Target Discovery and Validation Reviews and Protocols

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Pancreatic Cancer 81 in many ways. However, two different strategies are frequently used for separation of peptides. One setup uses two columns in tandem (a precolumn followed by an analytical column), whereas the second setup only uses a single analytical column. The tandem column setup is preferred when large volumes (10–40 µL) have to be analyzed or if the samples need to be desalted or washed extensively. This setup is very robust and can be used for most types of samples. One drawback is that one might loose sensitivity because of broader chromatographic peaks. Several column materials can be used for precolumns (e.g., YMC ODS-A, 5- to 15-µm beads; Kanamatzu USA Inc., New York, NY) and analytical columns (e.g., MS218, 5-µm beads; Vydac) and it is therefore strongly suggested that several trial runs including different reverse phase materials are tested for obtaining optimal separation and resolution during the LC-MS/MS analysis. The following sections describe a general strategy for building and packing reverse phase columns used for nano-LC-MS/MS analysis in addition to some general chromatographic guidelines (LC program, length of gradient, and context of mobile phase). 3.3.9. Preparation of Reverse Phase Column (Fig. 2) 1. The column is packed in a fused silica capillary tubing (375 µm od and 75 µm id) (52). A “frit” restrictor has to be generated inside the fused silica to block the reverse phase material during packing (the silica tubing is open in both ends). Mix 44 µL of Kvasil 1 with 8 µL of formamide and vortex for 1–3 min (the solution becomes viscous). 2. One end of a 20- to 25-cm fused silica is dipped in the solution for 1 to 2 s (the solution will move upward into the fused silica by capillary action). Excess solution is wiped off and the fused silica is left at room temperature o.n. to polymerize. The frit is checked under a microscope and should be approx 2–5 mm in lengths and has to present a V-shaped cone pointing outward. The frit can be cut if it is too long. 3. Insert the fused silica into a pressure vessel and wash it with 100% methanol. This process ensures that solvent can flow through the frit and that when pressure is applied, the frit is still affixed to the capillary. The reverse phase material (~4 mg) is placed in a glass microvial and resuspended in 500 µL of methanol. 4. To keep the column material in suspension a small magnetic stirring rod is added to the glass microvial before placing it in the pressure vessel. Place the pressure vessel on a stir plate and turn the magnetic stirrer at low speed. Turn the gas (He) pressure to 50 bar (800 psi), which initiates the packing of the column. The column should be 5–10 cm for precolumns and 15–20 cm for analytical columns. 5. The fused silica is connected to the LC system and flushed with mobile phase B (90% acetonitrile, 0.4% acetic acid, and 0.005% heptafluorobutyric acid in water) for 30 min and subsequently 30 min with mobile phase A (H2O with 0.4% acetic acid and 0.005% heptafluorobutyric [v/v]) to equilibrate the column.
82 Beaty et al. Fig. 2. Preparation of a reverse phase column for nano-LC-MS/MS analysis. A frit restrictor composed of a mix of Kvasil 1 and formamide is generated in one end of the fused silica capillary tubing (375 µm od and 75 µm id) to block the reverse phase material during packing. The frit restrictor should present a V-shaped cone and be 2–5 mm in length (A). The fused silica tubing with the frit restrictor is subsequently placed in small pressure vessel (frit restrictor pointing outward from the pressure vessel) containing a slurry of reverse phase material (B). The column is packed by applying a pressure (He) of 50 bar (800 psi) in the pressure vessel. The column should be approx 15–20 cm in length. 6. The performance of the generated reverse phase column is tested with several trial runs by a tryptic digest of a standard protein (e.g., 100–200 fmol of BSA). Elution time for the individual peaks in the chromatogram should be approx 15–20 s with 100 fmol of BSA and produce sequence coverage between 50 and 60% with a Mascot protein score of approx 1500 (see Subheading 3.3.11.).
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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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Page 65 and 66: 52 Imoto et al. Table 3 List of Roo
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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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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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214 Vijayaraj, Söhl, and Magin ulc
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216 Vijayaraj, Söhl, and Magin of
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218 Vijayaraj, Söhl, and Magin sug
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220 Vijayaraj, Söhl, and Magin abs
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222 Vijayaraj, Söhl, and Magin 1.2
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224 Vijayaraj, Söhl, and Magin Fig
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226 Vijayaraj, Söhl, and Magin gen
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228 Vijayaraj, Söhl, and Magin the
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230 Vijayaraj, Söhl, and Magin f.
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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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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272 Iversen and Sørensen Fig. 1. P
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274 Iversen and Sørensen the core
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 281 Fig.
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Overview of Immune System 283 then
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Overview of Immune System 285 expre
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Overview of Immune System 287 Once
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Overview of Immune System 289 solub
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Overview of Immune System 291 amino
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Overview of Immune System 293 (unre
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Overview of Immune System 297 the c
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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