Thesis final - after defense-7 - Jacobs University

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Chapter 4 4. Conclusions and Remarks 4.1. General conclusions and remarks This thesis explored for the first time the comparative analysis of hydrophobic ligands and base supports in hydrophobic interaction chromatography (HIC) as a function of complex cell proteome. Several protein properties were examined for their correlation with the protein retention behavior in HIC. The protein properties were also explored to determine their potential to differentiate among the ligands and base supports. The previous studies of HIC were based on the retention time of model proteins. Hence, a process proteomics approach was applied to explore the hidden underlying parameters in HIC. Although very laborious and experimentally demanding, the present approach has led to conclusions which are valid for a complex cell proteome and which refers to two families of hydrophobic adsorbents. This work has produced a first draft of the experimental data which can be used to predict the elution position of any target protein utilizing an in silico approach. 4.2. Influence of different ligands • The hydrophobicity of the ligands was comparatively investigated in the previous reports exploiting the retention time of model proteins. However, the hydrophobicity of the ligands has been investigated for the first time with the yeast cell proteome utilizing several protein properties. The protein properties have not fully differentiated among the ligands. This confirmed that protein properties have limited ability to differentiate among the ligands for their hydrophobicity. 4.3. Influence of different base supports • The influence of the base supports has been investigated for the first time with the proteome wide approach utilizing different protein properties. The results revealed that protein properties have limited ability to differentiate among the base supports. 113
Chapter 4 • The base supports revealed less difference at high salt concentrations than at low salt concentrations. In other words, the differences among the base supports were less at high hydrophobic conditions than at low hydrophobic conditions. The reason could be the effects of mobile phase hydrophobic conditions. 4.4. Effect of protein properties on separation behavior during chromatography • Several protein properties were investigated for the first time with the proteome wide approach for their effects on the protein retention behavior during HIC. • The average surface hydrophobicity (ASH), average hydrophobicity (AH) and average flexibility (AF) have revealed direct relationships with the retention behavior of the proteins in HIC. • The average bulkiness of the proteins did not show any relationship with the retention behavior during chromatography. • The polarity of the proteins exhibited an inverse relationship with the retention of the proteins. The higher the polarity of the protein, the less time it will stay on the adsorbent during chromatography and vice versa. • An indirect relationship was observed between polarity and hydrophobicity; proteins with high polarity were less hydrophobic in nature and vice versa. • An indirect relationship was also observed between polarity and flexibility; proteins with high polarity were less flexible in nature and vice versa. • Among all the properties, ASH was the only property which has a decisive role in the retention behavior of the proteins during HIC. • The salt and ASH ranges were defined for the first time during the chromatographic fractionation of the yeast cell proteome. The information gathered about ASH and the retention position during chromatography can be exploited for the chromatographic separation of recombinant proteins from host cell proteomes. 114
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A proteome wide approach to underst
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I also want to mention the efforts
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Dedication To my father Haji Sher A
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Table of Contents 1. Introduction .
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3.3.2.2. Protein distributions on 2
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Chapter 1 1. Introduction 1.1. Intr
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Chapter 1 cell proteome to facilita
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Chapter 1 proteins. It is this diff
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Chapter 1 Salt conc. decreasing (Gr
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Chapter 1 The ions at the start of
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Chapter 1 exposure of hydrophobic r
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Chapter 1 based on the surface hydr
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Chapter 1 1.5. Analysis of the yeas
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Chapter 1 molecular weight impuriti
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Chapter 1 followed by its introduct
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Chapter 1 protein towards purificat
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Chapter 2 EXPERIMENTAL APPROACH
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Chapter 2 Yeast cell proteome and c
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Chapter 2 ligands (Toyopearl-Hexyl,
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Chapter 2 number of spots was count
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Chapter 2 (http://www.matrixscience
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Chapter 3 RESULTS AND DISCUSSION
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Chapter 3 to the retention behavior
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Chapter 3 investigate the hydrophob
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Chapter 3 the interior of the prote
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Chapter 3 A B C Figure 9: The cryst
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Chapter 3 of the comparative analys
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Chapter 3 Figure 11: Represent the
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Chapter 3 Ether, some 2-D gels were
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Chapter 3 Figure 13: Represent 2-D
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Chapter 3 Table 5. List of the iden
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Chapter 3 Table 7. List of the iden
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Chapter 3 amino acids by Cowan-Whit
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Chapter 3 Figure 15 C: Represents t
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Chapter 3 Figure 16 A: Represents t
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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
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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
Page 93 and 94: Chapter 3 Table 14. List of the ide
Page 95 and 96: Chapter 3 r 2 values from the data
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Page 111 and 112: Chapter 3 3.3. Protein distribution
Page 113 and 114: Chapter 3 3.3.2.2. Protein distribu
Page 115 and 116: Chapter 3 Figure 29 B: Represent th
Page 117 and 118: Chapter 3 When the number of smalle
Page 119 and 120: Chapter 3 and hydrophobic interacti
Page 121: Chapter 4 CONCLUSIONS
Page 125 and 126: Chapter 4 additional understanding
Page 127 and 128: References 13. Lienqueo, M.E.; Lese
Page 129 and 130: References 32. Salgado, J.C.; Rapap
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Page 133 and 134: References 71. Xindu, G. and Regnie
Page 135 and 136: References 91. Alonso-Orgaz, S.; Mo
Page 137 and 138: References 112. Gu, Z. and Glatz, C
Page 139: References 132. Reubsaet, J.L.E. an
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Thesis final - after defense-7 - Jacobs University

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