Peptide Research Articles

8410 Lipid modification enhances the calcemic and bone building effects of injected PTH(1-14) and PTH(1-11) fragment peptides in mice

Abstract Disclosure: J. Höppner: None. H. Noda: None. A. Khatri: None. H. Jüppner: None. T.J. Gardella: None. PTH analogs with improved actions in vivo are of interest as potential treatment options for bone and mineral ion diseases such as hypoparathyroidism and osteoporosis. The efficacies in vivo of conventional PTH peptides are generally limited by rapid rates of clearance from the circulation (t1/2 = ∼ 5-30 minutes) and relatively short-dwell times on the receptor (PTH1R). This is especially true for small fragment analogs, such as M (modified)-PTH(1-14), and M-PTH(1-11), which have only been shown to be active in vitro. We explored whether PTH fragment peptide efficacy in vivo could be enhanced by addition of a lipid chain (e.g., a C15 palmitoyl chain (palm)) to either the peptide C-terminus or to the side chain of the amino acid residue at position 11 or 13. The C-tail lipid modifications were specifically designed to promote binding to serum albumin, and hence extend circulation half-life, while the lipid chains at sidechains of residue positions 11 and 13 were designed, based on structural models, to anchor the ligand to the receptor in situ -- i.e. by projection of the acyl chain between two adjacent transmembrane helices and into the surrounding plasma membrane -- to thereby extend receptor dwell time. Assessment of cAMP signaling potencies in HEK293/hPTH1R/glosensor cells revealed that both types of lipid modifications can preserve agonist potency, and that the lipid extensions at position 11 or 13 can indeed prolong receptor dwell times, as shown by extended durations of cAMP signaling after initial binding of Palm 11 or 13-modified M-PTH(1-14) and M-PTH(1-11) peptide fragment. Single subcutaneous injection of either Palm-13-M-PTH(1-14) or Palm-11-M-PTH(1-11) into mice resulted in prolonged increases in blood serum calcium levels, while 14 days of daily injection resulted in profound increases in bone mass, as measured by uCT, and marked increases in blood levels of the bone turnover markers CTX1 and P1NP, whereas non-lipidated M-PTH(1-14) control peptides were inactive in vivo. Peptide lipidation thus holds promise as a strategy to employ in the design of new PTH analogs with improved efficacies in vivo. Presentation: 6/1/2024

Ultrafast metaproteomics for quantitative assessment of strain isolates and microbiomes

BackgroundMicrobial communities are essential in human health and environmental regulation, but present a challenge for the analytical science due to their diversity and dynamic range. Tandem mass spectrometry provides functional insights on microbial life cycle, but is time-consuming. MALDI TOF excels in rapid species identification, but not functional assessment. To address critical challenges in human health and environmental sustainability, microbiology needs advanced mass spectrometry methods and bioinformatic tools enabling both rapid identification and accurate assessment of functional activity of microbial communities. ResultsWe show for the first time that both identity and functional activity of microorganisms and their communities can be accurately determined in experiments as short as 7 min per sample, using the basic Orbitrap MS configuration without peptide fragmentation. The approach was validated using strain isolates, mock microbiomes composed of bacteria spiked at known concentrations and human fecal microbiomes. Our new bioinformatic algorithm identifies the bacterial species with an accuracy of 95 %, when no prior information on the sample is available. Microbiome composition was resolved at the genus level with the mean difference between the actual and identified components of 12 %. For mock microbiomes, Pearson coefficient of up to 0.97 was achieved in estimates of strain biomass change. By the example of Rhodococcus biodegradation of n-alkanes, phenols and its derivatives, we showed the accurate assessment of functional activity of strain isolates, compared with the standard label-free and label-based approaches. SignificanceOur approach makes microbial proteomics fast, functional and insightful using the Orbitrap instruments even without employing peptide fragmentation technology. The approach can be applied to any microorganisms and can take a niche in routine functional assessment of microbial pathogens and consortiums in clinical diagnostics together with MALDI TOF MS and 16S rRNA gene sequencing.

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SignificanceIn this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

Synthesis of SARS-CoV-2 Spike Glycoprotein Peptide Fragments and Study of Their Binding to Human Blood Cells

Synthesis of SARS-CoV-2 Spike Glycoprotein Peptide Fragments and Study of Their Binding to Human Blood Cells

Prediction of Cardiovascular Events and Structural and Functional Remodeling of the Heart in Patients With Severe Mitral Regurgitation of Various Genesis Underwent Transcatheter Mitral Valve Repair "Edge-To-Edge".

