Thesis final - after defense-7 - Jacobs University

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Chapter 2 Table 2: The in-gel digestion protocol optimized specifically for the proteins fractionated by HIC Steps Description 1. Washing the gels The gel pieces were washed overnight, with 50% (v/v) methanol and 5% (v/v) acetic acids for the removal of salts and other impurities. 2. De-staining After washing, the gel pieces were incubated in 200 µl of 100 mM ammonium carbonate/acetonitrile (1:1 v/v) for 30 minutes to destain the gels. 3. Reduction After destaining, 50 µl of 10 mM DDT dissolved in 100 mM ammonium carbonate were added to gel pieces and incubated for 45 minutes at 56°C to reduce the disulfide bridges. 4. Alkylation Then 100 µl of 55 mM iodoacetamide was added to samples and incubated for 20 minutes in the dark to inhibit the reformation of disulfide bonds and alkylate the free cysteins. 5. Trypsin digestion Then 10 µl of trypsin and 90 µl of 25 mM ammonium bicarbonate in 9% acetonitrile solution were added to each sample and incubated in ice for 3 hours to absorb maximum enzymes. The samples were then incubated for 16 hours at 37°C in the digestion buffer for protein digestion. 6. Extraction of peptides 7. Concentration of the peptide extract a. 200 µl of Milli Q water was added to each sample and incubated for 15 minutes at 37°C to extract hydrophilic peptides. b. 200 µl of extraction buffer (1:2 v/v 5% formic acid/acetonitrile) was added to each sample and incubated for 15 minutes at 37°C to extract hydrophobic peptides. The peptide extract was concentrated and 15 µl of 0.5% v/v TFA was added to each sample, followed by centrifugation at 10,000 rpm for 10 minutes. The samples were then ready for identification by MALDI-ToF-MS. After digestion, the sample was collected as supernatant and 3 µl of the sample was spotted on the MALDI target plate followed by 0.5% TFA for washing. The sample was air dried at room temperature. All mass spectra were calibrated using peptides from the auto digestion of trypsin. The peak lists were searched using the Mascot search engine 30
Chapter 2 (http://www.matrixscience.com) against NCBI and SwissProt databases. The search parameters were carbamidomethylation and methionine oxidation entered as variable modifications and 1 missed trypsin cleavage site. In all protein identifications, the scores were greater than the score fixed by Mascot as significant with a p value < 0.05 (91). The identified proteins were further characterized and analyzed with computational tools. 2.2.8. Characterization of proteins utilizing Bioinformics The same procedure has been followed for the calculation of ASH as described by J.C. Salgado et al utilizing the Cowan-Whittaker scale (32, 92). Average surface hydrophobicity (ASH) of the proteins was calculated by writing a package in MATLAB, where it was mandatory to use the three-dimensional structure of a protein collected from protein databank (PDB, http://www.rcsb.org/pdb). STRIDE was used to generate a text file, which can determine the accessible surface area (ASA) of each amino acid at a particular position. After calculating the ASA, the ASH was measured by the package in MATLAB, programmed with the normalized values of Cowan-Whittaker hydrophobicity scale. According to this method, the ASH can be calculated by equation 1 as follows; Φ = ˆ rφ (1) suface ∑ i i∈A i Where Φ surface is the ASH of a protein, A is the collection of the 20 possible amino acids and ( φ i ) is the hydrophobicity of the amino acids of type i. The hydrophobicity of each amino acid was assigned according to Cowan-Whittaker scale. The fraction of superficial area ( r ∧ i) occupied by the amino acid i can be calculated by equation 2 as follows; 31
Page 1 and 2: A proteome wide approach to underst
Page 3 and 4: I also want to mention the efforts
Page 5 and 6: Dedication To my father Haji Sher A
Page 7 and 8: Table of Contents 1. Introduction .
