Question

You develop a scheme to purify the protein RANIN, and use your "sequencing toolbox" to identify it. You suspect RANIN has quaternary structure, but the polypeptides are not separated by extreme pH. Suggest another method SDS-PAGE samples run with and without reducing agent tefis you RANIN does not have intermolecular disulfide bonds, but it is made up of two distinct polypeptide chains. Draw the SDS-PAGE results and explain how you know. RANIN chain A has a pi of approximately 9, and chain B has a pi of approximately Choose the ion exchange column and buffer appropriate for separation of chains A and B. You digest RANIN chains A and B with trypsin and separate the following products. Deduce the sequence of each peptide from these products, or propose what to do next in order to deduce the sequences. Chain A; DLVQYR, LSMINAK, NYIMFR Chain B: AIQDEMR, EPHELLAR, STPSMNSVK, LSQAT

Answer 1

a) Proteins denature, losing their shape ans function when exposed to extremes of pH, temperature, detergent. RANIN's polypeptides did not separate at extreme pH, it possibly might be having a disulfide bond between the polypeptides that is not cleaved at extreme pH. So, another method to separate them would be to treat the protein to high temperature or detergent (SDS) in presence of a reducing agent such as beta-mercaptoethanol or Dithiothreitol, the polypeptides loses their structure and also disulfide bonds and thus can be separated.

b) If the protien's quarternary structure do not have disulfide linkages between the polypeptide chains, in a SDS_PAGE, it would give two bands either in presence or absence of reducing agent (See below figure). Since, SDS is a detergent, the protien is completely denatured loses its shape and appears as a primary sequence of aminoacids. So in SDS-PAGE you see two bands whether reducing agent is present or absent.   

Reducing agent

C)

The pI value can be calculated based on the primary sequence of the molecule. The choice of buffer pH then determines the net charge of the protein of interest.

In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge; therefore, a positively charged anion exchange resin is chosen to capture this protein.

In a buffer with a pH lower than the pI of the protein of interest, the protein will carry a positive net charge; thus a negatively-charged cation exchange resin is chosen.

When an ion exchange chromatography column is loaded with a sample at a particular pH, all proteins that are appropriately charged will bind to the resin. For example, if an anion exchange resin is chosen, all proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin. A good rule of thumb for choosing a buffer pH is the following:

• Anion exchanger — 0.5–1.5 pH units greater than the pI of the protein of interest
• Cation exchanger — 0.5–1.5 pH units less than the pI of the protein of interest

D) The peptides generated by tryptic digest is to be subjected to tandem mass spectrometry, where the peptide would be further cleaved as shown in figure below and the sequence can be deduced.

100%1 129.11 10% 85% y1 MMESAKPEDVSFQ GR (447-461) 75% 70% b2 b3 b4 b5 55% a»50% -45% 40% 35% hh2 263.10 a2 235.11 PE 227.13 y9 1034.53 25% 342.16 PEDVh yl 441.22 550.21 175.11 15% 10% 5% y5 hh3 16594.33 856.40 y10 1162.64 yl.1 693.40 808.44937.49 12 1233.691320.71 1449.73 479.20 y8 y13 100 200 300 400 500 600 700 800 000 1100 1200 1300 m/z