FR AB - Science Reference

ABRF 2001 ABSTRACTS
P73-S
Rapid and high throughput proteomics by protein capture chips
combined with MS analysis.
J. Chan1, P.T. Jedrzejewski1, P. Zwahlen2, P. Indermuhle2, P. Wagner1, S. Nock3; 1Zyomyx, Inc, 3911 Trust Way, Hayward, CA 94545,
2Zyomyx, Inc., Hayward, CA, 3Zyomyx, Inc.
The study of the proteome is a critical step towards the function determination
of each gene since proteins are the agents of life’s work. However,
robust, sensitive, and comprehensive methodologies for systemic approaches
to proteome analysis similar to genomics methods (e.g., gene arrays) are not
available. Current methodologies such as 2D-PAGE and multi-dimensional
chromatographic methods (e.g., LC-LC, LC-CE) suffer from fundamental limitations.
In order to overcome these limitations, we as well as others have
focused on the development of alternative methods (e.g., protein capture
chips) for proteomics.
Consisting of arrayed capture molecules, protein chips allow for rapid and
comprehensive micropurification and analysis of proteins. Protein chips may
be readily analyzed by optical methods; however, detailed data may be
obtained when arrays are interrogated by MS. Data on the post-translational
modifications (PTM), protein identification (especially important when complexes
are isolated), and quantitation may be obtained with a single detection
scheme without labeling of analytes. In this presentation, we will show
our efforts in this area.
We have been successful in microfabricating protein capture arrays, the accompanying
microfluidics devices, and implementing MS analysis. These mircrofluidic
devices offer a number of advantages (e.g., high density elements,
integration of operations). We have devised a microfluidic device capable of
performing a number of sample preparation operations prior to MS analysis.
In this presentation, we will demonstrate the functionality and utility of these
devices through the identification and characterization of proteins captured
from mixtures. Various assay types will be demonstrated (e.g., protein-protein,
small molecule-protein, protein-DNA). The overall system performance characteristics
will be presented: low femtomole sensitivity, high dynamic range,
and selectivity. The utility of rapid protein identification through database
searching and PTM characterization will be highlighted in various applications.
P75-T
Rapid protein identification of blocked proteins using dilute acid
cleavage and automated Edman sequencing.
S. Wong, A. Kishiyama, V. Pham, W.J. Henzel; Genentech
We have developed a method for rapidly cleaving and identifying proteins
electroblotted onto polyvinylidene difluoride (PVDF) membranes. Cleavage
is performed with 10% acetic acid in 7 M guanidine chloride at pH 2.5 for
1 hour at 90�C, resulting in fragmentation primarily at aspartyl-prolyl bonds.
Peptides resulting from non Asp-Pro cleavage are N-terminally blocked by
reaction with orthophthalaldehyde (OPA) prior to automated Edman degradation.
Reaction with OPA after cleavage blocks all amino acids containing
primary amino groups. Only peptides containing an N-terminal amino acid
with a secondary amino group (proline) will be available for reaction with
the Edman reagent. The sequences obtained are used for protein database
searching. Using this approach, proteins that are found to be N-terminally
blocked can be removed from the sequencer, cleaved with acetic acid,
blocked with OPA and reapplied to the sequencer. The protein can then be
identified from a database search using the sequence mixture obtained.
POSTER ABSTRACTS
206 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P74-M
Reflector in-source-decay MALDI TOF MS: a powerful tool for
N-terminal sequence characterization of proteins.
D. Suckau, A. Resemann, M. Witt; Bruker Daltonik GmbH, Bremen,
Fahrenheitstr. 4, Bremen 28359, Germany
MS-characterization of proteins can simply be initiated by intact molecular
weight determinations. If the experimental value doesn’t agree with the
anticipated structure, a closer investigation of the termini is a must—besides
modifications. In particular N-terminal modifications impose a significant
analytical problem, since Edman sequencing fails and MS/MS of digested
peptides generates a tremendous amount of (useless) information to address
this question and might even not contain the N-terminal peptide.
We introduce ISD measurements on a standard reflector MALDI-TOF instrument
to obtain near-N-terminal sequence information (residues �10–30).
C-type MS/MS fragment ions generated from intact proteins of up to 60 kDa
using this approach allow to read the sequence with a �0.04 Da mass accuracy.
This sequence information can directly be used for sequence database
searches. This information was sufficient to characterize the N-terminus, i.e.,
to identify the fusion protein system and the unexpected N-terminal Metmethylation,
which was confirmed by additional PSD and fingerprint analysis.
ISD seem to be an ideal technique to characterize the N-terminus of proteins
and in particual of N-terminally blocked proteins, provided they are available
on the 10 pmol level and not contaminated by other proteins in a mixture.
P76-S
Quantitation of gel separated proteins at the low picomole level.
S.W. Yuen, L.R. Zieske, K-L. Hsi, T.L. Schlabach; Applied Biosystems,
850 Lincoln Centre Drive, Foster City, CA 94404
Comparative proteomics depends on the ability to quantify changes in cellular
protein levels between the control state and the abnormal or treated
state. The most widely accepted method for protein quantitation is amino
acid analysis, but this method performs poorly at low picomole levels and
below, which are most common in proteomics studies.
Edman chemistry is a nearly stoichometric method for removing amino acid
residues from a protein with typical repetitive yields of greater than 90%. The
problem is the initial yield is not stoichometric and shows variability that is
dependent on protein sequence.
Cleavage of an unknown protein into numerous fragments reduces the
impact of variability in the initial yield of any one fragment and removes
much of variability associated with sequence dependence. We examine both
chemical and enzymatic digestion techniques for fragmentation.
We report in this presentation on the relative and absolute accuracy of protein
sequencing for determining protein concentration. Both solution and gel
isolated proteins will be studied and the results compared with sequence
analysis of intact proteins.