Page 9 and 10: 3.3.2.2. Protein distributions on 2
Page 11 and 12: Chapter 1 1. Introduction 1.1. Intr
Page 13 and 14: Chapter 1 cell proteome to facilita
Page 15 and 16: Chapter 1 proteins. It is this diff
Page 17 and 18: Chapter 1 Salt conc. decreasing (Gr
Page 19 and 20: Chapter 1 The ions at the start of
Page 21 and 22: Chapter 1 exposure of hydrophobic r
Page 23 and 24: Chapter 1 based on the surface hydr
Page 25 and 26: Chapter 1 1.5. Analysis of the yeas
Page 27 and 28: Chapter 1 molecular weight impuriti
Page 29 and 30: Chapter 1 followed by its introduct
Page 31 and 32: Chapter 1 protein towards purificat
Page 33 and 34: Chapter 2 EXPERIMENTAL APPROACH
Page 35 and 36: Chapter 2 Yeast cell proteome and c
Page 37 and 38: Chapter 2 ligands (Toyopearl-Hexyl,
Page 39: Chapter 2 number of spots was count
Page 43 and 44: Chapter 3 RESULTS AND DISCUSSION
Page 45 and 46: Chapter 3 to the retention behavior
Page 47 and 48: Chapter 3 investigate the hydrophob
Page 49 and 50: Chapter 3 the interior of the prote
Page 51 and 52: Chapter 3 A B C Figure 9: The cryst
Page 53 and 54: Chapter 3 of the comparative analys
Page 55 and 56: Chapter 3 Figure 11: Represent the
Page 57 and 58: Chapter 3 Ether, some 2-D gels were
Page 59 and 60: Chapter 3 Figure 13: Represent 2-D
Page 61 and 62: Chapter 3 Table 5. List of the iden
Page 63 and 64: Chapter 3 Table 7. List of the iden
Page 65 and 66: Chapter 3 amino acids by Cowan-Whit
Page 67 and 68: Chapter 3 Figure 15 C: Represents t
Page 69 and 70: Chapter 3 Figure 16 A: Represents t
Page 71 and 72: Chapter 3 The polarity value for th
Page 73 and 74: Chapter 3 Figure 17: Represents the
Page 75 and 76: Chapter 3 Figure 18: Represents the
Page 77 and 78: Chapter 3 published papers with mod
Page 79 and 80: Chapter 3 ligands revealed less dif
Page 81 and 82: Chapter 3 3.2. Influence of the dif
Page 83 and 84: Chapter 3 Table 10: The chemistry o
Page 85 and 86: Chapter 3 where approximately 50% o
Page 87 and 88: Chapter 3 3.2.3.2. The electrophore
Page 89 and 90: Chapter 3 Figure 22: Represent 2-D
Page 91 and 92:
Chapter 3 Table 12. List of the ide
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Chapter 3 Table 14. List of the ide
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Chapter 3 r 2 values from the data
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Chapter 3 Figure 24 C: Represents t
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Chapter 3 approximately 47%. The me
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Chapter 3 Figure 25 B: Represents t
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Chapter 3 values have been reported
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Chapter 3 towards conformational ch
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Chapter 3 Figure 28: Represents the
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Chapter 3 among the adsorbents were
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Chapter 3 3.3. Protein distribution
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Chapter 3 3.3.2.2. Protein distribu
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Chapter 3 Figure 29 B: Represent th
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Chapter 3 When the number of smalle
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Chapter 3 and hydrophobic interacti
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Chapter 4 CONCLUSIONS
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Chapter 4 • The base supports rev
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Chapter 4 additional understanding
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References 13. Lienqueo, M.E.; Lese
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References 32. Salgado, J.C.; Rapap
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References 52. B.H.J., H. Accessibl
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References 71. Xindu, G. and Regnie
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References 91. Alonso-Orgaz, S.; Mo
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References 112. Gu, Z. and Glatz, C
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References 132. Reubsaet, J.L.E. an
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Thesis final - after defense-7 - Jacobs University

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