Question

As part of development of a new test for detection of misuse of human erythropoietin (EPO) by world athletes, Sydney researchers have studied the signals arising from native and administered EPO within 2D gels of urine samples. “The amino acid sequence of endogenous human erythropoietin (EPO) and (rEPO) is identical and consist of 165 amino acids, with three N-glycosylation sites and one O-glycosylation site. This gives an apparent molecular weight of 34 kDa by SDS-PAGE of which approximately 40% by weight is sugar content. Different micro-heterogeneity in glycosylation profiles (in particular, the number of sialic acid residues attached and the length of the sugar chains) between EPO and rEPO gives each form of the protein a different series of isoelectric points (pI) and a small change in molecular mass. The experimentally determined pI ranges are approximately 3.7–4.7 and 4.4–5.1 for EPO and rEPO, respectively. …. Analysis of unknown urine samples was carried out by two-dimensional electrophoresis. Total separated urinary proteins were visualized on the gel with SYPRO Ruby (Molecular Probes, USA) per the manufacturer’s instruction. After cutting the protein spots, the protein was digested by adding trypsin dissolved in 50 mM ammonium bicarbonate and incubated at 30o C overnight. The peptide solution was desalted, concentrated and spotted onto a MALDI plate alpha-cyano-4-hydroxy-cinnamic acid dissolved in 70% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. MALDI-TOF MS was performed with an Axima CFR instrument (Shimadzu Kratos, UK), equipped with N2 laser (337 nm, 10 Hz repetition rate).”

Identification of other abundant urinary proteins pH range 3-7 is given below :

Tamm Horsfall glycoprotein (THP) – with mass (Da) 69715 and pI 5.05

Alpha-1-antichymotrypsin (AC)   - with mass (Da) 47651 and pI 5.3

Alpha-2-thiol proteinase inhibitor (PI) - with mass (Da)   71946 and pI 6.3

Alpha-2-HS-glycoprotein precursor (HSGP) - with mass (Da) 39325 and pI 5.4

Questions.

(a) Suggest the actual types of amino acid sidechains subject to glycosylation in EPO.

(b) Different rEPO products are currently available.

(i) Why might these products differ from one another in sugar content and structure?

(ii) Give two reasons why this chemical difference affects the 2D gel behaviour of the isoforms present

(c) Why are the protein components shown above have such a high precision of mass accuracy?

Note:

Please give explicit and comprehensive explanation .

Answer 1

a. Suggest the actual types of amino acid sidechains subject to glycosylation in EPO.

Ans. a. The actual sidechains of the protein will contain serine and threonine as they can be O-glycosylated. While the side chain with amino acid Asparagine and arginine are able to be N glycosylated. So the possible amino acids are among the Threonine. serine, arginine and asparagines.

b. Different rEPO products are currently available.

Ans. b. Many reputed companies are right now in the market of manufacture of the recombinant EPO. Some of them are as follows.

rEPO	Manufacturer
Epogen	Amgen, California, USA
Procit	Amgen, California, USA
Eprex	Cilag, Schaffhausen, Switzerland
Neo recomon	Roche, Basel, Switzerland
Ceriton	Ranbaxy, Newjersy, USA
Epofit	Intas Biopharmaceuticals Ltd., Ahmedabad, India
Erykine	Intas Biopharmaceuticals Ltd., Ahmedabad, India
Hemax	Hindustan antibiotics, Pune, India
Shanpoietin	Shanta Biotechnics, Hyderabad, India
Wepox	Wockhardt, Mumbai, India
Zyrop	Zydus Biogen, Ahmedabad, India

1. Why might these products differ from one another in sugar content and structure?

Depending upon the structure and the site of glycosylation, the biological efficacy of the product changed. For example the EPO with more branching at N linked sugar chain are much effective in expression of invivo biological activity. SO to improve and enhance the activity of rEPO there is different sugar content and structural variability.

1. Give two reasons why this chemical difference affects the 2D gel behaviour of the isoforms present

The chemical difference leads to the variation in the molecular weight as well as the rearrangement of the protein will lead to the formation of more globular of open structure that affect the mobility of the EPO in PAGE. Similarly different changes at side chains also affects the pH and hence isoelectric point also shifts leads to the variation in the position in 2 D gel electrophoresis.

(c) Why are the protein components shown above have such a high precision of mass accuracy?

Ans. The protein are undergone trypsin cleavage followed by purification of the peptide fragments. These peptides were then subjected to ionization in MALDI and passed through the TOF. And we know that MALDI TOF gives the molecular mass so accurately upto various points after decimal.

The MALDI TOF uses the uniform electric fields which allows “exact” calculations of flight times presented as mass-to-charge ratio. Thhis is why it is highly accurate.