Comparative Proteomic Analysis of Bone Marrow Plasma Cells By Using Mass Spectrometry Based Bottom up Proteomic Strategies
Authors: Beycan Ayhan , Ozel Demiralp Duygu Fatma , Dalva Klara , Beksaç Meral 
Published: October 2019

Abstract
Aim : As all other cancer types, multiple myeloma is a complex disease, yet its molecular mechanism that is not fully understood and needs to be furtherly investigated. Besides, the molecular mechanism responsible from the transition of healthy cells into malignant ones is still under investigation. In such comparative studies, human myeloma cell lines are preferred as positive control, while tonsillar plasma cells are used as negative control. The aim of this study is to perform a comparative proteomic analysis regarding the progression of Multiple Myeloma (MM) by using protein profiles of plasma cells obtained from MGUS, SMM, symptomatic myeloma patients, tonsil plasma cells obtained from healthy individuals and human myeloma cells of cell line origin to elucidate the underlying molecular mechanism involved in myeloma.
Material and Methods: Five groups were adopted in the experiment. Of these groups, MM patients were classified mainly into three groups according to marrow plasma cell content (PCC) validated by flow cytometry: group1: 0-9, group 2: 10-20 and group 3: >20 %. The fourth group consisted of human myeloma cell line (HMCL) RPMI 8226 and was designated as positive control, while tonsil plasma cells were used as negative control and specified as the fifth group. Marrow samples were collected from 30 patients newly diagnosed with Multiple Myeloma ( n: 28 symptomatic and n: 1 smoldering (SMM)) and MGUS (n: 1)).Tonsil plasma cells were isolated from healthy tonsil tissue treated with trypsin enzyme and were then confirmed by using Selection Kit microbead specific for EasySep Human CD138 marker. Protein profile maps of groups were obtained via 2D gel electrophoresis and comparative analysis and detection of up/down regulated protein spots was performed by using PDQuest 8.01 software. Proteins from differently expressed protein spots were then identified using Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI) by Peptide Mass Fingerprinting (PMF) analysis
Results: By using bottom up strategies, nine of the significantly expressed protein spots were identified with PMF analyses. The identified proteins are as follows:, Calreticulin (ERp60), Endoplasmin/ tumor rejection antigen (ERp99), MZB1(Marginal zone B and B1 cell specific protein/ pERp1), Actin cytoplasmic1 (ACTB), Thioredoxin domain-containing protein 5 (TXNDC5/ERp46), Protein disulfide isomerase (PDI) and HLA class I histocompatibility antigen (MHC class I antigen A*2). According to the results, calreticulin (which functions as chaperones and is responsible for Ca+2regulation and cell proliferation) showed a weak positive correlation with increase in patient PC content. The level was lowest in normal B cells. Endoplasmin/ tumor rejection antigen (which is assigned as chaperones and function in apoptotic process and Ca+2 regulation)declined by increase in PC content in patient samples. Level was lowest in HMCL. Marginal zone B and B1 cell specific protein (which functions as chaperones and is involved in apoptotic process, oxidoreductase activity, regulation of Ca+2, cell proliferation and activates B cell quantiity) correlated with PC percentage and was highest in HCML and lowest in normal B cells. Actin cytoplasmic1 protein (which functions in cell proliferation)synthesis showed a minimum decline in synthesis by increase in PC content .Level was highest in normal B cells. Thioredoxin domain-containing protein 5 which plays role in apoptotic process and shows oxireductase activity within MM/MGUS plasma cells also displayed a positive correlation but we were not able to detect a difference in HMCL and normal B cells which were synthesized less than the patient samples. Protein disulfide isomerase (which acts as chaperones and plays part in apoptotic process and shows oxireductase function) has a weak negative correlation with PC content and synthesis was lowest in HMCL. HLA class I histocompatibility antigen production did not correlate with PC content , it was higher in normal B cells compared to HMCL.
Conclusion: In conclusion in this study we were able to confirm our previous findings which were presented at ASH 2013 by comparing the results with normal tonsil CD138+ plasma cells and a HMCL. Our study results suggest that the proteins which are involved in Calcium metabolism, chaperone binding and oxidative stress are also associated with low vs. high proliferation profile in myeloma.

Disclosures
No relevant conflicts of interest to declare.