The goal of our research is to develop new methods and instrumentation for
the identification and structural characterization of peptides and proteins
in complex mixtures at the low femtomole or attomole level and to apply these
methods to important structural problems in cell biology, immunology and
neuroscience. Towards this end, we have developed automated "peak parking"
technology that uses nano-flow HPLC interfaced to a sheathless micro-ESI source
on either an ion trap or Fourier transform mass spectrometer. Briefly stated,
the approach involves the use of proteolytic enzymes to convert the protein or
group of proteins into a complex mixture of peptides, which are then fractionated
by nanoflow HPLC and eluted directly into the mass spectrometer. Protonated
peptides of a particular mass are selected from the mixture, stored in the ion trap,
fragmented on collision with helium atoms, and the resulting fragments are then
separated and mass analyzed. All of these steps are performed under control of
the mass spectrometer data system and require 1-2 sec to complete. Dissociation
of the peptide ions occurs more or less randomly at each of the amide bonds
in the molecules to produce a collection of fragments. The mass difference
between two fragments differing by a single amino acid defines the mass and
thus the identity of the extra residue in the longer fragment. The complete amino
acid sequence of the selected peptide is deduced by extending the above analysis
to all fragments observed in the spectrum. Samples present at the 50-100 attomole
level in complex mixtures can now be sequenced routinely with the above technology.
Our research focuses on two major applications of the above technology. The first
involves identifying peptides that trigger the immune system to kill diseased cells.
Cytotoxic T lymphocytes (CTL) or killer cells are an arm of the immune system
concerned with recognition of cells that express new antigens, proteins, as a result
of viral infection or cellular transformation (cancer). Cells convey their health
status to the immune system by generating fragments from each of the approximately
10,000 proteins being synthesized, loading them onto a protein carrier (MHC molecule),
and transporting them to the cell surface for screening by the killer cells. CTL
lyse those cells that display new fragments, antigens that are associated with a
particular disease states. Identification of these antigens is the first step in the
preparation of vaccines that promote immunity against the above diseases and our
laboratory has developed unique methodology to accomplish the above task. Four murine
tumor antigens, two human melanoma antigens, and a lung cancer antigen have been
identified with this approach. An additional fourteen peptides have been identified
as either bacterial antigens, viral antigens, minor histocompatibility antigens,
alloantigens or thymic self peptides involved in positive selection of cytotoxic
T-lymphocytes. Methodology for the identification of antigens presented to T-helper
cells in association with class II molecules is also under development. Two class II
antigens, one associated with human melanoma and the other with the onset of human
diabetes have been identified recently. Future research will focus on the identification
of additional tumor and minor histocompatibility antigens, characterization of class II
MHC epitopes, identification of alternate pathways for antigen presentation, and
characterization of post translational modificiations found in peptide presented by
class I molecules.
The second application involves research in the field of proteomics. DNA sequence
information on the human genome and that of selected organisms is now becoming available
at an ever increasing rate and will provide the starting point for development of novel
therapeutic interventions against many of the world's diseases. The next challenge is
at the level of proteomics, understanding the functions of proteins encoded by a
particular genome. Presently, we are using mass spectrometry to analyze all proteins
secreted by a particular cell type, to identify components of functionally active protein
complexes, to probe protein-protein and protein-DNA interactions, to locate post
translational modifications and covalently attached ligands, to compare proteins
expressed in healthy and diseased cells, and to characterize proteins in brain cells
that facilitate long termed memory. Also underway is research to characterize post
translational modifications on the androgen receptor and the associated proteins that
regulate its activity, to identify proteins that regulate development of the endoderm
(internal organs) in the nematode, C. elegans, to characterize proteins involved in spore
development in the bacterium, Bacillus subtilus, to identify proteins that constitute the
chloroplast, and to develop methods for characterizing proteins involved in signal
transduction cascades.
