Beckman Coulter Application (system support with verified methods)
Using the Biomek® 3000 Laboratory Automation Workstation to Interface 2D Liquid Chromatography with Mass Spectrometry for Multidimensional Proteome Profiling
Michael Simonian**, Matthew Cu**, Edna Betgovargez**, Keith Roby**
and Graham Threadgill**
Beckman Coulter, Inc.
**Indianapolis, IN and ***Fullerton, CA
The discovery stage of proteome profiling typically involves the comparison
of different states of a cell or tissue. One approach utilizes fractionation
of the proteome followed by mass spectrometry (MS). A two-dimensional,
liquid chromatographic fractionation system, the ProteomeLab® PF 2D,
followed by a third dimension with MALDI-TOF MS has been used for this
approach.
The first dimension separation is chromatofocusing where proteins are
separated by pI and collected in fractions based on pH intervals. Upwards
of 20 pI fractions are then separated in a second dimension by reversed-phase
chromatography. From each second dimension run, 80-90 fractions can be
collected. To accommodate the large number of fractions generated by the
first two dimensions for subsequent MS analysis, the Biomek 3000 Laboratory
Automation Workstation was used.
The Biomek 3000 Laboratory Automation Workstation prepared and spotted
the fractions from the second dimension runs along with the appropriate
matrix on a 384-well format MALDI target.
The third dimension measures the mass-to-charge ratio of the proteins.
Human plasma was analyzed with this multidimensional approach. The Biomek
3000 Laboratory Automation Workstation facilitated the complete analysis
of a fractionated proteome by removing the potential bottleneck resulting
from the large number of samples collected from the first two dimensions.
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