Subcellular Fractionation of Small Sample Amounts

Abstract

A central issue in proteomics is the generation of qualitative and quantitative protein inventories for a given tissue, organ, or organism. In many eukaryotes, this amounts to a formidable task. Mammals may express more than 200,000 different transcripts with a quarter of them encoding proteins (1). In addition, there exist several hundred different (2) posttranslational modifications, increasing the number of different protein species by an estimated factor of 10, compared to the number of different mRNAs (3, 4). Consequently, higher organism will display more than 1,000,000 different proteins (5). Considering as a lower, very conservative limit, the expression of 5–10% of the transcriptome in an organ, about 50,000–100,000 different protein species will be present in such a sample. Even in a single cell, > 10,000 different protein species likely exist. This number easily exceeds the potential of any known proteomic approach. Another important issue in protein analysis is the high range of protein abundance. Protein concentrations likely vary between 10⁶–10¹⁰, depending on the tissue analyzed (6, 7). This renders the identification of low abundant proteins impossible in the bulk of total protein extracts.