To search for predictors of adverse cardiovascular events after edge-to-edge transcatheter mitral valve repair (TMVR) in patients with severe mitral regurgitation (MR) of various origins with an assessment of structural and functional remodeling of the heart and left ventricular (LV) contractile function. The study included 73 patients (median age 71 [63; 80] years, 60.3% men) at a high surgical risk with severe MR of primary and secondary genesis, who underwent TMVR. The second-generation (58.9%) and fourth-generation (41.1%) clips were implanted. In addition to standard echocardiographic (EchoCG) indices, the parameters of left heart chamber longitudinal strain and LV myocardial function were assessed at baseline, on days 4-5, and at 6 and 12 months after the intervention. Also, the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) was assessed at baseline and on days 4-5 after TMVR. A significant decrease in MR was achieved during 12 months of follow-up. In the group with primary mitral regurgitation (PMR), MR decreased from 4.0 [3.4; 4.0] to 2.0 [1.5; 2.5] at one year of follow-up (p&lt;0.001). In the group with secondary mitral regurgitation (SMR), MR decreased from 3.5 [3.0; 3.9] to 2.0 [2.0; 2.5] at 12 months of follow-up (p&lt;0.001). This effect was associated with volumetric unloading of the left heart chambers evident as a significant decrease in the volumetric indices of the left chambers and an increase in the cardiac index. In the early postoperative period, the LV function was impaired as shown by decreases in the ejection fraction (EF), global longitudinal strain (GLS), LV myocardial function parameters, and an associated increase in NT-proBNP. By 12 months of follow-up, statistically significant improvements in global constructive work (GCW) and global work index (GWI) relative to baseline values were noted in both groups without significant changes in EF and LV GLS. A strong correlation was found between LV EF and GCW (r=0.812, p&lt;0.001) and GWI (r=0.749, p&lt;0.001). The overall survival was 89%, not differing between groups (p=0.72); the absence of hospitalization for decompensated heart failure (HF) was 79.5%, also without significant differences between the groups (p=0.78). According to multivariate regression analysis, the baseline GCW value was the strongest predictor of rehospitalization for decompensated HF (relative risk (RR) 0.997; 95% confidence interval (CI) 0.995-1.000; p=0.021) and the composite endpoint (CEP) (hospitalization for decompensated HF + all-cause mortality) (RR 0.998; 95% CI 0.996-1.000; p=0.033) in the cohort with PMR. In the group with SMR, the initial degree of MR was related with rehospitalization and the CEP (OR 12.252; 95% CI 2.125-70.651; p=0.005 and OR 16.098; 95% CI 2.944-88.044; p=0.001, respectively). The most significant predictor of overall mortality in the study population was the preoperative value of LV stroke volume (OR 0.824; 95% CI 0.750-0.906; p&lt;0.001). Edge-to-edge TMVR exerts a positive effect on the prognosis and structural and functional remodeling of the heart in patients with PMR and SMR. Myocardial function indices may be useful in assessing the LV contractile function in patients with severe MR of various origins. Identification of predictors for adverse cardiovascular events, including with new EchoCG technologies, may contribute to better patient stratification.

The potential role of galectin-4 in protecting the intestinal barrier in the lined seahorse Hippocampus erectus

Galectin-4 is a prominent antimicrobial peptide primarily expressed in the gastrointestinal tract of animals. It plays crucial roles in antimicrobial defense and in regulating the repair of intestinal epithelial cells. Given the unique immune system in seahorses, which are known for male pregnancy, the function of galectin-4 in their intestinal tract, which lack gut-associated lymphoid tissues, remains unknown. In this study, we identified a galectin-4 gene (HeGal-4) in the lined seahorse (Hippocampus erectus). The sequence length of the open reading frame (ORF) was 1041 bp, encoding 346 amino acids. The mRNA expression of HeGal-4 in the intestine was significantly higher than that in other internal organs. However, the synthesized peptide fragment (HeGal-4(302–315)) from the functional domain of HeGal-4 only showed moderate inhibitory effects against Escherichia coli, Klebsiella pneumoniae, and Micrococcus luteus. Interestingly, in situ hybridization showed that HeGal-4 was predominantly expressed in seahorse intestinal epithelial cells, while genes associated with galectin-4 in the repair of intestinal epithelium, such as β-catenins, were highly expressed in their intestines. Therefore, our results suggest that HeGal-4 may be crucial for regulating the repair of intestinal epithelial cells in seahorse intestinal epithelial cells, and that it possesses significant potential for the prevention and control of intestinal diseases in seahorse aquaculture.

