Biology Reference
In-Depth Information
In this context, blood still serves as the most prominent human
specimen for all kinds of analyses, even though it is characterized
by an enormous diversity of proteins covering a concentration
range of at least 10 orders of magnitude ( 1 ). The patients' good
acceptance of phlebotomy and its nearly unlimited availability
tempts one to underestimate the diffi culties accompanied with its
special composition.
Discovery and development of disease-related biomarkers,
which are defi ned as a “characteristic that is objectively measured
and evaluated as an indicator of normal biologic processes, patho-
genic processes, or pharmacologic responses to a therapeutic inter-
vention” ( 2 ), reveal a huge potential for substantial improvement
in the early diagnosis and prognosis of diseases. Nevertheless, the
complex and exhausting discovery and validation processes hinder
the availability of novel biomarkers for the implementation into
clinical practice, which is refl ected by the very low number of FDA-
approved biomarkers over the last couple of years. In view of these
complex challenges, continuous improvements in study design,
sample quality, reproducibility, as well as standardization of sample
processing and harmonized data analysis are needed to provide the
indispensable basis for driving biomarker usage into clinical practice.
Especially in face of the complexity of the workfl ow and the
enormous biological variability, it appears that an extraordinary
diligence in each step of the analysis is of great importance. This
begins with the fi rst experimental step, i.e., sample collection and
preparation, which is frequently underestimated. In this context,
the close collaboration between the different scientifi c disciplines
and the development of standard operation procedures is of
particular importance.
To pave the way for the identifi cation of novel disease-
dependent marker proteins from blood, extensive fractionation
procedures are often enforced. Several approaches have been
developed (for an overview see ref. ( 3 )), reducing sample complex-
ity or the amount of high-abundant proteins in order to overcome
suppression of the low-abundant ones, e.g., tissue leakage proteins.
However, some methods dilute the sample, lack specifi city, or
remove proteins of interest in an uncontrolled way eventually
leading to a loss of potentially important diagnostic information ( 4 ).
Here we introduce a valuable alternative “single-step” sample prep-
aration approach utilizing bead-based combinatorial hexapeptide
ligand libraries, being compatible with a broad spectrum of profi l-
ing techniques, including all kinds of gel electrophoresis and also
mass spectrometry-based approaches like SELDI or LC-MS.
Hexapeptides or other short sequences of amino acids offer non-
covalent binding sites for proteins due to the physicochemical
properties being defi ned by the type and sequence of the amino
acids (for an overview see ref. ( 5 )). If a hexapeptide library is cou-
pled to carrier beads and exposed to complex protein mixtures,
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