PMC

Comparative analysis of RNA and protein profiles. (A) Comparative analysis of the number of proteins detected by MS and RNA-seq in U-2 OS, respectively. Transcripts also detected at the protein level are colored according to the MS intensity as shown in . The data for A-431 and U-251 MG are presented in , respectively. (B) Venn diagram showing the overlap in gene products between the three methods for all genes studied by all three methods (n=3851) in U-251 MG cells. The overlap for the three methods in A-431 and U-2 OS cells is presented in . (C) Correlation between changes on protein (log2 SILAC ratio) and transcript levels (log2 RPKM ratio) for U-2 OS over A-431 cells. A two-fold change between the cell lines was used as cutoff for up-/down-regulation on RNA and protein levels. Data for the changes between the other cell lines are presented in . The Spearman correlation for randomized RNA and protein pairs is zero for all cell lines (). The correlation between RPKM and MS intensity levels are presented in .

The transcript profiles in the three human cell lines based on RNA sequencing (RNA-seq). (A) RNA-seq reads mapping to a part of the RPS19 gene (ENSG00000105372). The reads align almost exclusively to the exons of the gene. (B) Distribution of RPKM values for all protein-coding genes (blue) and a set of ≈3000 intergenic regions defined as a genomic region at least 1 kb away from a known gene or EST. To detect present genes, we used a RPKM cutoff value of 0.1 (red dashed line), which corresponds to false-positive and false-negative rates of 5% (). (C) Distribution of log2 RPKM values for all detected transcripts (n=14 064) in U-2 OS. (D) Venn diagram showing the number of transcripts detected by RNA-seq in each cell line and the overlap between the cell lines. (E) The number of genes present in the categories ‘similar', ‘slightly changed', ‘substantially changed', ‘cell-type specific' and ‘not detected'. (F) Distribution of log2 RPKM values for all transcripts detected in protein categories (see for details) in U-2 OS.

The protein profiles in the three human cell lines based on confocal microscopy and mass spectrometry analysis. The numbers of analyzed and detected genes for each platform are shown in . (A) The three cell lines used in the study: U-2 OS, U-251 MG and A-431 illustrated by confocal immunofluorescent images, where the microtubules (red), nuclei (blue) and an antibody (HPA024087) toward the human TUFM are staining mitochondria (green). (B) Venn diagram showing the number of proteins (%) detected by confocal IF in each cell line and the overlap between the cell lines. (C) Venn diagram showing the number of proteins detected by MS in each cell line and the overlap between the cell lines. (D) Distribution of log2 MS intensity for all detected proteins (n=5405) in U-2 OS. Bars are colored according to MS intensity, ranging from light yellow (low MS intensity) to dark red (high MS intensity). (E) The distribution of log2 MS intensity values for the protein categories (see for details) in U-2 OS. (F) Three-dimensional plot of log2 protein (SILAC) ratios between two cell lines performed for all three combinations. The genes are colored by the variation of expression between the three cell lines as: similar (less than two-fold difference between all cell lines in gray), slightly changed (two- to four-fold difference between two cell lines in light turquoise or between all cell lines in light purple) or substantially changed (more than four-fold difference between two cell lines in dark turquoise or between all cell lines in dark purple).