Theoretical investigation for the proline effect on peptide fragmentation induced by matrix-assisted laser desorption/ionization in-source decay with an oxidizing matrix

The use of an oxidizing matrix for matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) specifically induces the cleavage of Cα-C bonds on the peptide backbone, except for bonds located on the N-terminal side of proline residues. To examine the effect of Pro residues on bond cleavage induced by MALDI-ISD with an oxidizing matrix, the small dipeptides AcAA-NH2 and AcAP-NH2 were used as models. As the fragmentation is initiated by electron transfer from the peptide to the oxidizing matrix, the dissociation chemistry of the cation radical forms of the model peptides [AcAA-NH2]+• and [AcAP-NH2]+• was investigated using quantum chemistry calculations. The [AcAA-NH2]+• can produce fragment ions not only due to Cα–C bond cleavage, but also peptide bond cleavage. Quantum chemistry calculations indicated that peptide bond cleavage of [AcAA-NH2]+• occurs more slowly compared to Cα–C bond cleavage. Instead of Cα–C bond cleavage, the bond on the N-terminal side of Pro residue undergoes peptide bond cleavage during MALDI-ISD with an oxidizing matrix, due to the lack of an amide hydrogen in the Pro residue. The [AcAP-NH2]+• undergoes proton migration from the δ-carbon of the Pro residue. Depending on the proton binding site in the peptide cation radical, the peptide bond cleavage of [AcAP-NH2]+• results in the formation of either [b1]+, and [y1]•, or [a1]• and [y1]+. These theoretical results are consistent with experimental findings, and the newly proposed mechanism involving peptide cation radical formation followed by proton migration provides a more accurate model for the MALDI-ISD processes.

Mass Spectral Feature Analysis of Ubiquitylated Peptides Provides Insights into Probing the Dark Ubiquitylome.

Ubiquitylation is a structurally and functionally diverse post-translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ∼40% of ubiquitylation sites in the human proteome, i.e., "the dark ubiquitylome", including many important for human health and disease. In this meta-analysis of three large ubiquitylomic data sets, we performed a series of bioinformatic analyses to assess experimental features that could aid in uniquely identifying site-specific ubiquitylation events. Spectral predictions from Prosit were compared to experimental spectra of tryptic ubiquitylated peptides, revealing previously uncharacterized fragmentation of the diGly scar. Analysis of the LysC-derived ubiquitylated peptides reveals systematic, multidimensional peptide fragmentation, including diagnostic b-ions from fragmentation of the LysC ubiquitin scar. Comprehensively, these findings provide diagnostic spectral signatures of modification events that could be applied to new analysis methods for nontryptic ubiquitylomics.

Bioactive Peptides from Corn (Zea mays L.) with the Potential to Decrease the Risk of Developing Non-Communicable Chronic Diseases: In Silico Evaluation.

Studies have shown that corn (Zea mays L.) proteins, mainly α-zein, have the potential to act on therapeutic targets related to non-communicable chronic diseases, such as high blood pressure and type 2 diabetes. Enzymatic hydrolysis of proteins present in foods can result in a great diversity of peptides with different structures and possible bioactivities. A review of recent scientific research papers was performed to show evidence of the bioactive properties of corn peptides by in vitro assays. The α-zein amino acid sequences were identified in the UniProtKB protein database and then analyzed in the BIOPEP database to simulate enzymatic digestion and verify the potential biological action of the resulting peptides. The peptides found in the BIOPEP database were categorized according to the probability of presenting biological action using the PeptideRanker database. The aim was to use existing data to identify in silico the potential for obtaining biologically active peptides from α-zein, the main storage protein of corn. The analysis showed that the majority of peptide fragments were related to the inhibition of angiotensin-converting enzyme, followed by the inhibition of dipeptidyl peptidase IV and dipeptidyl peptidase III. Many drugs used to treat high blood pressure and type 2 diabetes work by inhibiting these enzymes, suggesting that corn peptides could be potential alternative agents. In vitro studies found that the primary bioactivity observed was antioxidative action. Both in vitro and in silico approaches are valuable for evaluating the bioactive properties resulting from protein hydrolysis, such as those found in α-zein. However, conducting in vitro studies based on prior in silico evaluation can be more efficient and cost-effective.

