Salivary gland tumors are highly variable in clinical presentation and histology. The World Health Organization (WHO) classifies 22 types of malignant and 11 types of benign tumors of the salivary glands. Diagnosis of salivary gland tumors is based on imaging (ultrasound, magnetic resonance imaging) and fine-needle aspiration biopsy, but the final diagnosis is based on histopathological examination of the removed tumor tissue. In this pilot study, we are testing a new approach to identifying peptide biomarkers in saliva that can be used to diagnose salivary gland tumors. The research material for the peptidomic studies was extracts from washings of neoplastic tissues and healthy tissues (control samples). At the same time, saliva samples from patients and healthy individuals were analyzed. The comparison of the peptidome composition of tissue extracts and saliva samples may allow the identification of potential peptide markers of salivary gland tumors in patients\' saliva. The peptidome compositions extracted from 18 tumor and 18 healthy tissue samples, patients\' saliva samples (11 samples), and healthy saliva samples (8 samples) were analyzed by LC-MS tandem mass spectrometry. A group of 109 peptides was identified that were present only in the tumor tissue extracts and in the patients\' saliva samples. Some of the identified peptides were derived from proteins previously suggested as potential biomarkers of salivary gland tumors (ANXA1, BPIFA2, FGB, GAPDH, HSPB1, IGHG1, VIM) or tumors of other tissues or organs (SERPINA1, APOA2, CSTB, GSTP1, S100A8, S100A9, TPI1). Unfortunately, none of the identified peptides were present in all samples analyzed. This may be due to the high heterogeneity of this type of cancer. The surprising result was that extracts from tumor tissue did not contain peptides derived from salivary gland-specific proteins (STATH, SMR3B, HTN1, HTN3). These results could suggest that the developing tumor suppresses the production of proteins that are essential components of saliva.

BACKGROUND: Association of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-β2 microglobulin (hβ2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype.
METHODS: Genetic interaction was studied between activin/transforming growth factor β (TGFβ) pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hβ2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFβ pathways induced by its ligands, and the transcript level of target genes of the TGFβ pathway, were evaluated.
RESULTS: In HLA-B27/hβ2m transgenic Drosophila, inappropriate signalling through the activin/TGFβ pathway, involving Baboon (Babo), the type I activin/TGFβ receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hβ2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFβ receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from B27 rats, showing evidence for deregulated TGFβ pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFβ exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern.
CONCLUSIONS: This study shows that HLA-B27 alters the TGFβ pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.

UNASSIGNED: With the aging of populations worldwide, Alzheimer\'s disease (AD) has become a concern due to its high prevalence and the continued lack of established treatments. Early diagnosis is required as a preventive intervention to modify the disease\'s progression. In our previous study, we performed peptidomic analysis of serum samples obtained from AD patients and age-matched healthy subjects to seek peptide biomarker candidates for AD by using BLOTCHIP-MS analysis, and identified four peptides as AD biomarker candidates.
UNASSIGNED: The objective was to validate the serum biomarker peptides to distinguish mild cognitive impairment (MCI) and AD in comparison to cognitively healthy controls using a new peptidome technology, the Dementia Risk Test.
UNASSIGNED: We enrolled 195 subjects with normal cognitive function (NC; n = 70), MCI (n = 55), and AD (n = 70), The concentrations of cognitive impairment marker peptides (Fibrinogen α chain (FAC), Fibrinogen β chain (FBC), Plasma protease C1 inhibitor (PPC1I), α2-HS-glycoprotein (AHSG)) were quantified by using a selected reaction monitoring assay based on liquid chromatography-MS/MS.
UNASSIGNED: The present study confirmed that three peptides, FAC, FBC, and PPC1I, were significantly upregulated during the onset of AD. This three-peptide set was both highly sensitive in determining AD (sensitivity: 85.7%, specificity: 95.7%, AUC: 0.900) and useful in distinguishing MCI (sensitivity: 61.8%, specificity: 98.6%, AUC: 0.824) from NC.
UNASSIGNED: In this validation study, we confirmed the high diagnostic potential of the three peptides identified in our previous study as candidate serum biomarkers for AD. The Dementia Risk Test may be a powerful tool for detecting AD-related pathological changes.

