute last?

D

If a protein has an isoelectric point (pI) of 3.2, which of the following amino acids is it likely to have a lot of?

D and E

The N-terminal sequence of a peptide can be determined by

Edman degradation

A heptapeptide was found to have an amino acid composition of asp. leu, lys, met,phe and tyr. Trypsin has no effect on the heptapeptide. One cycle of Edman degradation renders the product whose structure is shown below. Chymotrypsin treatment yields a dipeptide and a tetrapeptide, as well as a free amino acid. The tetrapeptide is known to contain asp, leu, lys, and met. Cyanogen bromide treatment generates a dipeptide that is determined to originate from the N-terminus of the heptapetide, a tetrapeptide, and free lys. The peptide sequence in single letter amino code is ___________________.

FMYDLMK

(Exam question 26) In affinity chromatography, molecules are separated based primarily on their charge.

False

(Exam question 30) When a protein is 'salted-in', it precipitates and is removed form a solution.

False

All mass spectrometers include an arc magnet to select certain ions.

False

GFP is an antibody.

False

IMAC chromatography separates molecules based on size.

False

NMR determination of protein structure requires that proteins be crystallized into ordered structures.

False

If a protein has an isoelectric point (pI) of 10.2, which of the following amino acids is it likely to have a lot of?

H and R

Which amino acid has an imidazole ring in its side chain?

Histidine

(Exam question 4)Which of the following proteins will elute first (go through the fastest) in size exclusion (also called molecular exclusion) chromatography?

Protein X (Molecular weight 149,000 g/mol, charge +1)

Cyanogen bromide will cleave the peptide KWNEMDAR

after the M

(Exam question 2) A basic amino acid has an R group that contains

an amine group

To make a monoclonal antibody, as opposed to a polyclonal antibody,

an antibody producing spleen cell is fused to a cancer cell

In 'Sandwich ELISA' the meat is _____________ and the bread is ______________.

antigen; antibody

Antibodies bind to

antigens

Which of the following amino acids has its isoelectric point at the highest pH?

arginine

(Exam question 28) Which amino acid has an amide group in its side chain?

asparagine

Which of the following amino acids has its isoelectric point at the lowest pH?

aspartic acid

(Exam question 22) Antibodies are useful because they

bind things

A quick way to start purifying a protein is to first fractionate cellular organelles and collect the one with the protein of interest. If you want to collect fractions that separately contain nuclei, mitochondra, endoplasmic reticulum, and cytosol, how is this most easily done?

centrifugation

Chymotrypsin

cleaves proteins after large hydrophobic amino acid residues

Dicyclohexylcarbodiimide is used to

couple amino acids (covalently bind them to each other)

In determining the following structure by NMR, the structure below would be specified by

crosspeaks in 2D NOESY spectra between protons 2 and 5

ELISA is a method that is used to

detect and quantify proteins and/or small molecules

A common reagent that activates amino acid carboxylates for solid phase protein synthesis is

dicyclohexylcarbodiimide

(Exam question 20) In protein x-ray crystallography, the crystal essentially serves as a

diffraction grating

(Exam question 19) When determining the structure of a protein by NMR the NMR experiments themselves primarily provide

distance constraints

In protein structure determination by NMR, the NMR provides

distance constraints

In SDS-PAGE, the SDS serves to

give proteins constant charge/mass and denature proteins

Which of the following amino acids has an isoelectric pH of 3.2?

glutamic acid

(Exam question 3) Which of the following amino acids has a side chain which contains a nonbasic nitrogen?

glutamine

(Exam question 23) An antibody

has two antigen binding sites

Which of the following is not an applicaiton of antibodies?

he Nuclear Overhauser Effect

(Exam question 24) A student is carrying out a sandwich ELISA. Their first step should be to _______________.

immobilize monoclonal antibody in the wells of a 96-well plate

(Exam question 15) The Edman Degradation

is a method for sequencing proteins from the N-terminus

In IMAC, proteins of interest form ___________ with the contents of the column immobilizing them until they are eluted.

metal-ligand bonds

When centrifuging sub-cellular structures, what organelles pellet out at 10,000 x g?