Nucleoprotein phase-separation affinities revealed via atomistic simulations of short peptide and RNA fragments.

Liquid-liquid phase separation of proteins and nucleic acids into condensate phases is a versatile mechanism for ensuring compartmentalization of cellular biochemistry. RNA molecules play critical roles in these condensates, particularly in transcriptional regulation and stress responses, exhibiting a wide range of thermodynamic and dynamic behaviors. However, deciphering the molecular grammar that governs the stability and dynamics of protein-RNA condensates remains challenging due to the multicomponent and heterogeneous nature of these biomolecular mixtures. In this study, we employ atomistic simulations of twenty distinct mixtures containing minimal RNA and peptide fragments to dissect the phase-separating affinities of all twenty amino acids in the presence of RNA. Our findings elucidate chemically specific interactions, hydration profiles, and ionic effects that synergistically promote or suppress protein-RNA phase separation. We map a ternary phase diagram of interactions, identifying four distinct groups of residues that promote, maintain, suppress, or disrupt protein-RNA clusters.

Direct mapping of tyrosine sulfation states in native peptides by nanopore.

Sulfation is considered the most prevalent post-translational modification (PTM) on tyrosine; however, its importance is frequently undervalued due to difficulties in direct and unambiguous determination from phosphorylation. Here we present a sequence-independent strategy to directly map and quantify the tyrosine sulfation states in universal native peptides using an engineered protein nanopore. Molecular dynamics simulations and nanopore mutations reveal specific interactions between tyrosine sulfation and the engineered nanopore, dominating identification across diverse peptide sequences. We show a nanopore framework to discover tyrosine sulfation in unknown peptide fragments digested from a native protein and determine the sequence of the sulfated fragment based on current blockade enhancement induced by sulfation. Moreover, our method allows direct observation of peptide sulfation in ultra-low abundance, down to 1%, and distinguishes it from isobaric phosphorylation. This sequence-independent strategy suggests the potential of nanopore to explore specific PTMs in real-life samples and at the omics level.

A Chemical Approach to Assess the Impact of Post-translational Modification on MHC Peptide Binding and Effector Cell Engagement.

The human major histocompatibility complex (MHC) plays a pivotal role in the presentation of peptidic fragments from proteins, which can originate from self-proteins or from nonhuman antigens, such as those produced by viruses or bacteria. To prevent cytotoxicity against healthy cells, thymocytes expressing T cell receptors (TCRs) that recognize self-peptides are removed from circulation (negative selection), thus leaving T cells that recognize nonself-peptides. Current understanding suggests that post-translationally modified (PTM) proteins and the resulting peptide fragments they generate following proteolysis are largely excluded from negative selection; this feature means that PTMs can generate nonself-peptides that potentially contribute to the development of autoreactive T cells and subsequent autoimmune diseases. Although it is well-established that PTMs are prevalent in peptides present on MHCs, the precise mechanisms by which PTMs influence the antigen presentation machinery remain poorly understood. In the present work, we introduce chemical modifications mimicking PTMs on synthetic peptides. This is the first systematic study isolating the impact of PTMs on MHC binding and also their impact on TCR recognition. Our findings reveal various ways PTMs alter antigen presentation, which could have implications for tumor neoantigen presentation.

Lipid Selectivity of Membrane Action of the Fragments of Fusion Peptides of Marburg and Ebola Viruses.

The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.

Comparison of casein glycations induced by lactose and lactulose

Effects of lactose and lactulose on the casein glycation process at different times and specific glycation sites were investigated. Lactulose treatment generated higher levels of initial glycation products fructoselysine (1.160 μg/mL) and methylglyoxal (0.266 mg/mL) than lactose treatment which had 0.959 μg/mL and 0.140 mg/mL, respectively. The concentrations of intermediate products Nε‐(Carboxymethyl) lysine (0.654 μg/mL) and Nε‐(Carboxyethyl) lysine (0.154 μg/mL) in the lactulose treatment were approximately 2.5 and 1.1 times higher than these in the lactose treatment, respectively. The glycation induced by lactulose targeted Lys residues and had more glycated peptide fragments and wide‐ranging glycation sites in αs1‐casein. The high degree of casein glycation in lactulose treatment was attributed by the fructose on lactulose which had different pathways in the Maillard reaction, compared with the glucose on lactose. The findings revealed the differences of casein glycations by the two sugars and during thermal processing. The information could be helpful in developing potential approaches to monitor and control glycation reactions in dairy products.

