Table I.
Results of iTRAQ analysis
| Rank | Protein ID | Protein name | Previously identified? |
Established CCV protein? |
Mascot score | Peptides quantified |
Control/mock ratio | SD | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Top 53 proteins | ||||||||||||||||
| 1 | gi|30582397 | AP-1 σ1A | ✓ | ✓ | 96 | 1 | 12.24 | 5.13 | ||||||||
| 2 | gi|32451593 | CHC17 | ✓ | ✓ | 1826 | 30 | 6.27 | 1.46 | ||||||||
| 3 | gi|30582933 | DNase II (lysosomal) | new | new | 63 | 1 | 5.01 | 1.09 | ||||||||
| 4 | gi|1363934 | Dynamin-2 | new | ✓ | 569 | 11 | 4.83 | 1.31 | ||||||||
| 5 | gi|41016993 | EpsinR/Enthoprotin | ✓ | ✓ | 478 | 10 | 4.59 | 1.31 | ||||||||
| 6 | gi|17028334 | AP-1 μ1A | ✓ | ✓ | 379 | 8 | 4.59 | 1.08 | ||||||||
| 7 | gi|56203491 | TPD52L1 | new | new | 104 | 1 | 4.45 | 1.24 | ||||||||
| 8 | gi|12643391 | AP-1 γ | ✓ | ✓ | 540 | 18 | 4.37 | 1.05 | ||||||||
| 9 | gi|3294548 | Cathepsin Z (lysosomal) |
new | new | 102 | 2 | 4.10 | 1.19 | ||||||||
| 10 | gi|38570101 | Unknown protein (RAB-GAP domain) |
new | new | 45 | 3 | 4.05 | 0.98 | ||||||||
| 11 | gi|1335854 | Clathrin heavy chain homologue(CHC22) |
✓ | new | 558 | 1 | 3.84 | 1.09 | ||||||||
| 12 | gi|17402231 | Clathrin light chain a | ✓ | ✓ | 231 | 9 | 3.81 | 1.02 | ||||||||
| 13 | gi|2143260 | PI 3-kinase C2α | ✓ | ✓ | 822 | 11 | 3.62 | 1.03 | ||||||||
| 14 | gi|55662275 | CI-Mannose 6- phosphate receptor |
✓ | ✓ | 668 | 14 | 3.53 | 0.85 | ||||||||
| 15 | gi|70608172 | TPD52 | new | new | 160 | 1 | 3.52 | 0.85 | ||||||||
| 16 | gi|25090897 | CALM | ✓ | ✓ | 258 | 5 | 3.43 | 0.94 | ||||||||
| 17 | gi|21903712 | Carboxypeptidase D | ✓ | ✓ | 406 | 7 | 3.41 | 0.93 | ||||||||
| 18 | gi|116505 | Clathrin light chain b | ✓ | ✓ | 171 | 9 | 3.39 | 0.77 | ||||||||
| 19 | gi|2226273 | TGN46 | new | ✓ | 163 | 1 | 3.30 | 0.75 | ||||||||
| 20 | gi|33150596 | AP-1 σ1B | new | ✓ | 49 | 1 | 3.20 | 0.74 | ||||||||
| 21 | gi|55663531 | Syntaxin 6 | ✓ | new | 121 | 1 | 3.15 | 0.78 | ||||||||
| 22 | gi|13477131 | Sorting nexin 9 | new | ✓ | 260 | 6 | 3.07 | 0.79 | ||||||||
| 23 | gi|56205909 | Rab4A | ✓ | ✓ | 319 | 4 | 2.78 | 0.71 | ||||||||
| 24 | gi|62287155 | NECAP-1 | ✓ | ✓ | 66 | 1 | 2.75 | 0.86 | ||||||||
| 25 | gi|182516 | Ferritin light subunit | ✓ | ✓ | 131 | 3 | 2.67 | 0.57 | ||||||||
| 26 | gi|14043007 | AP-1 β1 | ✓ | ✓ | 1020 | 8 | 2.60 | 0.73 | ||||||||
| 27 | gi|2827434 | Sorting nexin 2 | new | new | 403 | 4 | 2.58 | 0.54 | ||||||||
| 28 | gi|9716092 | Sortilin | new | ✓ | 125 | 2 | 2.53 | 0.77 | ||||||||
| 29 | gi|66932909 | Pumilio 1 | RNA binding | — | 105 | 1 | 2.46 | 0.74 | ||||||||
| 30 | gi|8546849 | CD-Mannose 6-phosphate receptor |
✓ | ✓ | 152 | 5 | 2.44 | 0.63 | ||||||||
| 31 | gi|67477390 | Inositolpolyphosphate 5-phosphatase OCRL-1 |
new | ✓ | 581 | 8 | 2.40 | 0.61 | ||||||||
| 32 | gi|2337920 | Syntaxin 7 | ✓ | new | 220 | 3 | 2.38 | 0.61 | ||||||||
| 33 | gi|47125326 | Ferritin heavy chain | ✓ | ✓ | 84 | 5 | 2.37 | 0.77 | ||||||||
| 34 | gi|55958410 | Argininosuccinate synthetase | metabolism | — | 176 | 5 | 2.37 | 0.62 | ||||||||
| 35 | gi|8922952 | Cappuccino | new | new | 73 | 1 | 2.36 | 0.79 | ||||||||
| 36 | gi|47086495 | BLOC-1, subunit 3 | new | new | 96 | 3 | 2.35 | 0.58 | ||||||||
| 37 | gi|56205243 | Auxilin | ✓ | ✓ | 150 | 3 | 2.33 | 0.63 | ||||||||
| 38 | gi|7920147 | NSF | ✓ | ✓ | 63 | 1 | 2.33 | 0.64 | ||||||||
| 39 | gi|4557469 | AP-2 β2 | ✓ | ✓ | 831 | 7 | 2.32 | 0.66 | ||||||||
