In the past decade, major developments in instrumentation and methodology have been achieved in proteomics. For proteome investigations of complex biological samples derived from cell cultures, tissues, or whole organisms, several techniques are state of the art. Especially, many improvements have been undertaken to quantify differences in protein expression between samples from, e.g., treated vs. untreated cells and healthy vs. control patients. In this review, we give a brief insight into the main techniques, including gel-based protein separation techniques, and the growing field of mass spectrometry.
As novel liquid chromatography–mass
spectrometry (LC-MS)
technologies for proteomics offer a substantial increase in LC-MS
runs per day, robust and reproducible sample preparation emerges as
a new bottleneck for throughput. We introduce a novel strategy for
positive-pressure 96-well filter-aided sample preparation (PF96) on
a commercial positive-pressure solid-phase extraction device. PF96
allows for a five-fold increase in throughput in conjunction with
extraordinary reproducibility with Pearson product-moment correlations
on the protein level of
r
= 0.9993, as demonstrated
for mouse heart tissue lysate in 40 technical replicates. The targeted
quantification of 16 peptides in the presence of stable-isotope-labeled
reference peptides confirms that PF96 variance is barely assessable
against technical variation from nanoLC-MS instrumentation. We further
demonstrate that protein loads of 36–60 μg result in
optimal peptide recovery, but lower amounts ≥3 μg can
also be processed reproducibly. In summary, the reproducibility, simplicity,
and economy of time provide PF96 a promising future in biomedical
and clinical research.
Background: Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII.
Objective:The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model.
Methods:A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models.Results: These hF12 KI mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12 KI mice in an arterial thrombosis model without affecting bleeding times.
Conclusion:These data establish the newly generated hF12 KI mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.
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