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J Biomol Tech. 2007 February; 18(1): 79.
PMCID: PMC2291843

ABRF Award Winner Innovative Technology for the Study of Cell Signaling

Abstract

This lecture will focus on three different aspects of cell signaling and the mass-spectrometry technologies developed to study them. Presented first will be results of studies to elucidate the “histone code,” complex patterns of post-translational modifications on histone proteins that regulate gene expression, gene silencing, DNA damage repair, and stem cell differentiation, and that block reprogramming of a somatic cell nucleus by an enucleated oocyte. The same modifications also facilitate heritable changes in phenotype that do not involve mutation of DNA (epigenetics).

To study these post-translational modifications, we constructed the prototype for the commercially available, tandem linear ion trap/FTMS instrument and also developed methodology that makes it possible to analyze intact proteins on a chromatographic time scale (1 protein/2–5 sec). Proteins are converted to gas-phase, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly, doubly, and triply charged ions are used to read a sequence of 15–60 amino acids at both the N- and C- termini of the protein. This information, along with the measured mass of the intact protein, is used to identify unknown proteins, to confirm the amino acid sequence of a known protein, to detect post-translational modifications, and to determine the presence of possible splice variants.

Part two of the presentation will describe results of studies to define sites of phosphorylation that regulate the formation of focal adhesions involved in cell migration. For this work, we employ immobilized metal affinity chromatography (IMAC ) to identify phosphorylaton sites present at levels as low as 0.1% of the parent protein. Stable isotopes are employed to follow changes in site usage as a function of cellular perturbation. Further information on this topic is available at cellmigration.org (site guide, phosphoproteomics).

Part three of the presentation will focus on signaling between cancer cells and cytotoxic killer cells by the class I antigen-processing pathway. Since signal transduction pathways in cancer cells are highly dysregulated, we hypothesized that this might manifest itself in the presentation of unique phosphopeptides to the immune system in association with Class I major histocompatibility complex molecules. By using a combination of IMAC, stable isotope labeling, and nano-flow HPLC-tandem mass spectrometry, we are now able to detect cancer-specific phosphopeptides present at levels as low as one to five copies per/cell. Results of studies to use these phosphopeptides as immunotherapeutics and/or vaccines against cancer will be discussed.


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