Table 2. Rat hypothalamus fixed tissue test-specific MS candidate proteins (selected from 49) with partial epitopes.
| Rat candidate no. | Accession no. | Name, description, gene | Citrate test score (area*) | RIPA test score (area*) | Triplet or better epitope match to KVKEFNNI | MW and match to westerns (∼7+, 35–45 or 60–65 kDa) | Identity (aa %) with fly candidate 6, Q9W2X8 |
|---|---|---|---|---|---|---|---|
| 1 | F1M5Z5 | Anoctamin; multi-pass membrane protein; ion channels; clotting; Ano6 | 4.25E7 | 5.87E6 | GxxxxxNNI, PLxxxxxxxNNI | 92.2 ✗ | 0.3 |
| 2 | D3Z822 | Protein Atr; kinase; Atr | 7.45E6 | 3.40E6 | KEF, EFN | 301.0 ✗ | 1.3 |
| 3 | D3ZZJ4 | Protein Ttc21a; cilium protein; Ttc21a | 4.10E6 | 1.03E6 | VKExN, LxxxVKE, KxxNNI | 148.6 ✗ | 13.8 |
| 4 | Q8CGL8 | Secretogranin II variant (fragment); neuroendocrine secreted protein; Scg2 | 3.09E6 | 2.52E6 | VxxxNNI | 37.1✓ | 5.2 |
| 5 | P63170 | Dynein light chain 1; motor protein; Dynll1 (also in hypothalamus unfixed control and PC12 unfixed test and control) | 1.98E6 | 6.73E6 | KEF | 10.4 ? | 1.5 |
| 6 | P85972 | Vinculin; membrane-cytoskeletal protein, actin binding; Vcl (Also in PC12 unfixed control) | 8.42E5 | 1.32E6 | VKE (×2) | 116.5 ✗ | 7.9 |
| 7 | D3ZBB8 | Cytoplasmic dynein 2 heavy chain 1; motor protein; Dync2h1 | 0** | 5.95E7 | VKE (×2), NNI | 491.0 ✗ | 5.2 |
| 8 | D4AAG8 | Protein Smchd1; ATP binding, nuclear; Smchd1 | 8.97E6 | – | KVK, VKE, VxxFNNI | 225.9 ✗ | 5.1 |
| 9 | F1M1M4 | Protein Dst (fragment); dystonin, adhesion junction protein; deleted from UniProt database | 6.31E6 | – | KVKE, NNI | 300.0 ✗ | 9.2 |
| 10 | F1M7V4 | Protein piccolo (fragment); synaptic vesicles; Pclo | – | 1.31E8 | KVK, MxxxxxxxKEF | 426.1 ✗ | 6.8 |
| 11 | F1M2I5 | Opioid-binding protein/cell adhesion molecule; Opcml | – | 5.76E6 | PxxxKVKxxxN | 22.5 ✗ | 4.8 |
| 12 | Q4V8E7 | Protein Tex21; ‘Testis expressed gene 21,’ coiled coil; Tex21 | – | 3.36E6 | KVKxxxN, VKE (×2) | 60.3 ✓ | 10.9 |
Notes:
UniProt database (http://www.uniprot.org/) used for search: Rattus norvegicus.
E7 = ×107, etc.
Detected by machine without area assignment.
MW, molecular weight, kDa. Amino acid identity (%) established via EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). The first seven candidates were all the test specifics found in both citrate and RIPA antigen retrieved samples. The rest are a selection from a further 42 test specifics found in one sample only having above-triplet partial epitopes. Candidates were identified on the basis of single peptides, except candidate 5 (two peptides in both samples) and candidate 7 (1 in citrate, 2 in RIPA). FDR 5%. Filtering at 1% FDR removes over half of the above candidates leaving 5, 6, 8, 9 and 11. MS peptides unexhibited, except that for candidate 4, a 7mer in both samples comprising residues 11–17, IILEALR (coverage 2.17%), scores 1.71/1.98 (citrate/RIPA).