TABLE 1.
Mass spectrometric analysis of KPC1 shows that it is ubiquitinated in FR3T3 cellsa
| Accession no. | Results for protein in indicated molecular mass range (kDa) of gel sample
|
MW | Annotation | |||||
|---|---|---|---|---|---|---|---|---|
| 100-200
|
200-300
|
>300
|
||||||
| No. of pept. | AI | No. of pept. | AI | No. of pept. | AI | |||
| KPC1-expressing cells | ||||||||
| NP_001029102.1 | 3 | 25 | 4 | 25 | 5 | 56 | 8 | Ubiquitin |
| NP_071347.2 | 28 | 17 | 20 | 11 | 26 | 17 | 148 | Ring finger protein 123 (KPC1) |
| NP_001605.1 | 0 | 0 | 0 | 0 | 3 | 3.6 | 42 | Actin, gamma 1 propeptide |
| NP_001393.1 | 0 | 0 | 3 | 3 | 3 | 3 | 50 | Eukaryotic translation elongation factor 1 alpha 1 |
| NP_001093241.1 | 0 | 0 | 0 | 0 | 2 | 0.8 | 121 | Protein encoded on chromosome 2, expressed in prostate, ovary, and testis |
| NP_001077007.1 | 0 | 0 | 0 | 0 | 2 | 0.8 | 121 | Protein expressed in prostate, ovary, testis, and placenta 2 |
| NP_001120959.1 | 0 | 0 | 2 | 0.3 | 3 | 0.5 | 287 | Gamma filamin isoform b |
| NP_001449.3 | 0 | 0 | 2 | 0.3 | 3 | 0.5 | 291 | Gamma filamin isoform a |
| NP_001448.2 | 0 | 0 | 0 | 0 | 2 | 0.4 | 278 | Filamin B, beta (actin binding protein 278) |
| NP_005518.3 | 0 | 0 | 2 | 1.4 | 0 | 0 | 70 | Heat shock 70 kDa protein 1-like |
| NP_000468.1 | 0 | 0 | 2 | 1.4 | 0 | 0 | 69 | Albumin preproprotein |
| NP_002464.1 | 0 | 0 | 5 | 1.1 | 0 | 0 | 226 | Myosin, heavy polypeptide 9, nonmuscle |
| NP_001933.2 | 2 | 0.9 | 0 | 0 | 0 | 0 | 114 | Desmoglein 1 preproprotein |
| Control cells | ||||||||
| XP_001721581.1 | 2 | 0.5 | 0 | 0 | 0 | 0 | 190 | Predicted to be similar to hornerin |
| NP_001014364.1 | 2 | 0.4 | 0 | 0 | 0 | 0 | 248 | Filaggrin family member 2 |
| NP_001009931.1 | 2 | 0.4 | 0 | 0 | 0 | 0 | 282 | Hornerin |
a
FR3T3 cells were transfected with plasmid expressing HA-tagged KPC1 or empty vector. Lysates were prepared under denaturing conditions and then subjected to immunoprecipitation with anti-HA antibodies. The eluates from the pellets were electrophoresed on 9% SDS-PAGE gels, and the indicated gel slices subjected to proteomic analysis as described in Materials and Methods. Shown are the number of peptides (pept.) identified for each protein and the abundance index (AI), which estimates the relative abundance by normalizing the number of spectra assigned to the protein to its molecular mass (see Materials and Methods for details).