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| Institute: |
Lab or Branch |
| Gyros US Inc, Monmouth, NJ |
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| Title: |
| High-Speed Protein Digestion and MALDI
Peptide Mapping on a CD Microlaboratory |
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| Authors: |
| M. Holmquist, A. Palm, J. Engström,
U. Selditz, P. Andersson |
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| Abstract: |
| Peptide mapping by MALDI-ToF-MS is considered
key for protein identification in high-throughput proteomics.
Recently, we presented a microfluidic platform based on
a rotating CD that allows for integrated sample peptide
concentration, desalting, crystallization and MALDI-MS
detection directly on the CD; Thus, offering routinely
high detection sensitivity of peptides by mass spectrometry.
Current limitations in peptide mapping include extensive
manual sample preparation steps, and low proteolytic digestion
rates, particularly with dilute protein samples. Here
we demonstrate a CD microlaboratory that allows for integrated,
high-speed multiplex (96 samples) protein digestion and
peptide mass mapping of proteins from dilute solutions.
Efficient digestions of low micromolar to nanomolar
concentrations of protein were performed. Bovine serum
albumin (BSA) was used as a model protein substrate.
The CD contains 96 identical microstructures, each packed
with a 10 nl reversed-phase chromatographic (RPC) column.
The generated peptides were adsorbed in RPC columns,
eluted together with matrix and crystallized onto small
(~500 µm diameter) MALDI target areas on the CD
for subsequent MALDI-MS analysis. A robotic workstation
was developed that enables fully automated sample and
reagent transfer from microplates to CD's. High-speed
protein digestion was performed in CD microstructures
either by means of capturing the protein substrate on
a solid support or by using immobilized trypsin, and
results from these analyses are presented. We expect
these techniques to facilitate routine generation of
multiplex high quality peptide mass maps from dilute
protein samples, thus providing high fidelity tools
for efficient proteome exploration. |
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