5 research outputs found
The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts
The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts. June 4-7, 2019, Szczyrk, Polan
Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm
Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables
Mass spectrometry versus conventional techniques of proteindetection : Zika virus NS3 protease activity towards cellular proteins
Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process
Advances in the Study of Aptamer–Protein Target Identification Using the Chromatographic Approach
Ever since the development of the
process known as the systematic
evolution of ligands by exponential enrichment (SELEX), aptamers have
been widely used in a variety of studies, including the exploration
of new diagnostic tools and the discovery of new treatment methods.
Aptamers’ ability to bind to proteins with high affinity and
specificity, often compared to that of antibodies, enables the search
for potential cancer biomarkers and helps us understand the mechanisms
of carcinogenesis. The blind spot of those investigations is usually
the difficulty in the selective extraction of targets attached to
the aptamer. There are many studies describing the cell SELEX for
the prime choice of aptamers toward living cancer cells or even whole
tumors in the animal models. However, a dilemma arises when a large
number of proteins are being identified as potential targets, which
is often the case. In this article, we present a new analytical approach
designed to selectively target proteins bound to aptamers. During
studies, we have focused on the unambiguous identification of the
molecular targets of aptamers characterized by high specificity to
the prostate cancer cells. We have compared four assay approaches
using electrophoretic and chromatographic methods for “fishing
out” aptamer protein targets followed by mass spectrometry
identification. We have established a new methodology, based on the
fluorescent-tagged oligonucleotides commonly used for flow-cytometry
experiments or as optic aptasensors, that allowed the detection of
specific aptamer–protein interactions by mass spectrometry.
The use of atto488-labeled aptamers for the tracking of the formation
of specific aptamer–target complexes provides the possibility
of studying putative protein counterparts without needing to apply
enrichment techniques. Significantly, changes in the hydrophobic properties
of atto488-labeled aptamer–protein complexes facilitate their
separation by reverse-phase chromatography combined with fluorescence
detection followed by mass-spectrometry-based protein identification.
These comparative results of several methodological approaches confirmed
the universal applicability of this method to studying aptamer–protein
interactions with high sensitivity, showing superior properties compared
with pull-down techniques