The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

The chemical analysis of biological molecules has provided the code and structural basis to understand the general molecular logic of life. As a Bioanalytical Group, we continue this tradition of translating analytical advance into biological discovery. Specifically, we search for novel covalent modifications to proteins and study the mechanisms of large enzymes (>100 kDa) with covalently-bound intermediates. Developing Fourier-Transform Mass Spectrometry (FTMS) with on-line separations to spearhead the research, our program focuses on correlating protein fragmentation data to fully sequenced genomes to scan for chemically-altered gene products and the cellular alteration machinery. Additionally, the biosynthesis of several clinically-used antibiotics and immunosuppressants occurs on large enzymes that act as supports for covalent catalysis, ideal for study by mass spectrometry.

I. Microbial Proteomics. Bacterial genomes encode all possible virulence determinants, vaccine candidates, and potential drug targets. Further, a completed genomic sequence establishes a basis for high throughput analysis of the proteins expressed (i.e., the proteome). Respiratory pathogens have been among the first to have their genomes entirely sequenced.

Mycoplasma pneumoniae harbors the second smallest genome of any self-replicating life form and encodes 679 putative proteins. These genome-predicted proteins will be correlated with those actually present, detecting any biological event that generates a protein of different molecular composition than that predicted. These include sequence or reading frame errors, imprecise bioinformatics, co- or post-translational modifications, and mutational or proteolytic strategies for antigenic variation.

II. Instrumentation and Bioinformatics. In addition to our commercial 4.7 Tesla instrument, we are currently building a 9.4 Tesla FTMS at the National High Magnetic Field. Completion of this project in the Fall of 2000 will outfit our lab with one of the highest performance mass spectrometers in the world. On the front end of these instruments, we will couple capillary electrophoretic and chromatographic separations to enable submicrogram sampling and on-line analyses. Also, back end algorithms for computerized protein identification and correlation to raw genomic sequences will be required to achieve high protein analysis rates.

III. Natural Product Biosynthesis on MegaEnzymes. The common enzyme uses non-covalent interactions to bind and chemically alter the structure of its substrate. However, there are several families of enzymes that act upon their substrates via covalent catalysis, where building blocks of natural products are all bound covalently and then assembled while on the enzyme. Due to the covalent attachment of all biosynthetic intermediates, we can measure the mass and relative abundances of them vs. time and "watch" the intermediates "crawl" along the enzyme as they grow. This will yield detailed insight into how this molecular machine works. The high performance of FTMS is critical for extracting such kinetic data from complex proteolytic mixtures of large enzymes.