In proteomic studies, separate experimental protocols have been necessary
to identify proteins, determine their function, and predict their three-dimensional
structure. In this study, a function-based separation of proteins was conceived to
fractionate proteins prior to enzymatic digestion. In the initial demonstration of
this technique, a DNA substrate was used to separate the DNA-binding proteins
from the rest of the proteins in a lysate in order to identify protein function and to
simplify the complex mixture of proteins. A total of 232 putative DNA-binding
proteins and over 540 proteins in all were identified from E. coli. Hypothetical or
unknown proteins were found, some of which bind to DNA. As a part of this
demonstration, changes in protein expression caused by different environmental
conditions (aerobic and anaerobic atmospheres) were observed. In a second
demonstration, aimed at determining the three-dimensional structure of the DNA
binding proteins, binding sites were blocked with oligonucleotides, and the
modified proteins were purified, enzymatically digested, and subjected to tandem
mass spectrometry. The amino acids in the DNA-binding domains of three
proteins were determined.
In a final application of function-based separation, DNA-binding proteins
were digested with trypsin and the resulting peptides were separated using HPLC
and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments
to study the complementary nature of the two ionization techniques, taking into
account the differences between the mass analyzers. Based on the analysis of a
large data set containing hundreds of peptides and thousands of individual amino
acids, some of the currently held notions regarding the ionization processes were
confirmed. ESI tends to favor the analysis of hydrophobic amino acids and
peptides while MALDI is disposed toward mainly basic and aromatic species.
These tendencies in ionization account in large part for the complementary nature
of the peptides and proteins identified by the ESI and MALDI instruments and
make it necessary to employ both types of instruments to gain the most
information out of a given sample in a proteomics study. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30578 |
| Date | 01 October 2003 |
| Creators | Stapels, Martha Degen |
| Contributors | Barofsky, Douglas F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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