omation mbers - Society for Laboratory Automation and Screening

TP031
Alex Berhitu
Spark Holland, Inc.
666 Plainsboro Road, Suite 1336
Plainsboro, New Jersey 08536
alex.berhitu@sparkholland.com
161
Co-Author(s)
Steven Eendhuizen, Emile Koster,
Martijn Hilhorst, Bert Ooms
XLC-MS: Sample Extraction and LC Separation Merged Into a Single Automated
Front-end System
The universal applicability of MS as detection technique has been a great help in streamlining the lab organization.
For sample preparation, though just as desirable, such a universal approach has not evolved so far, hampering
the total automation and integration of front-end sample prep and LC-MS. To circumvent this problem, sample
extraction is integrated with LC to permit direct injections of “raw” biological samples without prior filtration,
centrifugation or protein precipitation. With this approach, LC, on-line SPE-LC (“XLC”) and method development
can be performed without any change of hardware or MS connections. The potential of this new sample
introduction approach for MS is demonstrated by examples of the various operating modes. In LC mode, the
system can analyze standards or extracts obtained from validated LLE, protein precipitation and SPE procedures.
XLC mode can be used to analyze untreated biological samples such as plasma directly. The benefits of this
automated technique are fully explored, which means that, e.g., denaturing wash procedures were developed for
a very efficient automated on-cartridge protein precipitation. Denaturing proteins on-cartridge minimizes matrix
interferences such as ionization suppression, and also omits off-line protein precipitation and centrifugation.
Also a generic XLC method is developed to eliminate method development (MD). For more demanding assays
an automated protocol has been developed to simplify MD. The obtained data gives information on recovery,
breakthrough and tubing adsorption of the drug to be analyzed. Moreover, the obtained data can be used to point
out ionization suppression without applying post-column infusion experiments.
TP032
Morten Egeberg
Dynal Biotech ASA
Molecular Systems
P.O. Box 114 Smestad
Oslo, 0309 Norway
morten.egeberg@dynalbiotech.com
Co-Author(s)
Ingrid Manger, Tommy Rivrud,
Tine Borgen, Stine Bergholtz,
Dag Lillehaug
Magnetic Bead Based Automated Isolation of Polyhistidine-Tagged Proteins for Purification
and Target Screening
We have developed automated protocols for the isolation of recombinant polyhistidine-tagged proteins from
bacterial lysates on ThermoLabsystem’s KingFisher 96 magnetic particle processor system, in addition to Tecan’s
Genesis RSP and Beckman Coulter’s Biomek FX liquid handling robots. This method employs cobalt-based
Immobilized Metal Affinity Chromatography (IMAC) using Dynal Biotech’s super-paramagnetic beads (Dynabeads ® )
to which the BD Talon chemistry has been immobilized. Compared to technologies that employ nickel-based
IMAC, Dynabeads ® Talon are able to bind polyhistidine-tagged proteins with an enhanced selectivity. In addition,
bound polyhistidine-tagged proteins are able to be eluted using conditions less stringent than those needed when
using nickel-based IMAC. The polyhistidine-tagged proteins can be eluted from the beads, or bound to the beads
they can be used directly in downstream applications such as phage display screening, protein-protein interaction
studies and drug-target screens. By setting up this protocol on a variety of robotic systems, covering medium to
high throughput applications, we show that Dynabeads ® Talon have properties making them highly suitable for
automation. Moreover, our results demonstrates the flexibility and amenability of the Dynabeads ® Talon.
POSTER ABSTRACTS