Ion exchange chromatography separates proteins according to their net charge. Why is pH an important consideration during this technique?

Consider that you wish to separate a mixture of histidine, serine and aspartate by ion exchange chromatography.

Using a cation exchange column at pH 5, which amino acid would you expect to elute first and which last?

pKa and pI values are as follows:

Histidine: pK₁ = 1.82, pK₂ = 9.17, pK_R = 6.00, pI = 7.59

Serine: pK₁ = 2.21, pK₂ = 9.15, pI = 5.68

Aspartate: pK₁ = 1.88, pK₂ = 9.60, pK_R = 3.65, pI = 2.77

In attempting to define the subunit composition of an unknown protein, you decide to subject the protein to SDS-PAGE in the presence or absence of the reducing agent DTT. The results of this analysis are shown below.

Lane 1: molecular weight marker ladder.

Lane 2: protein sample without DTT

Lane 3: protein sample with DTT.

From this analysis, what is the subunit composition of the original native protein?

The following peptide sequence was deemed too long to be easily assessed by mass spectrometry. Therefore, it was first digested with trypsin. In the box below, enter the amino acid sequence (use the single letter code; do not enter spaces or non-amino acid characters) of any ONE possible tryptic peptide that may have been detected by the mass spectrometer.

VQSYEKEEDLLQAWSTFIRIMDPDVITGYNIQNFDLPYLISRAQTLKVQTFPF

Amino acids have characteristic titration curves. A plot of the titration of an amino acid with a strong base resulted in just two distinct regions of greatest buffering power. This would likely be indicative of an amino acid with:

The image below is a representation of an acid-base titration of a diprotic amino acid, labelled at specific points (i through v).

Which region represents where this amino acid's net charge is zero?