omation mbers - Society for Laboratory Automation and Screening

WP058
Michael Simonian
Beckman Coulter, Inc.
Biomedical Research Division
P.O. Box 3100
Fullerton, California 92834-3100
mhsimonian@beckman.com
Using the Biomek ® 3000 Laboratory Automation Workstation to Interface 2D Liquid
Chromatography With Mass Spectrometry for Multidimensional Proteome Profiling
220
Co-Author(s)
Matthew Cu
Edna Betgovargez
Graham Threadgill
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 and serum were compared with this multidimensional approach. Although albumin
constitutes 55% of blood proteins, lower abundant proteins and proteins of 4-12 kD in the same pI fraction as
albumin were detected by MALDI-TOF MS. 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.
WP059
Katja Sippola
PerkinElmer Life and Analytical Sciences, Wallac Oy
Biochemistry
P.O. Box 10
TurkuFIN-20101 Finland
katja.sippola@perkinelmer.com
Co-Author(s)
Joni Helenius, Sanna Rönnmark,
Maija-Liisa Mäkinen, Jari Suontausta,
Christel Gripenberg-Lerche, Satu Kovanen
Automated DELFIA ® Filtration Assays in 96- and 384-well Format for GPCR Studies
Time-resolved fluorescence enhancement technique DELFIA ® enables development of highly sensitive assays
for high throughput screening. We have developed a family of Europium-labeled peptides and proteins designed
for ligand receptor binding assays, as well as a Europium-labeled GTP binding kit for functional assays. These
products provide an excellent non-radioactive alternative that is both stabile and sensitive. Ligand binding assays
based on filtration are the most widely used assays to characterize drug candidates for specific receptors. The
procedure requires liquid handling, incubation and filtration steps that are typically performed manually. We have
automated the Europium-label utilizing binding assays both in 96- and 384-well format using an assay workstation
that allows fully hands-free operation. The results demonstrate comparable or superior performance to manual
assays with greatly increased through-put.