Nickel Affinity Chromatography

Question 1

How is the stationary phase in resin functionalized?

Answer 1

With a chemical group specific to the tag that is fused to protein of interest

Question 2

How can nickel affinity chromatography be used?

Answer 2

To isolate protein of interest in a singe step (column or batch method)

Question 3

How is nickel affinity chromatography eluted?

Answer 3

Using a variety of methods (competitive ligand most common)

Question 4

What is done after nickel affinity chromatography?

Answer 4

Mass spectrometry to unambiguously identify purified proteins

Question 5

What do mass spectrometry instruments do?

Answer 5

Produce, separate, and detect the m/z ratio of ionized molecules present in the gas phase (held at vacuum)

Question 6

What are a couple of key features of mass spectrometry?

Answer 6

• High mass accuracy and sensitivity
• Capable of elucidating chemical composition

Question 7

What is key to mass determination?

Answer 7

Ionization since only ions in the gas phase can be accurately measured

Question 8

What did ionization techniques become in the late 1980’s?

Answer 8

“Soft enough” to study large macromolecules such as proteins and oligonucleotides

Question 9

What is protein mass spectrometry used for?

Answer 9

• Protein identification
• Mapping of post-translational modifications
• Quantitative proteomics
• Clinical chemistry
• Structure-function insights including protein conformation dynamics

Question 10

What are the steps for protein mass spectrometry?

Answer 10

1) Trypsin hydrolyzes peptide bond C-terminal to Arg and Lys residues
2) Tryptic peptides are measured, and a mass fingerprint is generated
3) Individual tryptic peptides are isolated and fragmented to produce amino acid sequence
4) Collectively, multiple peptide sequences leads to protein identification using database processing