Neil L. Kelleher

Research

Research

Our laboratory has three main areas of research: custom instrumentation for Fourier Transform Mass Spectrometry (FTMS), Nuclear Signaling and Natural Products. More specifically, our main interests lie in the enzymology of natural product biosynthesis, mass spectrometric-based studies of the "Histone Code," and development of Fourier Transform Mass Spectrometry (FTMS) for Top Down Proteomics (i.e. analyzing intact proteins directly; no proteases).

A core activity is measuring chemical modifications to proteins in both hypothesis-driven and discovery modes. Our pioneering efforts in "Top Down" proteomics involve fragmenting intact protein ions in the gas phase and developing custom bioinformatics to characterize unexpected post-translational modifications (PTMs) in methane-producing microbes, yeast, and human cancer cells. In both human cell biology and antibiotic biosynthesis, key proteins harbor over 20 PTMs that present a "code" of biological logic written in the language of protein modifications. We construct, automate, and apply custom mass spectrometry and algorithms to detect and decode this logic.

Group Facilities and Instrumentation

Cell Growth

Our cell growth facility is located on the first floor of Roger Adams Lab (RAL). Here we can grow batches of cells with custom plasmids for the over-expression of enzymes involved in natural product and antibiotic synthesis.

Separations

We employ a wide range of separation methods for fractionating complex mixtures of proteins. Some of these include continuous elution gel electrophoresis (used with and acid-labile surfactant in the place of SDS), ion exchange chromatography, hydrophilic interaction chromatography (HILIC), free flow electrophoresis, and reversed phase chromatography. For nanoLC applications, we employ an Eksigent 1D system and an Advion TriVersa robot.

Mass Spectrometry

Our lab has a total of four FTMS instruments at three different locations on campus. In the main instrument bay (37 RAL), we house a custom built 8.5 Tesla Q-FTMS and a 12 Tesla LTQ-FT hybrid mass spectrometer (picture below). We also have a 7 Tesla LTQ-FT in 357 Noyes Laboratory and another 12 Tesla LTQ-FT (coming soon) in the Institute for Genomic Biology.

Bioinformatics

The new field of Top Down Proteomics has required the development of new informatic and software tools. To meet this need, we have focused thousands of hours of labor on the building and refining of the ProSight suite of software.

Publications

Garcia, B.A., Pesavento, J.J., Mizzen, C.A., Kelleher, N.L. "Pervasive Combinatorial Modification of Histone H3 in Human Cells", Nat. Methods, 2007, 4, 487-489.

Hansen, D.B., Bumpus, S.B., Aron, Z.D., Kelleher, N.L., Walsh, C.T. "The Loading Module of Mycosubtilin: An Adenylation Domain with Fatty Acid Selectivity." J. Am. Chem. Soc., 2007, 129, 6366-6367

Jiang, L., Smith, J.N., Mizzen, C.A., Kelleher, N.L. "Global Assessment of Combinatorial Post-translational Modification of Core Histones in Yeast Using Contemporary Mass Spectrometry", J. Biol. Chem., 2007, 282, 27923-27934.

Calderone, C.T., Kowtoniuk, W.E., Kelleher, N.L., Walsh, C.T., Dorrestein, P.C., "Convergence of Isoprene and Polyketide Biosynthetic Machinery: Isoprenyl-S-carrier Proteins in the pksX Pathway of Bacillus subtilis", Proc. Natl. Acad. Sci. USA, 2006, 103, 8977-8982.

Dorrestein, P.C., Bumpus, S.D., Calderone, C.T., Garneau-Tsodikova, S., Aron, Z.D., Straight, P.D., Kolter, R., Walsh, C.T., Kelleher, N.L., "Facile Detection of Acyl and Peptidyl Intermediates on Thiotemplate Carrier Domains Via Phosphopantetheinyl Elimination Reactions During Tandem Mass Spectrometry", Biochemistry, 2006, 45, 12756-12766.

Hicks, L.M., Moffitt, M.C., Beer, L.L., Moore, B.S., Kelleher, N.L. "Structural Characterization of In Vitro and In Vivo Intermediates on the Loading Module of Microcystin Synthetase", ACS Chem. Biol., 2006, 1, 93-102.

Du, Y., Parks, B.Z., Sohn, S., Kwast, K.E., Kelleher, N.L. "Top Down Approaches for Measuring Expression Rations of Intact Yeast Proteins using Fourier-Transform Mass Spectrometry", Anal. Chem., 2006, 78, 686-694.

Awards

• Pfizer Award in Enzyme Chemistry from the ACS Division of Biological Chemistry, 2007
• Presidential Early Career Award
• Alfred P. Sloan Fellow
• Packard Fellow
• NSF CAREER Award
• Lilly Analytical Chemistry Award
• Burroughs Wellcome Fund Young Investigator
• Searle Scholar
• Fulbright Scholar

Highlights

1. First-of-a-kind Mass Spectrometers now up and running.

Both 12 Tesla LTQ FT mass spectrometers are now on-line and producing prodigious amounts of data for both small molecules (natural product discovery) and proteins (studying human cancer cells as they enter anti-proliferative states of growth arrest and controlled cell death).

2. "Top Down" Mass Spec. goes high throughput.

In a series of two articles in Analytical Chemistry, we have demonstrated the capability to accomplish large scale analysis of intact proteins from yeast cells in culture and human white blood cells. HIGH-RESOLUTION tandem mass spectrometry of intact proteins is now feasible on a chromatographic time scale, with all the associated software ready for even larger scale Top Down Proteome Projects.