As part of development of a new test for detection of misuse ofhuman erythropoietin (EPO) by world athletes, Sydney researchershave studied the signals arising from native and administered EPOwithin 2D gels of urine samples. “The amino acid sequence ofendogenous human erythropoietin (EPO) and (rEPO) is identical andconsist of 165 amino acids, with three N-glycosylation sites andone O-glycosylation site. This gives an apparent molecular weightof 34 kDa by SDS-PAGE of which approximately 40% by weight is sugarcontent. Different micro-heterogeneity in glycosylation profiles(in particular, the number of sialic acid residues attached and thelength of the sugar chains) between EPO and rEPO gives each form ofthe protein a different series of isoelectric points (pI) and asmall change in molecular mass. The experimentally determined pIranges are approximately 3.7–4.7 and 4.4–5.1 for EPO and rEPO,respectively. …. Analysis of unknown urine samples was carried outby two-dimensional electrophoresis. Total separated urinaryproteins were visualized on the gel with SYPRO Ruby (MolecularProbes, USA) per the manufacturer’s instruction. After cutting theprotein spots, the protein was digested by adding trypsin dissolvedin 50 mM ammonium bicarbonate and incubated at 30o C overnight. Thepeptide solution was desalted, concentrated and spotted onto aMALDI plate alpha-cyano-4-hydroxy-cinnamic acid dissolved in 70%(v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. MALDI-TOFMS 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 3-7is 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 toglycosylation in EPO.

(b) Different rEPO products are currently available.

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

(ii) Give two reasons why this chemical difference affects the2D gel behaviour of the isoforms present

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

Note:

Please give explicit and comprehensive explanation .