Thesis final - after defense-7 - Jacobs University

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Chapter 1 factors were mostly studied with model proteins; however their applicability to a natural host still needs to be explored. 1.4.2. Effect of stationary phases in HIC Stationary phases are usually composed of small hydrophobic groups (e.g. butyl, phenyl and hexyl) attached to the hydrophilic polymer base supports. The different types of stationary phases in HIC differ in the degree of ligand substitutions on the base support. The base supports can also differ in the chemical nature and particle size (51). The availability of the hydrophobic adsorbents is increasing in the market, due to the increasing applications of HIC as a purification method. The adsorbents determine the protein adsorption selectivity in HIC according to the nature of ligands and base supports. The straight chain alkyl ligands were reported for pure hydrophobic character while aryl ligands (phenyl) showed mixed mode behavior due to the presence of aromatic interactions in addition to the hydrophobic interactions (52-55). The polyether’s ligands such as polyethylene glycols and polypropylene glycols were also reported for their milder hydrophobic nature than alkyl ligands (56). The retention of cytochrome c, ribonuclease A, lysozyme and ovalbumin has been reported on polyvinyl alcohol, oligoamino alcohol, and polyoxyethylene silica supports in descending gradient of ammonium sulfate buffer. It has been observed that hydrophobic interactions between proteins and adsorbents increased with increasing chain lengths of the ligand (55, 57). However, resolution of the chromatography decreased when ligands of the higher chain lengths have been used (51). The effects of adsorbent hydrophobicity on the conformation of proteins were also reported. The elution profiles of lactalbumin have shown different behavior, when separated by polyether stationary phases having methyl and ethyl terminating groups. The lactalbumin has given one and two peaks for the adsorbents with methyl and ethyl ligands, respectively. The first peak was the native one while the second peak was the denatured peak of the lactalbumin. The second peak has been retained longer due to the 10
Chapter 1 exposure of hydrophobic residues usually buried in the interior of proteins. Other protein such as lysozyme when applied has shown single peak on both adsorbents due to the stable nature of the protein (46). The charged type HIC adsorbents have shown different behavior during chromatographic separations. Ionic interactions will occur between proteins and adsorbents in the presence of charges, which favor the use of charge free adsorbents in HIC. The choice of ligand type is hard to interpret and could be established case to case basis depending on the biomaterials to be separated (53, 54). The retention behavior of the proteins can be changed by adjusting the ligand substitution on the base support. The protein binding capacity has been increased by an increase in the substitution degree of the ligand. Once the degree of substitution reaches beyond the sufficient level, the binding capacity of proteins remains constant, although the strength of interaction increases. At very high substitution of the ligands, multipoint attachments occur between proteins and adsorbents resulting in stronger binding. Thus harsh conditions are required to elute the product which may also increase the chances of protein denaturation. Some studies have preferred low ligand density in case of the unstable proteins to preserve the native structures of proteins (58-60). There are different types of base supports such as hydrophilic polysaccharide gels (e.g., agarose and dextran), synthetic polymers (polymethacrylate and polystyrene divinyl benzene) or modified silica gel commonly used in HIC. It has been recently investigated with model proteins, that the adsorbent chemistry and up to some extent its particle size have effects on the retention behavior during chromatography (61, 62). The total number of water molecules released upon adsorption was found proportional to the hydrophobic area of the adsorbent (44). All the above findings were based on the retention data of model proteins and have been never investigated with the process proteomics approach. 11
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: Chapter 1 The ions at the start of
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 and 40: Chapter 2 number of spots was count
Page 41 and 42: Chapter 2 (http://www.matrixscience
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
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Chapter 3 Figure 17: Represents the
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Page 77 and 78:
Chapter 3 published papers with mod
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Chapter 3 ligands revealed less dif
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Chapter 3 3.2. Influence of the dif
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Chapter 3 Table 10: The chemistry o
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Chapter 3 where approximately 50% o
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Chapter 3 3.2.3.2. The electrophore
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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 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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