Target Discovery and Validation Reviews and Protocols

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Immunoproteomics 329 by “shot gun” analysis by using tandem mass spectrometry (MS/MS). In both methods, spectra are used for database searches, leading to protein identification. It should be noted that MS/MS mass spectrometry allows the identification of a protein from a single peptide. Indeed, after fragmenting that peptide, the second spectrum (MS/MS or MS 2 ) is used for determining its sequence or a sequence tag. Recent advances in proteomics have been realized by coupling liquid chromatography with mass spectrometry (LC-MS/MS). LC-MS/MS is a powerful tool for analyzing the composition of complex mixtures (12). 3. 2D Gel-Based Western Blot Approach 3.1. Strategy A proteomic-based approach, using 2DE followed by Western blot experiments with sera from cancer patients, has enabled the identification of tumor-associated antigens that elicit a humoral response. The methodology to identify proteins eliciting humoral response in cancer patients involves two essential steps: first, the screening of autoantigens after serological analysis; and second, the identification of the target molecule. The use of 2D-PAGE allows separating simultaneously several thousand individual cellular proteins from tumor tissue or tumor cell lines. Separated proteins are transferred onto membranes. Sera from cancer patients obtained at time of diagnosis are used as primary antibody for Western blot analysis and are screened individually for antibodies that react against separated proteins. The comparison of series of Western blot obtained with sera from cancer patients with those performed with sera from healthy individuals led to determination of the occurrence of relevant autoantigens. An important advantage of this methodology is that isoforms can be separately analyzed. Notably, some modifications, such as glycosylation, can be immunogenic. For identification, the recurrent protein spots that specifically react with sera from cancer patients are located on silver-stained 2D gels after superimposition with the blot. The proteins of interest are extracted from the gel and identified by mass spectrometric analysis. To confirm the identification of TAAs, a validation step is usually necessary. This step can be performed using classical methods such as Western blot experiments when antibodies are available. The relevance to cancer in terms of expression and location may be investigated using ELISA, immunohistochemistry, or by other techniques (Fig. 1). 3.2. Practical and Technical Aspects 2D gels are performed with 100–200 µg of proteins from whole cell extracts of cell lines or biopsies (see Subheading 6.). The first dimension can be realized using different pH gradients on different sizes of strips. A cocktail of 7 M urea, 2 M thiourea, and 2% CHAPS can be used as a regular buffer for
330 Le Naour Fig. 1. To illustrate the proteome-based approach for targeting autoantigens in cancer, an example is given from a study on breast cancer (15). The proteins from a cancer cell line (SUM-44) were solubilized and separated by two-dimensional electrophoresis (2DE). Proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes. The screening of autoantigens was performed by Western blot using sera from cancer patients or healthy individuals as the primary antibody. The comparison of the patterns allowed visualization of specific protein spots in cancer. In our example, three protein spots were specifically reactive (white arrows). For identification, the protein spots were located on the gel after staining of the proteins and superimposition with the blot. The proteins of interest were cut out and in-gel digested. The resulting peptides were analyzed by mass spectrometry. The spectra were
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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182 Houdebine Fig.7. Example of a v
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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192 Houdebine interference of the g
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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206 Vijayaraj, Söhl, and Magin Fig
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208 Vijayaraj, Söhl, and Magin fil
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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270 Iversen and Sørensen witnessed
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14 An Overview of the Immune System
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Page 332 and 333: 15 Potential Target Antigens for Im
Page 334 and 335: Tumor Antigen Discovery 321 develop
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Page 344 and 345: Immunoproteomics 331 isoelectric fo
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Page 356 and 357: Protein Arrays 343 The ideal proteo
Page 358 and 359: Protein Arrays 345 18. Cekaite, H.
Page 360 and 361: Protein Arrays 347 49. Yuko Kawahas
Page 362 and 363: Index 349 Index A Acetyl-CoA carbox
Page 364 and 365: Index 351 conditional by using FRT
Page 366 and 367: Index 353 Recombinant tumor antigen
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