Starvation is a complex physiological state that induces changes in protein expression to ensure survival. The insect midgut is sensitive to changes in dietary content as it is at the forefront of communicating information about incoming nutrients to the body via hormones. Therefore, a DIA proteomics approach was used to examine starvation physiology and, specifically, the role of midgut neuropeptide hormones in a representative lepidopteran, Manduca sexta. Proteomes were generated from midguts of M. sexta fourth-instar caterpillars, starved for 24 h and 48 h, and compared to fed controls. A total of 3047 proteins were identified, and 854 of these were significantly different in abundance. KEGG analysis revealed that metabolism pathways were less abundant in starved caterpillars, but oxidative phosphorylation proteins were more abundant. In addition, six neuropeptides or related signaling cascade proteins were detected. Particularly, neuropeptide F1 (NPF1) was significantly higher in abundance in starved larvae. A change in juvenile hormone-degrading enzymes was also detected during starvation. Overall, our results provide an exploration of the midgut response to starvation in M. sexta and validate DIA proteomics as a useful tool for quantifying insect midgut neuropeptide hormones.

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the β-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Millions of people worldwide currently suffer from chronic kidney disease (CKD), requiring kidney replacement therapy at the end stage. Endeavors to better understand CKD pathophysiology from an omics perspective have revealed major molecular players in several sample sources. Focusing on non-invasive sources, gut microbial communities appear to be disturbed in CKD, while numerous human urinary peptides are also dysregulated. Nevertheless, studies often focus on isolated omics techniques, thus potentially missing the complementary pathophysiological information that multidisciplinary approaches could provide. To this end, human urinary peptidome was analyzed and integrated with clinical and fecal microbiome (16S sequencing) data collected from 110 Non-CKD or CKD individuals (Early, Moderate, or Advanced CKD stage) that were not undergoing dialysis. Participants were visualized in a three-dimensional space using different combinations of clinical and molecular data. The most impactful clinical variables to discriminate patient groups in the reduced dataspace were, among others, serum urea, haemoglobin, total blood protein, urinary albumin, urinary erythrocytes, blood pressure, cholesterol measures, body mass index, Bristol stool score, and smoking; relevant variables were also microbial taxa, including Roseburia, Butyricicoccus, Flavonifractor, Burkholderiales, Holdemania, Synergistaceae, Enterorhabdus, and Senegalimassilia; urinary peptidome fragments were predominantly derived from proteins of collagen origin; among the non-collagen parental proteins were FXYD2, MGP, FGA, APOA1, and CD99. The urinary peptidome appeared to capture substantial variation in the CKD context. Integrating clinical and molecular data contributed to an improved cohort separation compared to clinical data alone, indicating, once again, the added value of this combined information in clinical practice.

Snake venom peptidomes are known to be a large source of molecules with different pharmacological properties. The complexity and variability of snake venoms, the presence of proteinases, and the lack of complete species-specific genome sequences make snake venom peptidome profiling a challenging task that requires especial technical strategies for sample processing and mass spectrometric analysis. Here, we describe a method for assessing the content of snake venom peptides and highlight the importance of sampling procedures, as they substantially influence the peptidomic complexity of snake venoms.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Peptidomics is the comprehensive characterization of peptides from biological sources instead of heading for a few single peptides in former peptide research. Mass spectrometry allows to detect a multitude of peptides in complex mixtures and thus enables new strategies leading to peptidomics. The term was established in the year 2001, and up to now, this new field has grown to over 3000 publications. Analytical techniques originally developed for fast and comprehensive analysis of peptides in proteomics were specifically adjusted for peptidomics. Although it is thus closely linked to proteomics, there are fundamental differences with conventional bottom-up proteomics. Fundamental technological advancements of peptidomics since have occurred in mass spectrometry and data processing, including quantification, and more slightly in separation technology. Different strategies and diverse sources of peptidomes are mentioned by numerous applications, such as discovery of neuropeptides and other bioactive peptides, including the use of biochemical assays. Furthermore, food and plant peptidomics are introduced similarly. Additionally, applications with a clinical focus are included, comprising biomarker discovery as well as immunopeptidomics. This overview extensively reviews recent methods, strategies, and applications including links to all other chapters of this book.

Salt stress significantly impedes plant growth and the crop yield. This study utilized de novo transcriptome assembly and ribosome profiling to explore mRNA translation\'s role in rice salt tolerance. We identified unrecognized translated open reading frames (ORFs), including 42 upstream transcripts and 86 unannotated transcripts. A noteworthy discovery was the role of a small ORF, Ospep5, in conferring salt tolerance. Overexpression of Ospep5 in plants increased salt tolerance, while its absence led to heightened sensitivity. This hypothesis was corroborated by the findings that exogenous application of the synthetic small peptide Ospep5 bolstered salt tolerance in both rice and Arabidopsis. We found that the mechanism underpinning the Ospep5-mediated salt tolerance involves the maintenance of intracellular Na+/K+ homeostasis, facilitated by upregulation of high-affinity potassium transporters (HKT) and Na+/H+ exchangers (SOS1). Furthermore, a comprehensive multiomics approach, particularly ribosome profiling, is instrumental in uncovering unannotated ORFs and elucidating their functions in plant stress responses.