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

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2.1.4.5. Mass Spectrometry Mass Spectrometry (MS) is an analytic technique for the determination of the elemental composition of a molecule. Its principle basically depends on separating molecular ions according to their mass-to-charge ratio (m/z). The main purposes of MS studies are molecular mass determination, structure characterization, quantification at trace levels, and mixture analysis. In 1897, cathode ray tube experiments carried out by Sir J.J. Thomson cause the rising a new field named MS (Thomson, 1897), but it came into prominence with the exploration of soft ionization techniques. Electrospray ionization (ESI) was introduced by Fenn and his co-workers (Fenn et al., 1989) and matrix-assisted laser desorption/ionization (MALDI) was put into the service of science by Karas and Hillenkamp (Karas and Hillenkamp, 1988). MS currently plays a key role in the identification of proteins. High sensitivity (low attomole levels), rapidity, versatility, and the accuracy of the method in the field of biological science enable MS the most attractive properties of MS most comprehensive and used analytical technique. Mass spectrometer is fundamentally composed of three essential parts, namely the ionization source, the mass analyzer, and the detector (Figure 2.6). Figure 2.6. Basic Components of a Mass Spectrometer, Premier Biosoft. The ionization source is the first component of the MS. In MS analysis, the first vital step is to turn the analyte which is neutral into gas phase ionic species to be able to check their motion. If the energies higher than ionization energies, fragmentation happens. Next, ions are accelerated towards a mass analyzer, which is the second component of the MS, through a voltage difference. Then, the charged fragments of the molecular ions are separated according to their m/z ratio. The last component of MS is the detector (photomultiplier, micro-channel plate, electron multiplier) which detects the 37
ions resolved by the mass analyzer and it transforms the ion beam into a signal. Each of these three parts of mass spectrometer is under vacuum-pump systems. This high vacuum (10 -4 – 10 -8 torr) is required to allow the ions move freely in space without colliding or interacting with other species. 2.1.4.5.1. Ionization Methods Sample ionization is of great significance in mass spectrometric analysis as the compound is required to be charged and ionized. The conversion of neutral molecules into gas phase ionic species is achieved by ionization sources, so that electric and magnetic fields can be used to exert forces on charged particles in a vacuum-pump system for mass analysis. The most common techniques for ionization are matrix- assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) which are soft ionization techniques. The result of ionization, ion separation and detection is a mass spectrum that can provide structural information. 2.1.4.5.1.1. Matrix-Assisted Laser Desorption/Ionization (MALDI) Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) was first introduced by Karas and Hillenkamp in 1988 (Karas and Hillenkamp 1988) as mentioned above. MALDI is a method that allows for the ionization and transfer of a sample from a condensed phase to the gas phase. This technique has gained a wide acceptance for analysis of biological macromolecules with masses up to 500 kDa. In this approach, the sample molecules have to be co-crystallised with a high molar excess of an appropriate matrix, which is a low-molecular weight energy- absorbing organic acid, in a ratio 1:1000, respectively. The amount of matrix must be much more than analyet to both absorb all ot the radiation energy and prevent the analyte from fragmentation. 38
Page 1 and 2: CHANGES IN PROTEIN PROFILES IN BORT
Page 3 and 4: ACKNOWLEDGEMENTS First of all, with
Page 5 and 6: ÖZET BORTEZOMĠB UYGULANAN MULTĠP
Page 7 and 8: 2.1.4.1.2. Two-Dimensional Gel Elec
Page 9 and 10: LIST OF FIGURES Figure Page Figure
Page 11 and 12: LIST OF TABLES Table Page Table 1.1
Page 13 and 14: When normal cells in the body begin
Page 15 and 16: Figure 1.2. Development of Cancer S
Page 17 and 18: the myeloma cells does not help fig
Page 19 and 20: A rare form of MM called Nonsecreto
Page 21 and 22: 1.2.3. Risk Factors for MM A risk f
Page 23 and 24: of the mineralizations by adjusting
Page 25 and 26: To measure the levels of red cells,
Page 27 and 28: The levels of blood urea nitrogen (
Page 29 and 30: By using the system introduced by D
Page 31 and 32: 1.2.7.2. Biology of The Proteasome
Page 33 and 34: Figure 1.10. Ubiquitin-Proteasome P
Page 35 and 36: Figure 1.12. Effect of Bortezomib o
Page 37 and 38: encompasses the investigation of pr
Page 39 and 40: of modification. Ubiquitin is a sma
Page 41 and 42: the given cells. On the other hand,
Page 43 and 44: is-acrylamide (N,N'-methylene-bisac
Page 45 and 46: 2.1.4.2. Detection of Protein Spots
Page 47: ange of 600-2500 Da. This is the id
Page 51 and 52: 2.1.4.5.2. Mass Analyzers Figure 2.
Page 53 and 54: There is no doubt that different ma
Page 55 and 56: mask low-quantity proteins that may
Page 57 and 58: 3.1. Materials 3.1.1. Medias CHAPTE
Page 59 and 60: 3.2.5. Cell Proliferation Assay Ant
Page 61 and 62: After 72 hour incubation period at
Page 63 and 64: eceptors of the Tumor Necrosis Fact
Page 65 and 66: The preparation of stock BSA Standa
Page 67 and 68: 3.2.6.3. Detection of the Loss of M
Page 69 and 70: centrifugation, the supernatant was
Page 71 and 72: selected symmetrically to load the
Page 73 and 74: approximately 35 ml, the volume of
Page 75 and 76: After electrophoresis was finished,
Page 77 and 78: 7 Day Two: Reduction, Alkylation, a
Page 79 and 80: CHAPTER 4 RESULTS AND DISCUSSION 4.
Page 81 and 82: % Changes in Cytoplasmic/Mitochondr
Page 83 and 84: Table 4.1. Magnified Segments of Di
Page 85 and 86: Table 4.1. Magnified Segments of Di
Page 87 and 88: After labeling, black and red color
Page 89 and 90: 78 7 8 Y2S Y3S Table 4.2. Results o
Page 91 and 92: 80 12 S4Ca Table 4.2. Results of Di
Page 93 and 94: 82 27 Y5S Table 4.2. Results of Dif
Page 95 and 96: Spot 4, Ubiquitin-conjugating enzym
Page 97 and 98: is a transcriptional repressor, but
Page 99 and 100:
activity and loss of mitochondrial
Page 101 and 102:
distributed, but correlate with nat
Page 103 and 104:
DeMartino, G. N. and Slaughter, C.
Page 105 and 106:
proteomic analyses of a systematica
Page 107 and 108:
Mahindraa, A., Hideshimab, T. and A
Page 109 and 110:
Richardson, P. G., Hideshima, T. an
Page 111 and 112:
Terpos, E. 2008. Bortezomib Directl
Page 113 and 114:
A.1. RPMI-1640 Growth Medium APPEND
Page 115 and 116:
Table B.1. Chemicals and Reagents U
Page 117 and 118:
B.4.3. Standard Curve for BSA Table
Page 119 and 120:
Urea (8M) 4.8 g CHAPS (%2) 0.2 g DT
Page 121 and 122:
E. F. G. STOCK REAGENT PREPARATION
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