Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots

Abstract

We have outlined various aspects and limitations of the collective analysis of protein species of a cell (lymphocyte). We have indicated research directions that, in to our opinion, deserve more attention. We have evaluated mainly the approach used in our laboratory and we recognize that a bulk of important research on the interface of proteomics and genomics remains to be dealt with. It is of great value that we can proceed in our quest by trial and error. But as much as the human genome initiative was not implemented by trial and error, but by formulating new technological approaches, we hope that our approach can be incorporated in the mainstream of proteomics. We need several integrating research directions, some of which are outlined in this communication, namely the use of ordered cDNA libraries, cell-free expression systems, high density filter hybridization, identification of two-dimensional (2-D) gel spots in terms of their amino acid composition through biosynthetic labeling and identification of restriction sites in the corresponding coding sequences. In the accompanying paper the cDNA ordered library approach will be described in some detail.

The hepatic transcriptome in human liver disease

“…A prototype human cell such as a lymphoblast has a mass of 670 pg, contains 67 pg of protein (a total of 10 9 polypeptide molecules representing 4,000 different proteins) and 0.024 pg of mRNA (4 × 10 4 transcripts representing 5,000 different mRNA) [20]. Rare mRNA transcripts present at a frequency of one copy per cell may well be missed based on the cell population frequency and size of the mRNA pool being analysed [20].…”

“…Rare mRNA transcripts present at a frequency of one copy per cell may well be missed based on the cell population frequency and size of the mRNA pool being analysed [20]. This is an important consideration for hepatologists utilizing biopsy specimens where the non-parenchymal cell subpopulation abundance is low and subject to sampling error [21-23].…”

“…Detection of protein expression differs as the analytically threshold is the limiting factor. Currently proteomic detection requires picomole amounts of protein (10 12 – 10 13 peptides) [20]. In a homogenous cell population 90% of the cellular protein mass is due to the 100 most abundant proteins and a further 1200 proteins account for another 7% of the protein mass that is detectable on typical proteomic analysis (from a lysate of 10 6 cells) [20].…”

“…Currently proteomic detection requires picomole amounts of protein (10 12 – 10 13 peptides) [20]. In a homogenous cell population 90% of the cellular protein mass is due to the 100 most abundant proteins and a further 1200 proteins account for another 7% of the protein mass that is detectable on typical proteomic analysis (from a lysate of 10 6 cells) [20]. However, the remaining 3% of the protein mass includes 2800 proteins (over 50% of the different protein species) that fall below the threshold of detection for typical proteomic analysis [20].…”

“…In a homogenous cell population 90% of the cellular protein mass is due to the 100 most abundant proteins and a further 1200 proteins account for another 7% of the protein mass that is detectable on typical proteomic analysis (from a lysate of 10 6 cells) [20]. However, the remaining 3% of the protein mass includes 2800 proteins (over 50% of the different protein species) that fall below the threshold of detection for typical proteomic analysis [20]. Therefore, differential protein expression or proteomics can document changes in protein expression but is currently restricted to moderate to high abundance proteins as well as being technically demanding.…”