New targets and designed inhibitors of ASAP Arf-GAPs derived from structural characterization of the ASAP1/440-kD ankyrin-B interaction

ASAP1 and its paralog ASAP2 belong to a PI4,5P2-dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a non-canonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, β-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a Kd ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes.

Proteome-Wide Assessment of Protein Structural Perturbations under High Pressure

One of the planet's more understudied ecosystems is the deep biosphere, where organisms can experience high hydrostatic pressures (30–110 MPa); yet, by current estimates, these subsurface and deep ocean zones host the majority of the Earth's microbial and animal life. The extent to which terrestrially relevant pressures up to 100 MPa deform most globular proteins—and which kinds—has not been established. Here, we report the invention of an experimental apparatus that enables structural proteomic methods to be carried out at high pressures for the first time. The method, called high-pressure limited proteolysis (Hi-P LiP), involves performing pulse proteolysis on whole cell extracts brought to high pressure. The resulting sites of proteolytic susceptibility induced by pressure are subsequently read out by sequencing the peptide fragments with tandem liquid chromatography–mass spectrometry. The method sensitively detects pressure-induced structural changes with residue resolution and on whole proteomes, providing a deep and broad view of the effect of pressure on protein structure. When applied to a piezosensitive thermophilic bacterium, , we find that approximately 40% of its soluble proteome is structurally perturbed at 100 MPa. Proteins with lower charge density are more resistant to pressure-induced deformation, as expected; however, contrary to expectations, proteins with lower packing density (i.e., more voids) are also more resistant to deformation. Furthermore, high pressure has previously been shown to preferentially alter conformations around active sites. Here, we show this is also observed in Hi-P LiP, suggesting that the method could provide a generic and unbiased modality to detect binding sites on a proteome scale. Hence, data sets of this kind could prove useful for training emerging artificial intelligence models to predict cryptic binding sites with greater accuracy. Published by the American Physical Society 2024

Magnetic mesoporous silica nanoparticles loaded with peptides for the targeted repair of cavernous nerve injury underlying erectile dysfunction

Erectile dysfunction (ED) is a common male sexual disorder characterized by repeated or persistent difficulty in achieving or maintaining an erection. It can arise from various factors, with cavernous nerve injury (CNI) from radical prostatectomy being a predominant cause of iatrogenic ED, posing significant clinical concerns. The complexity of cavernous tissue damage in CNI-induced ED (CNIED) often results in poor efficacy and resistance to conventional vascular ED treatments. To address CNI-induced ED, this study developed a system of magnetic mesoporous silica nanoparticles (MSNs) loaded with peptides for targeted treatment. Core-shell Fe3O4-coated MSNs were used as drug carriers and loaded with RADA16-I/RAD-RGI peptides (PD) to create a neurotrophic microenvironment to treat peripheral nerve defects. Furthermore, the neuro-targeting peptide HLNILSTLWKYR (PT) was grafted onto MSNs. The in vivo therapeutic effect was evaluated using a rat bilateral cavernous nerve injury (BCNI) model. The results showed that the neuro-targeted Fe3O4@SiO2-PT-PD nanoparticles significantly promoted regeneration of the cavernous nerve and restored erectile function. This promising strategy offers significant clinical potential for treating CNI-induced ED. Nanomedicine technology has the potential to not only improve treatment outcomes but also reduce side effects in healthy cells, paving the way for more accurate targeted repair of cavernous nerve damage.

Pathogenesis of human atheroma necrotic core: degradation of connective tissue fibers and possible involvement of cathepsin K