| 40 | gi|57162630 | AP-3 μ3A | new | new | 90 | 1 | 2.31 | 0.46 | ||||||||
| 41 | gi|15489411 | AP-2 μ2 | ✓ | ✓ | 121 | 4 | 2.31 | 0.67 | ||||||||
| 42 | gi|54695838 | Rab5C | ✓ | ✓ | 311 | 5 | 2.29 | 0.55 | ||||||||
| 43 | gi|62751805 | D19 | new | new | 88 | 2 | 2.29 | 0.47 | ||||||||
| 44 | gi|15214676 | Unknown protein (putative Rab interactor) |
new | new | 62 | 1 | 2.28 | 0.50 | ||||||||
| 45 | gi|4433649 | Syntaxin 8 | ✓ | new | 122 | 2 | 2.21 | 0.52 | ||||||||
| 46 | gi|13543973 | IMP (inosine monophosphate dehydrogenase) 2 | metabolism | — | 510 | 15 | 2.17 | 0.55 | ||||||||
| 47 | gi|9557955 | Sorting nexin 5 | new | new | 206 | 3 | 2.15 | 0.61 | ||||||||
| 48 | gi|17375734 | cyclin G–associated kinase/auxilin2 |
✓ | ✓ | 288 | 3 | 2.12 | 0.64 | ||||||||
| 49 | gi|15214696 | Glucosamine (N-acetyl)- 6-sulfatase (lysosomal) |
new | new | 162 | 4 | 2.11 | 0.58 | ||||||||
| 50 | gi|13431563 | Huntingtin-interacting protein 1 related (Hip1R) |
✓ | ✓ | 210 | 1 | 2.09 | 0.81 | ||||||||
| 51 | gi|1184699 | tyrosyl-tRNA synthetase | RNA-binding | — | 47 | 1 | 2.06 | 0.61 | ||||||||
| 52 | gi|30582345 | Snapin | new | new | 48 | 1 | 2.05 | 0.53 | ||||||||
| 53 | gi|19913414 | AP-2 α | ✓ | ✓ | 388 | 7 | 2.03 | 0.46 | ||||||||
| Selected additional proteins | ||||||||||||||||
| 88 | gi|23512245 | AP-3 β3A | new | new | 438 | 9 | 1.47 | 0.34 | ||||||||
| 94 | gi|12654697 | Transferrin receptor | ✓ | ✓ | 953 | 20 | 1.46 | 0.38 | ||||||||
| 114 | gi|32880009 | AP-3 σ3A | new | new | 57 | 1 | 1.33 | 0.46 | ||||||||
| 155 | gi|5442368 | AP-4 σ4 | — | — | 76 | 1 | 1.20 | 0.29 | ||||||||
| 192 | gi|37675283 | AP-4 ɛ | — | — | 90 | 3 | 1.11 | 0.22 | ||||||||
| 196 | gi|6580116 | Glycogen-debranching enzyme |
metabolism | — | 1898 | 98 | 1.10 | 0.26 | ||||||||
| 197 | gi|56204938 | AP-4 β4 | — | — | 69 | 2 | 1.10 | 0.26 | ||||||||
| 252 | gi|976227 | 26S proteasome subunit p45 |
proteasome | — | 905 | 28 | 1.01 | 0.24 | ||||||||
| 270 | gi|31108 | EF-2 | ribosome | — | 544 | 13 | 0.98 | 0.22 | ||||||||
| 279 | gi|16306837 | TCP1, subunit 5 (ɛ) | TCP complex | — | 689 | 26 | 0.95 | 0.21 | ||||||||
| 520 | gi|15990478 | Major vault protein (MVP) |
Vault complex | — | 838 | 20 | 0.15 | 0.03 | ||||||||
Control and mock CCV fractions were prepared as in Fig. 1 and analyzed by iTRAQ (Fig. 3). Identified proteins were ranked based on their relative abundance in control and mock CCVs; a high rank corresponds to a high ratio of control/mock CCVs and, thus, to candidate CCV proteins. Shown are the top-ranking 53 proteins (ratio >2.0), some representative contaminants (ratios near 1), and some other proteins of interest. The fourth column indicates whether a protein had been previously identified by CCV proteomics in Blondeau et al. (2004) or Girard et al. (2005). The Mascot score reflects the confidence with which a protein was identified; a score >35 indicates >95% confidence of identification. “Peptides quantified” indicates how many iTRAQ-labelled peptides were used for the quantification. “Control/mock ratio” corresponds to the relative abundance of a protein in control and mock CCVs; the ratio is the mean of two technical replicates. The proteins can be found in the National Center for Biotechnology Information database. Likely false positives among high ranking proteins are in italics. See Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200607164/DC1) for a complete list of the proteins identified.