changes in protein profiles in bortezomib applied multiple myeloma ...

After labeling, black and red colored spots were cut out from Control group
while the others (green and blue ones) from Bortezomib applied group for enzymatic
digestion by using trypsin. Then samples were analyzed by MALDI-TOF-TOF Mass
Spectrometry and mass spectrometric data was compared to the protein database for
sequence matches. If the amino acid sequence of a peptide could be identified, it would
be used to find out the protein from which it was derived. In addition to this, in order to
identify an unknown protein spot, it was necessary for a mass spectrometric analysis to
match more than two peptide’s sequences. In our experiments, protein spot
determination was carried out thanks to parameters such as isoelectric point, molecular
mass, sequence coverage, and mascot mowse score, but the desired peptide matches
were not observed. Although we cannot say that the results obtained mass spectrometric
analysis are definite, they are the most probable results because of their parameters and
functions. These results were shown in Table 4.2.
Table 4.2 summarizes the 37 differentially expressed proteins that were
characterized. The proteins were identified as follow:
Sixteen proteins were not identified due to some personal and instrumental
errors during the experiment. These were Spot 1, 13, 16, 17, 19, 20, 21, 24, 26, 28, 30,
31, 32, 33, 36 and 37, namely M1S, S4Cb, K3C, K4C, M6S, M7S, M8S, K6C, S8C,
S10C, M10Sc, M10Sb, M10Sa, K8Cb and K8Ca, respectively.
Spot 2, Caspase-3 is involved in the activation cascade of caspases responsible
for apoptosis execution. At the onset of apoptosis, it proteolytically cleaves poly (ADP-
ribose) polymerase (PARP) at a '216-Asp-|-Gly-217' bond and activates sterol
regulatory element binding proteins (SREBPs) between the basic helix-loop-helix
leucine zipper domain and the membrane attachment domain. Additionally, it cleaves
and activates caspase-6, -7 and -9. Caspase-3 found in many cell lines, highest
expression in cells of the immune system. Because of its function and role in apoptosis,
after applying Bortezomib (17 nM) to MM U-266 cells, the increasing expression level
of this protein was expected results.
Spot 3 were signed to uncharacterized protein C7orf46. Although its function
does not know exactly, it is thought that this protein may be involved in alternative
splicing.
Spot 18, C-type lectin domain family 5 member A has a function as a positive
regulator of osteoclastogenesis. Thus, it was upregulated after the cells exposed to
Bortezomib.
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