E.coli Proteomics: Subcellular localization, abundance, protein sequence, ESI-MS, 2D Gels

  • Back to the Church Lab Home Page



     Text and Data from both Andy Link's 1994 Harvard University Thesis, "Experimental Tools for the Analysis of Genomes"...

    " Link, A.J., Robison, K. and Church, G.M. (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli." Electrophoresis 18:1259-1313 (special proteome issue)."

    The experimental tools of two-dimensional electrophoresis and N-terminal protein sequencing are combined to survey the protein content of E. coli. The observed isoelectric point and molecular weight are precisely measured from the mobilities of proteins on 2-DE gels, and the relative abundance of each protein is estimated from the intensity of the stained spot. Using cellular fractionation techniques allows the cellular location of proteins to be determined. Limited sequence of the amino terminus of each protein produces a query sequence for identifying the protein and marks the mature protein start site. A second estimate of protein abundance is obtained from the initial yield of each sequence tag.

     Another protein characterization of E. coli has been underway for over a decade to create an index linking a gene to its protein product on a reference 2-DE gel and to monitor changing global protein expression as a function of cellular environment (Neidhardt et al., 1983). This group has been annotating the proteins displayed on 2-DE gels by cataloging the observed isoelectric point, molecular weight, abundance, and regulatory information, and have primarily used migration of known proteins with radiolabeled total cell extracts to identify the spots on the reference 2-DE gel (Block et al., 1980; Phillips et al., 1980; Neidhardt et al., 1983; Phillips et al., 1988; VanBogelen et al., 1990; VanBogelen and Neidhardt, 1991; VanBogelen et al., 1992).

     Our project uses protein sequencing to identify and quantitate the protein products of the E. coli genome. The protein data complements the genomic DNA sequence by identifying open reading frames being expressed and measuring the accumulated protein in E. coli. Using protein sequencing of 2-DE spots, the colinearity of DNA sequence and protein sequence is compared to look for deviations from the code. The observed isoelectric point and molecular weight of each identified protein is compared to values predicted by genomic sequence data to identify proteins with unexpected electrophoretic mobilities. The abundance of identified proteins is compared to the expected protein content of the cell.

    Protein Mass-spectrometry links:

    This page was last updated 20-Jan-1999 by GMC. reyes@arep.med.harvard.edu