Table 2.
ICAT and MS/MS spectral counting quantification of zonal centrifugation-purified proteasomes from the liver of rats fed-ethanol (E) and from the liver of pair-fed controls (C): Proteasome subunits, proteasome regulators, proteasome-associated deubiquitinases and proteins from the UPP
| Name | Accession # | ICAT quantification | Spectral counting quantification | |||||
|---|---|---|---|---|---|---|---|---|
| # quantifying peptides | Ratioa E/C | CV (%)b | CV (%)c | # MSMS | Ratiod E/C | |||
| Control | Ethanol | |||||||
| 20S Proteasome subunits | ||||||||
| α1 | P60901 | 6 | 0.8 | 11.2 | 14.0 | 45 | 29 | 0.8 |
| α2 | P17220 | 3 | 1.2 | 5.6 | 5.9 | 40 | 28 | 0.8 |
| α3 | P21670 | 5 | 0.8 | 23.7 | 11.6 | 22 | 24 | 1.3 |
| α4 | Q9Z2U0 | 1 | 1.0 | / | 6.8 | 41 | 33 | 1.0 |
| α5 | P34064 | 6 | 0.9 | 7.0 | 2.2 | 34 | 26 | 0.9 |
| α6 | P18420 | 5 | 1.1 | 13.7 | 5.1 | 34 | 37 | 1.3 |
| α7 | P18422 | 1 | 0.9 | / | 0.0 | 27 | 30 | 1.3 |
| β1 | P28073 | 3 | 0.8 | 17.1 | 0.8 | 14 | 11 | 0.9 |
| β2 | Q9JHW0 | 1 | 1.0 | / | 0.7 | 20 | 14 | 0.8 |
| β3 | P40112 | 2 | 1.1 | 2.4 | 2.7 | 30 | 30 | 1.2 |
| β4 | P40307 | 2 | 1.0 | 5.6 | 6.9 | 28 | 20 | 0.9 |
| β5 | P28075 | NQ | NQ | / | / | 13 | 10 | 0.9 |
| β6 | P18421 | 3 | 1.0 | 23.4 | 6.9 | 31 | 24 | 0.9 |
| β7 | P34067 | 1 | 1.2 | / | 9.8 | 20 | 18 | 1.1 |
| β1i | P28077 | NQ | NQ | / | / | 18 | 14 | 0.9 |
| β2i | Q4KM35 | NQ | NQ | / | / | 15 | 11 | 0.9 |
| β5i | P28064 | NQ | NQ | / | / | 38 | 24 | 0.8 |
| 19S regulator subunits | ||||||||
| Rpn1 | Q8VDM 4 | 7 | 0.9 | 15.8 | 2.1 | 112 | 83 | 0.9 |
| Rpn2 | O88761 | 9 | 0.9 | 13.7 | 2.6 | 106 | 94 | 1.1 |
| Rpn3 | Q5U2S7 | 2 | 0.9 | 18.3 | 15.9 | 48 | 42 | 1.0 |
| Rpn5 | Q5XIC6 | NQ | NQ | / | / | 8 | 8 | 1.2 |
| Rpn6 | Q8BG32 | 2 | 1.1 | 18.2 | 1.8 | 44 | 43 | 1.2 |
| Rpn7 | Q99JI4 | 2 | 0.8 | 9.6 | 1.2 | 52 | 41 | 0.9 |
| Rpn8 | P26516 | 2 | 1.5 | 30.5 | 10.3 | 20 | 23 | 1.4 |
| Rpn9 | Q9WVJ2 | 2 | 0.9 | 6.4 | 8.0 | 56 | 43 | 0.9 |
| Rpn10 | O35226 | 7 | 0.8 | 18.4 | 6.8 | 14 | 15 | 1.3 |
| Rpn11 | O35593 | 1 | 0.7 | / | 3.7 | 58 | 26 | 0.5 |
| Rpn12 | Q9CX56 | 5 | 0.8 | 29.0 | 10.7 | 29 | 28 | 1.2 |
| Adrm1 (Rpn13) native form (40 to 50 kDa) split form (25 to 30 kDa) |
Q9JMB5 | 11 7 4 |
1.3 0.4 2.6 |
90.8 21.1 9.8 |
4.6 13.7 3.7 |
21 15 6 |
13 5 8 |
0.7 0.4 1.6 |
| Rpt1 | Q63347 | 7 | 1.1 | 17.3 | 0.2 | 47 | 36 | 0.9 |
| Rpt2 | P62193 | 4 | 0.9 | 14.2 | 0.2 | 65 | 73 | 1.3 |
| Rpt3 | Q63570 | 4 | 1.0 | 9.9 | 4.6 | 38 | 33 | 1.0 |
| Rpt4 | P62334 | 9 | 0.9 | 9.4 | 4.4 | 59 | 49 | 1.0 |
| Rpt5 | Q6P6U2 | 2 | 0.9 | 16.3 | 13.0 | 63 | 62 | 1.2 |
| Rpt6 | P62198 | 7 | 1.1 | 11.7 | 5.6 | 60 | 63 | 1.3 |
| Other regulators | ||||||||
| PA28α | Q63797 | NQ | NQ | / | / | 8 | 3c | 0.4 |
| PA28β | Q63798 | 2 | 0.3 | 7.6 | 1.9 | 9 | NI | <0.1 |
| PA200 | Q5SSW2 | NQ | NQ | / | / | 35 | 4e | 0.1 |
| Proteasome-associated Deubiquitinases | ||||||||
| Usp14 | Q5U2N2 | 4 | 0.6 | 7.7 | 19.7 | 36 | 22 | 0.7 |
| UCHL5 | Q5HZY3 | NQ | NQ | / | / | 19 | 11 | 0.7 |
| Other proteins from the UPP found to be varied by ICAT and MS/MS spectral counting | ||||||||
| TPP-II | Q64560 | 2f | 0.3 | 8.1 | 5.8 | 6d | NI | <0.1 |
| Ecm29 | Q6PDI5 | 1f | 0.1 | / | 24.4 | 60 | 8 | 0.1 |
Proteins listed in the table were purified as described in the experimental section and identified by nanoLC-ESI-QqTOF MS/MS analysis and database searching. Criteria for acceptation of proteins identification are described in the experimental part. More information on the quantification process using ICAT and MS/MS spectral counting is also provided in the experimental section. ICAT and MS/MS spectral counting quantification were obtained by independent experiments.
NQ : not quantified; NI : not identified; E: Ethanol; C: Control
: Normalized ratio (normalization factor = 1.10)
: coefficient of variation on quantifying peptides
: coefficient of variation on technical replicates
: Normalized ratio (normalization factor = 0.84)
: Top ranking peptide with a Mascot expectation value of less than 0.01
: Top ranking peptide with a Mascot expectation value of less than 0.05