Peptide Mapping Proteomics ZipChip...
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1 ZipChip Applications Proteomics & Peptide Mapping Bottom-up Applications Peptide Mapping Proteomics
Transcript of Peptide Mapping Proteomics ZipChip...
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ZipChip Applications
Proteomics & Peptide Mapping Bottom-up Applications
Peptide Mapping
Proteomics
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// Peptide Mapping
The ZipChip Advantage
• Fast (≥5x reduction in run time)
• Performs well with very polar or very
hydrophobic peptides
• Sample stacking techniques improve
sensitivity
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// Peptide Mapping of mAb
0.4 mg/mL Sigma mAb tryptic digest (2 ng injected) ~600 V/cm Thermo QE HF Biopharma
• ZipChip separation of tryptic peptides
• Complete in ~15 minutes
• Peak capacity of ~130 (FWHH)
Data courtesy of Ben Orsburn
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// Peptide Mapping of mAb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Q S A L T Q P R S V S G S P G Q S V T I S C T G T S S D I G G Y N F V S W Y Q Q H P G K A P K L M I
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
Y D A T K R P S G V P D R F S G S K S G N T A S L T I S G L Q A E D E A D Y Y C C S Y A G D Y T P G
101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
V V F G G G T K L T V L G Q P K A A P S V T L F P P S S E E L Q A N K A T L V C L I S D F Y P G A V
151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200
T V A W K A D S S P V K A G V E T T T P S K Q S N N K Y A A S S Y L S L T P E Q W K S H R S Y S C Q
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218
V T H E G S T V E K T V A P T E C S
SiluLite_heavy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
E V Q L V E S G G G L V Q P G G S L R L S C V A S G F T L N N Y D M H W V R Q G I G K G L E W V S K
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
I G T A G D R Y Y A G S V K G R F T I S R E N A K D S L Y L Q M N S L R V G D A A V Y Y C A R G A G
101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
R W A P L G A F D I W G Q G T M V T V S S A S T K G P S V F P L A P S S K S T S G G T A A L G C L V
151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200
K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q4.6
4.3
201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250
T Y I C N V N H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L F P P K
251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y
301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350
N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P
351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400
Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450
V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G
Colorcodeforsignalintensity
4.9 2.7
>4.7e+006 >9.2e+005 >1.8e+005 >3.6e+004 >7.0e+003
3.3 4
5.4 2.93.1
4
3
4 5.44 4.2
4.6 4.2 6.2
4.9 4.64.7
4.64
3.3
4 3.2 46.5 3.6 4
3.9 5 4.64.1 3.5
4.6
3.6 42.8 5.3 4.7 6.5
3.93.83.4
2.8
3.6 3.9 4.3
4.4 4.64.34.44.34.2
4.4 3.94.8 4.64.5 5.8
3.4
5 3.64 4.8 2.8
4.34.44.3
4.23.93.8
7.1 4.46.3 4.8
4.54.4
5.7 4.15.5
57.9 4
4.5 4.3 4.66.2 4.4 4.3
7.8 4.44.3 6.5
3.7 4
3.93.4
3.5 2.8 4.7 4.5
3.75.3 5.3
8.9 2.98
4.1
3.84.1
3.5
5.6 4.1
5 5.2 4.14.9 3.5
4.7
5.9 55.2
5.8 4.1 4.2 3.8
3 5.92.9 5.2
8.2 3.8 5.6 5.8
3.43.33.1
4 2.8 8.2
6.9 4.5 45.4
4.84.5
• Nearly maximum theoretical coverage
• Many sequences are far too small to detect
with Trypsin K/R fragments
Data courtesy of Ben Orsburn
Sequence Coverage Map
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// Resolution of Oxidized and Deamidated Peptides 6.82
6.58 7.84 7.18
7.61 6.99 6.41 8.07 8.40 7.41 9.69 10.03
9.36 10.45 8.69 11.23 8.91
8.40
NL: 3.58E8
NL: 3.24E8
8.54 NL: 8.60E6
20 40 60 80 100 8.67
RT (min)
NL: 6.11E6 DSLYLQMNSLR N83 deamidation
DSLYLQMNSLR M82 oxidation
0
20 40 60 80 100
0
20 40 60 80 100
0
20 40 60 80 100
0
• Oxidized and deamidated peptides
• Resolved with ZipChip prior to MS analysis
Data courtesy of Ben Orsburn
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// Proteomics
The ZipChip Advantage:
• Fast analysis times allow rapid generation of
replicates
• Combine multiple runs for data processing
• Sharp peaks from ZipChip separation minimize
isolation interference
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// ZipChip Bottom-up Proteomics
Sigma Yeast Digest ZipChip HR ZipChip Peptide Kit ~600 V/cm QE HF Biopharma
Data courtesy of Ben Orsburn
Triplicates
2x injection volume
• DDA analysis
• Excellent reproducibility between triplicate runs
• Triplicate runs and 1 run at 2x injection volume
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// ZipChip Bottom-up Proteomics
Data processing: Uniprot, Swissprot databases, SequestHT, Percolator
Processing results in good agreement between 3 replicate runs
Data courtesy of Ben Orsburn
Processing results, 4 runs: Proteins
• 752 high confidence • 89 medium confidence
Peptides • 2549 high confidence • 919 medium confidence
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