Search

Naturally occurring peptides, including growth factors, hormones, and neurotransmitters, represent an important class of biomolecules and have crucial roles in human physiology. The study of these peptides in clinical samples is therefore as relevant as ever. Compared to more routine proteomics applications in clinical research, peptidomics research questions are more challenging and have special requirements with regard to sample handling, experimental design, and bioinformatics. In this review, we describe the issues that confront peptidomics in a clinical context. After these hurdles are (partially) overcome, peptidomics will be ready for a successful translation into medical practice. [ABSTRACT FROM AUTHOR]

Background: The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays. Results: We established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts. Conclusions: The described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants. [ABSTRACT FROM AUTHOR]

Despite decades of research, no early-onset biomarkers are currently available for Alzheimer's disease, a cureless neurodegenerative disease afflicting millions worldwide. In this study, transgenic Caenorhabditis elegans were used to investigate changes in the metabolome after induced expression of amyloid-β. GC- and LC-MS-based platforms determined a total of 157 differential features. Some of these were identified using in- house (GC-MS) or public libraries (LC-MS), revealing changes in allantoin, cystathionine and tyrosine levels. Since C. elegans is far better suited to metabolomics studies than most other model systems, the accordance of these findings with vertebrate literature is promising and argues for further use of C. elegans as a model of human pathology in the study of AD. [ABSTRACT FROM AUTHOR]

Immunopeptidomics is becoming an increasingly important field of study. The capability to identify immunopeptides with pivotal roles in the human immune system is essential to shift the current curative medicine towards personalized medicine. Throughout the years, the field has matured, giving insight into the current pitfalls. Nowadays, it is commonly accepted that generalizing shotgun proteomics workflows is malpractice because immunopeptidomics faces numerous challenges. While many of these difficulties have been addressed, the road towards the ideal workflow remains complicated. Although the presence of Posttranslational modifications (PTMs) in the immunopeptidome has been demonstrated, their identification remains highly challenging despite their significance for immunotherapies. The large number of unpredictable modifications in the immunopeptidome plays a pivotal role in the functionality and these challenges. This review provides a comprehensive overview of the current advancements in immunopeptidomics. We delve into the challenges associated with identifying PTMs within the immunopeptidome, aiming to address the current state of the field. [ABSTRACT FROM AUTHOR]

In differential peptidomics, peptide profiles are compared between biological samples and the resulting expression levels are correlated to a phenotype of interest. This, in turn, allows us insight into how peptides may affect the phenotype of interest. In quantitative differential peptidomics, both label-based and label-free techniques are often employed. Label-based techniques have several advantages over label-free methods, primarily that labels allow for various samples to be pooled prior to liquid chromatography-mass spectrometry (LC-MS) analysis, reducing between-run variation. Here, we detail a method for performing quantitative peptidomics using stable amine-binding isotopic and isobaric tags.

The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the "peptidome" of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC-MALDI-TOF MS and on-line 2D-nanoLC-Q-TOF MS/MS) and the necessary instrumentation are described.

Endocrine tissues like the pituitary, hypothalamus and islets of Langerhans are rich in bioactive peptides. These are used for intercellular signalling and are involved in regulation of almost all physiological processes. Peptidomics is the comprehensive analysis of peptides in tissues, fluids and cells. Peptidomics applied to (neuro-)endocrine tissues aims therefore to identify as many bioactive peptides as possible. Peptidomics of (neuro-)endocrine tissues requires an integrated approach that consists of careful sample handling, peptide separation techniques, mass spectrometry and bioinformatics. Here we describe the methods for isolation and dissection of endocrine tissues, the extraction of bioactive peptides and further sample handling and identification of peptides by mass spectrometry and hyphenated techniques. We also present a straightforward method for the comparison of relative levels of bioactive peptides in these endocrine tissues under varying physiological conditions. The latter helps to elucidate functions of the bioactive peptides.