Abstract:
Analysis of erythrocyte membrane associated proteins (ERMBPs) of Polycythemia Vera (PV) and
healthy individuals in Sri Lanka are presented in this thesis. The main aim of this research is
identification and precise quantification of ERMBPs of PV patients and healthy controls employing
mass spectrometry based proteomics to understand better the molecular mechanism that underline
the PV disease via the information provided by identified ERMBPs.
Blood samples were obtained from five PV patients with the JAK2 V617F mutation and five healthy
volunteers. All 5 patients were on hydroxycarbamide and low dose aspirin for over 6 months with
stable blood counts and had also been subjected to multiple therapeutic venesections by the time of
collection. All the fresh blood samples were kept at +4 °C for 3 days in order to mature
reticulocytes to erythrocytes. ERMBPs were isolated by osmotic lysis, followed by several
centrifugation and washing steps. The isolated membranes were solubilized by 4% Triton X-100 (in
lOmM Tris-HCl, pH 8.8). Then the protein concentration was measured, equal amount of proteins
were loaded on to the polyacrylamide gel, subjected to electrophoresis, and silver staining was used
to visualize the separated proteins. Gel bands 40-55 KDa were dissected out from gels, digested by
trypsin, resulting peptides were extracted, the supernatant containing tryptic peptides was dried by
vacuum centrifugation, were dissolved in 2% formic acid, and the solution was analyzed by Liquid
Chromatography Mass Spectrometry (LC-MS/MS), QStarElite. The experimentally generated
singly charged ions of peptide mass fragments were compared with the theoretical mass values
obtained in-silico digestion and identification was made using Mascot database. Quantification of
proteins was performed by spectral counting, and quantification of proteins based on precursor ion
intensities. The outcomes were validated by targeted quantitative proteomic technique, Selected
Reaction Monitoring (SRM).
4 ERMBPs, PDIA1, PDIA6, TXND5 and ERp44, showed statistically significant, 1.5 - 3.5 fold
increase in protein abundance in PV patients compared to healthy controls. Characterization of
PDIA1, PDIA6, TXND5 and ERPp4 was performed using databases and software. The thioredoxin
domains (Trxs) were located by sequence alignments, which were carried out using BioEdit
Sequence Alignment Editor and comparing with the structures obtained from PDB. The ribbon
cartoons for PDIA1, PDIA6, TXND5 and ERp44 were generated using PyMOL. The Trxs and
CGHC motifs were visualized. All 4 proteins belong to the same family called protein disulphide
isomerase (PDI). They consist of at least one Trx, which is known to cause formation, breakage and
rearrangement of disulphide bonds in proteins related to oxidation stress.
In view of the above findings, as erythrocytes are constantly exposed to oxidative stress, therefore it
is hypothesized that increased abundance of 4 PDI proteins containing Trxs could be considered as
an inhibiting factor of stress induced apoptosis in erythrocytes and therefore contribute towards the
increased erythrocyte mass in PV patients. Hence thioredoxin domain containing PDIAI, PDIA6,
TXND5 and ERp44 proteins in PV proteins could be identified as possible targeted by drugs to treat
PV disease.