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“Evolving Role of Tandem Mass Spectrometry in Contemporary Protein Science” Neil L. Kelleher, Ph.D. Associate Professor of Chemistry, University of Illinois Urbana, IL. (bio)
ABSTRACT Fragmentation of gas phase ions allows a type of fingerprinting that has been used for years in mass spectrometry to identify and characterize molecules in complex mixtures. Over the decades, the mass of ions that amenable to direct fragmentation for tandem mass spectrometry has been increasing steadily. From small molecules to small peptides to small proteins and intact protein complexes now are analyzed by high resolution tandem mass spectrometry. This historical progression of technology now allows a "top down" philosophy of molecular analysis to be implemented for intact proteins to capture any mass shift occurring on protein molecules, such as coding polymorphisms, alternative splicing, and post-translational modifications. Recent progress along this general line of investigation will be reviewed for non-mass spectrometrists, with illustrations of an integrated approach incorporating protein separations, new mass spectrometers, and novel database curation/search strategies to allow initial implementation of Top Down Proteomics for yeast and human cells. Summary
The top-down approach is used for comprehensive characterization of a protein, in contrast to proteomic profiling, which tabulates as many proteins as possible present in a sample . Analysis of intact proteins, or large fragments, can distinguish "when some are glycosylated and some are acetylated, from when some are glycosylated and acetylated", i.e. when modifications are correlated. In the bottom- up proteomic approach proteins are cleaved into fragments, typically less than 5 kDa. But why throw out the stellar technical achievement we have worked so hard for, of being able to analyze larger fragments -- especially if the most important biological information is in it? Bottom-up typically gives only 5-50% sequence coverage -- it indicates a gene is expressed, but cannot reliably discern which isoform of the gene is expressed or post-translational modifications are made -- a stated goal of proteomics.
All of the histone modification states seen with top-down have been tabulated. 170 modification states -- specific combinations of modifications -- have been characterized and catalogued, which has enabled shotgun proteomics of histone modifications. Also, noting that only 10% of the peptides in bottom-up proteomic analysis are identifiable, Dr. Kelleher has added the posttranslational modifications to a database search algorithm. ProSight PTM is available on the web.
Neil L. Kelleher, Ph.D. - Professor Kelleher received a B.S. and B.A. from Pacific Lutheran University in 1992, a Fulbright Fellowship the following year, and a Ph.D. from Cornell University in 1997. After a NIH Postdoctoral Fellowship at Harvard Medical School with Chris Walsh, Kelleher joined the faculty at University of Illinois at Urbana- Champaign in 1999 as a bioanalytical chemist. Kelleher has interest in Mass Spectrometry-based enzymology of natural product biosynthesis and development of Fourier Transform Mass Spectrometry (FTMS) for “Top Down” proteomics using intact proteins (no proteases) for efficient detection of their post-translational modifications (such as those found in chromatin). Kelleher has received several awards including a Packard Fellowship, an Alfred P. Sloane Fellowship, a Presidential Early Career Award, the National Science Foundation CAREER Award, the Lilly Analytical Chemistry Award, and support from the Burroughs Wellcome, the Searle, and the Dreyfus Foundations.
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The
size limit of mass spectrometry has been steadily increasing since its
inception in 1901: from ions, to molecules, to proteins of 10-30 kDa. We should anticipate 100 kDa. In addition to
getting the molecules "to fly", the high resolution of FTMS
is needed to resolve isotopic envelopes. 
The
value of top down is illustrated in an analysis of histones.
There are 30 million nucleosomes in the genome; all are the
same 4 histone proteins, but the histones have tails that hang out and
can be modified to signal whether the DNA inside should be expressed
or is silenced. Deciphering and reading this histone code is
crucial to understanding gene expression. Methylation at lysine 4 is
thought to be a signal; several sites of acetylation also
occur. It was an open question whether the
methylation and acylation are cause-and-effect; some say these
modifications are mutually exclusive. But the MS data shows
acetylation correlates with, is not antagonistic to, methylation.
He
has also contributed a top-down analog of Yates' mudPIT. In mudCAT (multi-dimensional
characterized by top-down), intact proteins are separated over
tandem chromatographic columns, with anion exchange steps eluted
directly onto sequential RPLC separations. Once characterized, a
protein can be identified quickly in subsequent samples from its exact
mass, without repeating the detailed MS/MS analysis and database
search. 
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