BackgroundAtheroma is a serious atherosclerotic lesion related to plaque rupture, thrombosis, and ischemic disease. To clarify the pathogenesis of human atheroma, particularly the formation of the liponecrotic tissue in the necrotic core, the distribution of connective tissue fibers, such as collagen and elastic fibers, and related substances was investigated in this study.MethodsAtherosclerotic lesions in human coronary arteries were classified into three categories: pathologic intimal thickening (PIT), atheroma with lipid core (ALC), and atheroma with necrotic core (ANC). PIT and ALC consisted of the lipid pool and the lipid core, respectively. The necrotic core of ANC was composed of a lipid core-like region and liponecrotic tissue containing amorphous materials and lacking cells and connective tissue fibers. The distribution of collagen type I, elastin, C-terminal crosslinked telopeptide of collagen type I (CTX-I), and cathepsin K (CatK) was investigated through immunohistochemistry. CTX-I is a fragmented peptide consisting of the C-terminal region of collagen type I that is generated by CatK. The distribution of denatured and unfolded collagen chains was also investigated through collagen hybridizing peptide staining.ResultsCollagen type I, denatured and unfolded collagen chains, and elastin were positively stained in the PIT, ALC, and lipid core-like region of ANC. However, they were negatively stained in the liponecrotic tissue of ANC. CTX-I and CatK were positively stained in all four regions, and their grade of staining appeared to show a positive relationship. Both CTX-I and CatK demonstrated higher staining grades in the liponecrotic tissue. These findings suggested that pre-existing collagen type I was severely degraded by CatK, resulting in the production of CTX-I in the liponecrotic tissue, and that pre-existing elastin was also degraded in this region. CatK was mainly found in macrophage foam cells, many of which were localized within and around the liponecrotic tissue.ConclusionsThis study suggests that the liponecrotic tissue in the necrotic core of human atheroma is formed by severe degradation of pre-existing connective tissue fibers and consequent collapse of tissue structure. This degradation is likely caused by proteolytic enzymes, such as CatK, secreted by macrophage foam cells.

NovoBoard: A Comprehensive Framework for Evaluating the False Discovery Rate and Accuracy of De Novo Peptide Sequencing

De novo peptide sequencing is one of the most fundamental research areas in mass spectrometry (MS) based proteomics. Many methods have often been evaluated using a couple of simple metrics that do not fully reflect their overall performance. Moreover, there has not been an established method to estimate the false discovery rate (FDR) of de novo peptide-spectrum matches (PSMs). Here we propose NovoBoard, a comprehensive framework to evaluate the performance of de novo peptide sequencing methods. The framework consists of diverse benchmark datasets (including tryptic, nontryptic, immunopeptidomics, and different species), and a standard set of accuracy metrics to evaluate the fragment ions, amino acids, and peptides of the de novo results. More importantly, a new approach is designed to evaluate de novo peptide sequencing methods on target-decoy spectra and to estimate and validate their FDRs. Our FDR estimation provides valuable information to assess the reliability of new peptides identified by de novo sequencing tools, especially when no ground-truth information is available to evaluate their accuracy. The FDR estimation can also be used to evaluate the capability of de novo peptide sequencing tools to distinguish between de novo PSMs and random matches. Our results thoroughly reveal the strengths and weaknesses of different de novo peptide sequencing methods, and how their performances depend on specific applications and the types of data.

O67 URINARY PROTEOMIC PROFILING REVEALS MOLECULAR CUES UNDERLYING HIGHER HYPERTENSION SUSCEPTIBILITY IN BLACKS THAN WHITES

Background and Objective: Compared with Whites, Blacks are more prone to hypertension and salt-sensitivity. The objective was to investigate whether sequenced urinary peptide fragments derived from proteins with a key role in cardiovascular or renal structure or function have different levels in sub-Saharan Blacks compared with Whites. Methods: This individual-participant meta-analysis included individuals enrolled in African-PREDICT (476 Blacks and 483 Whites), FLEMENGHO (720 Whites), PROVALID-Austria (467 Whites) and UPRIGHT-HTM (106 Nigerian Blacks and 61 Polish and Slovenian Whites). Urinary peptides were quantified by capillary electrophoresis combined with mass spectrometry. Results: Among 2313 participants (49.7% women), median age was 40.9 years. Of 513 urinary peptides with 70% prevalence, 300 had Bonferroni-corrected significantly different levels among Black and White South Africans sharing the same environment. Analyses contrasting 582 Blacks vs 1731 Whites, sub-Saharan Blacks (582) vs European Whites (1248) replicated the findings. Combining these results with information from public databases, we identified COL4A1, COL4A2, CD99, FAT1, FGA, FXYD2, MGP, MYOCD, PCDH7, PROC, and UMOD as most likely candidates underlying the different susceptibility to hypertension and salt sensitivity between Blacks and Whites. The most relevant enriched pathways were related to hemostasis, platelet activity, collagens and biology of the extracellular matrix, and protein digestion and absorption. Disease Ontology terms comprised small-vessel disease and coronary and macrovascular atherosclerotic disease. Conclusions: MGP and MYOCD being involved in cardiovascular function, FGA in clot formation, FYXD2 and UMOD in salt homeostasis, and COL4A1 and COL4A2 as major component of the glomerular basement membrane are proteins deserving further exploration in molecular and human studies as potential targets for intervention to reduce the excess hypertension risk and associated complications in Blacks.