Proteomic analysis of mouse thymoma EL4 cells treated with bis(tri-n-butyltin)oxide (TBTO)

Figures & data

Figure 1.  An illustration of nano-LC/MS/MS analysis of tryptic peptides obtained from TBTO-treated cells. (A) Total ion chromatogram; (B) Tandem mass spectra of the peak at retention time 28.58 min.

Figure 2.  Number of identified proteins common for control and TBTO-exposed sample, as well as the number proteins detected in either the control or TBTO-exposed cells.

Table 1.  Proteins altered upon exposure to TBTO.

Accession Number	Protein Name	P (pro)	Sf score	XC	Mr (Da)	Peptides^a
IPI 00112448.1	40S ribosomal S10	1.26E-07	1.884887	20.1931	18915.6	6
IPI 0012318.2	myosin-9	2.22E-14	11.84001	132.2659	226223.5	37
IPI 00381239.3	plectin-8	3.21E-07	10.26928	128.222	517327.5	14
IPI 00224784.3	Prothymosin alpha (ProTα)	3.42E-08	3.85521	40.23136	12122.82	7
IPI 00671411.1	Predicted: similar to ProTα	3.35E-08	3.638016	32.17696	11064.82	4
IPI 00114560.4	Ras-related protein Rab1	2.55E-07	2.14366	30.2248	22546.41	5
IPI 00121892.7	spectrin beta 2	3.02E-06	1.926793	20.21993	251181.6	14
IPI 00396802.1	Chromodomain helicase DNA Binding protein 4	3.23E-07	2.8445776	30.23672	217748.4	6
IPI 00120691.3	Nuclear RNA helicase II	0.000441	2.563277	36.14079	93581.66	5
IPI 00121135.4	Splicing factor arginine/serine rich 2	7.43E-12	1.896528	20.22739	25344.99	3
IPI 00118678.1	T-complex 1 subunit alpha A	7.6E-07	1.652765	30.17544	60340.25	3
IPI 00421179.1	Isoform 1 of eukaryotic translation initiation factor 4 gamma 1	4.35E-11	7.382142	104.2432	93581.66	11

^aDifferent peptides identified.

A change of 1.5-fold or greater in protein expression level and consistency in the direction of this change for a protein in all three replicate experiments were applied as criteria for attributing a specific effect to TBTO on protein expression. Pro designates the probability that a protein’s identification has come about by chance. The parameter Sf (final score) indicates how good the protein match is. XC (X-correlation) indicates how well the experimental mass spectrum matches the theoretical spectrum generated in silico.

Table 2.  TBTO-altered protein-fold changes compared to the corresponding mRNA ratio.

Gene symbol	Protein Name	Protein-fold change (± SD)	mRNA ratio
RPS10	40S ribosomal S10	0.38 ± 0.05	0.715
Myh9	myosin-9	0.33 ± 0.11	0.787
Plectin 1	plectin 8	2.20 ± 0.37	0.770
Ptma	Prothymosin-alpha (ProTα)	0.13 ± 0.03	0.669
N/A^a	Predicted: similar to ProTα	0.16 ± 0.01	N/A
Rab1	Ras-related protein Rab1	2.20 ± 0.51	0.769
Spnb2	spectrin beta 2	0.36 ± 0.09	0.792
Chd4	Chromodomain helicase DNA Binding protein 4	0.22 ± 0.05	0.424
Ddx21	Nuclear RNA helicase II	0.36 ± 0.13	0.519
Sfrs2	Splicing factor arginine/ serine rich 2	0.39 ± 0.10	0.557
Tcp1	T-complex 1 subunit alpha A	0.28 ± 0.05	0.785
Eif4g1	Isoform 1 of eukaryotic translation initiation factor 4 gamma 1	0.27 ± 0.16	0.615

N/A^a This newly identified protein has not gene symbol.

Comparison of the fold changes of the differentially expressed proteins with their corresponding mRNA expression data. The fold changes of each protein level is the average value of the ratios of the peptides identified for the same protein in control and exposed samples of triplicate experiments (± SD).

Figure 3.  Protein extracts of TBTO-treated and non-treated cells (70 μg each; obtained as described in Materials and Methods) were separated by 10% SDS-PAGE, transferred onto Hybond-P PVDF membrane, and probed with rabbit anti-prothymosin-α polyclonal IgG. Detection was performed using goat anti-rabbit IgG conjugated with horseradish peroxidase and ECL Western blotting detection reagents. The numbers on the left indicate the molecular weights of the ECL markers.