Presentation 1 - Agilent’s advanced solutions for front end proteomic
sample preparation and proteomics research
By Dr. Rudolf Grimm
Abstract:
In recent years proteomics has emerged as an essential field within the systems biology
approach to understand complex pathways and mechanisms in living organisms.
Although proteomics is now widely applied to solve biological questions it still suffers
from many technical and methodological limitations.
Typical proteomics samples such as human body fluids are characterized by their
incredible complexity and by proteins concentrations that span 10-12 orders of
magnitude. Furthermore, post-translational modifications present additional significant
challenges to scientists as the variability in the degree of protein modification can result
in significant microheterogeneities. As the majority of proteins are post-translationally
modified such as by phosphorylation or glycosylation, efficient protein prefractionation
with high-resolution protein separation techniques is mandatory.
In this presentation a new solution-based isoelectric focusing technique (OGE) for the
efficient prefractionation of peptides and proteins as well as a new high-resolution HPLC
column (mRP) for peptide and protein separations will be discussed in detail. The mRP
column also can be applied for the efficient separation of intact membrane proteomics
samples as well as for the efficient delipidation of complex protein samples. In addition,
the latest developments in our immunodepletion column technology will be presented.
After successful protein prefractionation protein digests are further characterized with
new advanced mass spectrometry techniques. Ion trap mass spectrometers with HPLCChip interface and rapid CID-ETD switching capabilities allow the sensitive and
comprehensive analysis of post-translational modifications with precise determination of
even multiple phosphorylation and/or glycosylation sites. High mass accuracy and high
resolution peptide sequencing with ion trap comparable sensitivity on various samples
will be demonstrated with the new Agilent QTOF mass spectrometer.
Presentation 2 - OFFGEL Electrophoresis and LC-MS : A New
Multidimensional Approach to Mining of Low Abundant Proteins in
Complex Samples
By Dr. Chen Wei
Abstract:
Sample complexity and a large dynamic range of protein concentrations present major
challenges for today’s proteomics scientists and strategies to simplification of samples
are emerging. OFFGEL electrophoresis is a novel prefractionation technique jointly
developed by Agilent Technologies and Diagnoswiss which fractionates proteins or
peptides according to their isoelectric point (pI). It is based on the high resolving power
and proven technology of immobilized pH-gradient (IPG) gel but provides analytes in
solution after fractionation. This makes the technology directly compatible with LC-MS
and upfront sample preparation techniques, e.g. immunodepletion and mRP-C18. In
addition, the pI is a useful parameter for validation of peptide hits.