FR AB - Science Reference

ABRF 2001 ABSTRACTS
P105-T
Innovations in proteome analysis: biomolecular interaction
analysis mass spectrometry.
R.W. Nelson, D. Nedelkov; Intrinsic Bioprobes Inc., 625 S. Smith Rd.
Suite 22, Tempe, AZ 85281
The relative ease and fast pace by which the genomic data has been gathered
stands out against the complexity of the proteome world and the
involvedness required for protein characterization. Even though significant
strides have been made lately in several protein characterization techniques,
novel technologies and multiplexation of the existing ones are required to
fully address the many sides of the proteome.
Biomolecular Interaction Analysis Mass Spectrometry (BIA/MS) is a twodimensional
chip-based analytical technique geared toward quantitative and
qualitative detection and analysis of small volumes of biological samples. In
the first (functional) dimension, BIA/MS takes a form of micro-scale planaraffinity
chromatography performed on a sensor-active surface. Surface plasmon
resonance (SPR) is used for detection of biorecognition events that
occur at a sensor surface/solution interface between an immobilized biomolecule
(the ligand) and its interacting partner (the analyte) present in the
sample solution. For the second (structural) dimension, BIA/MS employs
MALDI-TOF MS analysis. With minimal physical modifications and thorough
application of MALDI matrix, SPR-active sensor surfaces are converted to
amenable MALDI target. The ensuing mass spectrometry analysis serves the
purpose of validating the SPR sensing data by providing the molecular mass
of the retained analyte and it yields other qualitative information about the
SPR-monitored interaction, such as identification of non-specific binding,
binding of analyte variants/fragments and multi analyte binding. Presented
here are results from the utilization of BIA/MS in detection of a number of
biological markers found in complex biological fluids. Small volumes of
human plasma and urine were analyzed for cystatin C, beta-2-microglobulin,
urinary protein 1, retinol binding protein and transthyretin, exploring the
effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers.
Issues such as limit of detection, recognition of protein complexes
and delineation of non-specific binding were also explored.
P107-M
Miniature integrated surface plasmon resonance biosensor for
characterization of protein-protein interactions.
M.L. Stolowitz, G. Li, K.P. Lund, J.P. Wiley; Prolinx, Inc.,
22322 20th Avenue SE, Bothell, WA 98021
The utility of a miniature integrated surface plasmon resonance biosensor is
described. The disposible device is approximately the size of a thumbnail and
houses all of the optics and electronics needed to acquire surface plasmon
resonance sensorgrams. The gold sensor surface has been modified so as to
minimize nonspecific binding and utilizes Versalinx Chemical Affininty Tools
to facilitate the immobilization of macromolecular targets for binding studies.
The utility of the biosensor is demonstrated in conjunction with a prototype
data acquisition interface and a simple orbital shaking device.
POSTER ABSTRACTS
214 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P106-S
Dissecting structure-function relationships in RNA/protein
interaction using biocore.
I.A. Laird-Offringa1, P.S. Katsamba1, D.G. Myszka2; 1Univ. of Southern
California, 1441 Eastlake Ave., Los Angeles, CA 90089-9176, 2Univ. of Utah
RNA-binding proteins play critical roles in gene expression and regulation at
the post-transcriptional level. While much is known about the various naturally
occurring RNA-binding motifs, and co-crystal structures of a number of
RNA/protein complexes are available, very little is known about the dynamics
of RNA/protein interactions. We have used the spliceosomal protein U1A
and its RNA target in the U1 small nuclear RNA (U1hairpinII or U1hpII) as a
model to study the kinetics of RNA/protein interaction. Using the previously
solved structure of the U1A/U1hpII complex, we have engineered a series of
mutants designed to probe the roles of electrostatics, hydrogen bonding, aromatic
stacking, and RNA loop length, all of which have been implicated in
formation of the U1A/U1hpII complex. The effects of these mutations on the
binding dynamics were studied using BIACORE, which yielded high quality
kinetic data about the interaction. We determined that neutralization of positive
charges on the protein slows the association rate and reduces the deleterious
effect of salt on complex formation. In contrast, removal of hydrogenbonding
or stacking interactions within the RNA/protein interface, or
reducing the size of the RNA loop, increases the dissociation rate. Our data
support a mechanism of binding consisting of a rapid initial association
based on electrostatic interactions and a subsequent locking step based on
the hydrogen bonding and stacking interactions that occur during the
induced fit of RNA and protein. Our results demonstrate the power of BIA-
CORE to dissect the functional differences between structural features of two
interacting macromolecules.
P108-T
The study of peptide-peptide interaction by ion-mobility MALDI.
A.S. Woods1, J. Koomen2, M.A. Huestis1, K.J. Gillig2, D.H. Russell2, A.J. Schultz3, K. Fuhrer3, M. Gonin3; 1NIDA, NIH, 5500 Nathan Shock
Drive, Baltimore, MD 21224, 2Texas A&M Univ., 3Ionwerks Inc.
We showed in previous work that MALDI could be used to study peptidepeptide
interactions. Matrices such as ATT [pH 5.4] do not disrupt non-covalent
interactions, while more acidic matrices such as CHCA [pH 2.0] do. Our
study found that one peptide had to have 2 adjacent Arg [RR] or an Arg-Lys-
Arg [RKR] motif and the other had to have a minimum of two adjacent Glu
[EE] or Asp [DD] in order to form a complex.
In this work we used MALDI/mobility/TOF mass spectrometry to further
study the formation of these non-covalent complexes [NCX]. The instrument
consists of a short drift tube (the ion-mobility cell) in which is applied an
electric field which causes the MALDI ions to drift through a Helium carrier
gas (2 torr) into an orthogonal TOF mass spectrometer. Ion-mobility [IM] separates
gas phase ions on the basis of their collision cross section-to-charge
ratio, when combined with mass spectrometry it can be a powerful instrument
for structural studies to determine the conformations of biomolecules.
Dynorphin (a 17 a.a. peptide that contains an RR motif) and several other
peptides containing two RR or RKR motifs formed NCX with acidic peptides
(containing 2 or more adjacent Glu or Asp) and were detected by IM and
TOF MS. We discuss the capabilities and limitations of this new instrument
when applied to the gas phase conformational study of MALDI desorbed
NCX, as well as the advantages of looking at peptide mixtures by IM in addition
to MALDI-TOF MS