mitochondria

Which of the following amino acids does not contain an aromatic R-group?

none of the above

When a disulfide linkage is formed, the compound containing this new linkage has been __________.

oxidized

(Exam question 25) A reagent that oxidizes cystines (cysteine disulfides) into cysteic acids that can no longer form disulfide bonds is

performic acid

Which is easier to produce?

polyclonal antibodies

Which of the following amino acids has its α-carbon as part of a 5-membered ring?

proline

Which of the following proteins would elute first from a size exclusion chromatography column?

protein Z (MW 250,000 g/mol; charge +3)

The main drawback of protein X-ray crystallography is that

proteins must first be crystallized and conditions may never be found to crystallized the protein of interest

(Exam question 11) Dialysis is carried out on a solution containing a 100 kD red protein, a 10 kD yellow protein, and a 2,000 kD blue dye by placing the mixture in the dialysis bag. If the dialysis bag has a pore size of 50 kD, what color will the solution in the bag be after several equilibrations and buffer exchanges?

red + blue = purple

Dithiothreitol (DTT) is used to

reduce disulfide bonds

(Exam question 5) Ion exchange chromatography

separates molecules based on charge

(Exam question 21) Dialysis

separates small molecules from large molecules

(Exam question 18) In SDS-PAGE, proteins separate based primarily on

size

Gel-filtration chromatography largely separates proteins (or other molecules) based upon

size

In normal SDS-PAGE, separation is based on

size

Cyanogen bromide's specificity for cleaving amide bonds after methionine residues results from the formation of a required ______________ intermediate.

sulfonium salt

In the Edman Degradation, phenyl isothiocyanate reacts with ___________________.

the N-terminal amino acid of a peptide

(Exam question 12) To purify an enzyme, liver cells are homogenized in a Dounce tube and centrifuged at 500g for 10 minutes. The supernatant is centrifuged further for 10,000g for 20 minutes, then the resulting supernatant is centrifuged at 100,000g for one hour. The resulting pellet is resuspended in buffer and used for enzyme kinetics. The enzyme being studied must be found in

the endoplasmic reticulum

In MALDI mass spec

the protein is embedded in a 'matrix' to help it 'desorb'

(Exam question 10) In MALDI-TOF mass spec, the mass selector is

the time it takes to traverse a flight tube

At a pH > 9, the zwitterion of glycine will have

a net negative charge.

A Western Blot is essentially

a way of staining SDS-PAGE gels for specific proteins

(Exam question 7) We can now follow movement of proteins in live cells using advances in microscopy, antibody methods, and fluorescent proteins.

True

(Exam question 8) New high resolution brain maps exist in part due to advances in protein-based cell imaging.

True

Advances in computers have increased our ability to image proteins in cells at very high resolution.

True

Advances in techniques for the determination of protein structures by Cryo Electron Microscopy won the Nobel Prize in 2017.

True

Denatures protein will usually not bind to an affinity column as well as normal, folded protein.

True

Mass spec can be used to detect cancer cells.

True

Peptides can be synthesized using the techniques of Organic Chemistry.

True

Some nuclear hormone receptors wait in the cytosol for their hormone to arrive.

True

When we purify protein, we are going against the natural tendency toward disorder (we are fighting against entropy).

True

You can sequence a protein using mass spec (along with some other tools, like enzymes to cut the protein into smaller pieces).

True

(Exam question 1) Which amino acid has a phenol in its side chain?

Tyrosine

(Exam question 29) When the protein bands in an SDS-PAGE gel are transferred to paper and stained with antibody, it is called a

Western Blot

(Exam question 6)The role of dicyclohexylcarbodiimide in peptide synthesis is

to activate carboxyl groups, giving them a better leaving group

The most common method used to determine the sequences of proteins today is

translation of DNA sequences in computers using the genetic code

a TOF mass spec

uses the time of transit through a flight tube as the mass selector

Antibodies bind to antigens via

weak interactions

Immobilized metal affinity chromatography (IMAC) will retain proteins

with 4-6 consecutive histidine residues