Omics to Unveil Diabetes Mellitus Pathogenesis and Biomarkers: Focus on Proteomics, Lipidomics, and Metabolomics.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by elevated blood sugar levels, resulting from either body's inability to produce or effectively utilize insulin. There are several types of DM, but the most common are type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes mellitus (GDM). DM is a complex disease and a global health concern, and the current clinical markers, such as fasting glucose, are helpful in the diagnosis of DM, but are not specific and sensitive, especially when measured on the beginning of the pathogenesis. Therefore, there is a pressing need to discover new early biomarkers that can provide an early diagnosis. Omics is an important field for the discovery of potential new biomarkers, especially proteomics, metabolomics, and lipidomics, where techniques such as liquid chromatography, mass spectrometry, and nuclear magnetic resonance are utilized to identify novel DM biomarkers and their pathways. In this review, we report papers that applied omics in the context of DM to identify new markers and their relationship with this disease, with the aim of elucidating new diagnostic techniques for the main types of DM.

Cardioproteomics: Insights on Cardiovascular Diseases.

Cardiovascular diseases (CVDs) remain a global health challenge and are the leading cause of deaths worldwide. Proteomics has emerged as a valuable tool for unraveling the complex molecular mechanisms underlying CVDs, offering insights into biomarker discovery, drug targets, and personalized medicine. This review explores key breakthroughs in proteomic applications related to CVDs, mainly coronary artery disease (CAD), ischemic heart diseases such as myocardial infarction (MI), and cardiomyopathies. Notable findings include potential biomarkers, therapeutic targets, and insights into disease pathogenesis. The review highlights the importance of proteomics in advancing our understanding of CVDs and shaping future therapeutic approaches.

Proteomic-Based Studies on Memory Formation in Normal and Neurodegenerative Disease-Affected Brains.

A critical aspect of cognition is the ability to acquire, consolidate, and evoke memories, which is considerably impaired by neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. These mnemonic processes are dependent on signaling cascades, which involve protein expression and degradation. Recent mass spectrometry (MS)-based proteomics has opened a range of possibilities for the study of memory formation and impairment, making it possible to research protein systems not studied before. However, in the context of synaptic proteome related to learning processes and memory formation, a deeper understanding of the synaptic proteome temporal dynamics after induction of synaptic plasticity and the molecular changes underlying the cognitive deficits seen in neurodegenerative diseases is needed. This review analyzes the applications of proteomics for understanding memory processes in both normal and neurodegenerative conditions. Moreover, the most critical experimental studies have been summarized using the PANTHER overrepresentation test. Finally, limitations associated with investigations of memory studies in physiological and neurodegenerative disorders have also been discussed.

Neuroproteomics: Unveiling the Molecular Insights of Psychiatric Disorders with a Focus on Anxiety Disorder and Depression.

Anxiety and depression are two of the most common mental disorders worldwide, with a lifetime prevalence of up to 30%. These disorders are complex and have a variety of overlapping factors, including genetic, environmental, and behavioral factors. Current pharmacological treatments for anxiety and depression are not perfect. Many patients do not respond to treatment, and those who do often experience side effects. Animal models are crucial for understanding the complex pathophysiology of both disorders. These models have been used to identify potential targets for new treatments, and they have also been used to study the effects of environmental factors on these disorders. Recent proteomic methods and technologies are providing new insights into the molecular mechanisms of anxiety disorder and depression. These methods have been used to identify proteins that are altered in these disorders, and they have also been used to study the effects of pharmacological treatments on protein expression. Together, behavioral and proteomic research will help elucidate the factors involved in anxiety disorder and depression. This knowledge will improve preventive strategies and lead to the development of novel treatments.

Proteomic analysis reveals stage-specific reprogramed metabolism for the primary breast cancer cell lines MGSO-3 and MACL-1.

Breast cancer is the most prevalent cancer in women worldwide. Its molecular subtypes are based on the presence/absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MACL-1 and MGSO-3 are cell lines derived from primary tumor sites of patients diagnosed with luminal A subtype carcinoma (ER+/PR+/HER2-) and ductal carcinoma in situ (ER-/PR-/HER2+), respectively. However, these cell lines lost the expression of these markers over cell culturing, and both have triple-negative phenotypes (ER-/PR-/HER2-), which has the poorest prognosis. Here, we sought to study the proteome signature of MGSO-3 and MACL-1, comparing them with the epithelial cell line MCF-10A and the well-established metastatic-derived breast cancer cell line MDA-MB-231. Our results showed that proteins associated with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) were upregulated in MGSO-3 and MACL-1 cells. These cell lines also showed upregulation of pro-apoptotic proteins when compared with MDA-MB-231. The molecular differences highlighted in this study may clarify the molecular basis behind cancer cells functioning and may reveal novel signatures across the breast cancer cell models.

Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

Assessing the effects of Leishmania (Leishmania) infantum and L. (L.) amazonensis infections in macrophages using a quantitative proteome approach.

Leishmania (Leishmania) infantum is the causative agent of visceral leishmaniasis, while L. (L.) amazonensis is associated with localized cutaneous and diffuse leishmaniasis, which can affect different organ tissues leading to visceral manifestations in some hosts. The wide range of clinical manifestations of leishmaniasis depends on host factors such as the immune response and on the species of Leishmania involved in the infection. Macrophages are the main infected cells in the vertebrate host, and proteins play a pivotal role in Leishmania-macrophage interactions. Here, we performed difference gel electrophoresis (DIGE) and shotgun quantitative mass spectrometry-based proteomics by means of tandem mass tags (TMT) isobaric peptide labeling followed by LC-MS/MS to investigate differentially abundant proteins in BALB/c macrophages infected with these Leishmania species. Using DIGE for comparison, we found that 2.34% spots (29/1240) were differentially intense in infected murine macrophages. Leishmania (L.) infantum and L. (L.) amazonensis induced similar changes in the host cells; 11 spots were selected as differentially intense in each species and seven in the uninfected control group. Using TMT, 5939 Mus musculus proteins were identified, of which 410 and 433 were differentially abundant in L. (L.) infantum and L. (L.) amazonensis infections, respectively, while 170 proteins were commonly regulated by both the species. Gene ontology enrichment analysis indicated that Leishmania infection interfered with apoptotic mechanisms in macrophages and induced epigenetic changes that may affect gene transcription. Moreover, downregulation of proteins such as PYCARD and MyD88 seemed to influence the inflammatory process in L. (L.) amazonensis infection, whereas upregulation of TAP1 and ERAP1 was involved in the adaptive immune response in L. (L.) infantum infection. Differentially abundant proteins identified in this study may contribute to a better understanding of the factors that determine the course of infection. Our results suggest several possible targets for vaccines, drugs, and diagnosis of leishmaniasis.

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

ACE2 in the renin-angiotensin system.

In 2020 we are celebrating the 20th anniversary of the angiotensin-converting enzyme 2 (ACE2) discovery. This event was a landmark that shaped the way that we see the renin-angiotensin system (RAS) today. ACE2 is an important molecular hub that connects the RAS classical arm, formed mainly by the octapeptide angiotensin II (Ang II) and its receptor AT1, with the RAS alternative or protective arm, formed mainly by the heptapeptides Ang-(1-7) and alamandine, and their receptors, Mas and MrgD, respectively. In this work we reviewed classical and modern literature to describe how ACE2 is a critical component of the protective arm, particularly in the context of the cardiac function, coagulation homeostasis and immune system. We also review recent literature to present a critical view of the role of ACE2 and RAS in the SARS-CoV-2 pandemic.

Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Delineation of the pan-proteome of fish-pathogenic Streptococcus agalactiae strains using a label-free shotgun approach.

BACKGROUND: Streptococcus agalactiae (GBS) is a major pathogen of Nile tilapia, a global commodity of the aquaculture sector. The aims of this study were to evaluate protein expression in the main genotypes of GBS isolated from diseased fishes in Brazil using a label-free shotgun nano-liquid chromatography-ultra definition mass spectrometry (nanoLC-UDMSE) approach and to compare the differential abundance of proteins identified in strains isolated from GBS-infected fishes and humans. RESULTS: A total of 1070 protein clusters were identified by nanoLC-UDMSE in 5 fish-adapted GBS strains belonging to sequence types ST-260 and ST-927 and the non-typeable (NT) lineage and 1 human GBS strain (ST-23). A total of 1065 protein clusters corresponded to the pan-proteome of fish-adapted GBS strains; 989 of these were identified in all fish-adapted GBS strains (core proteome), and 62 were shared by at least two strains (accessory proteome). Proteins involved in the stress response and in the regulation of gene expression, metabolism and virulence were detected, reflecting the adaptive ability of fish-adapted GBS strains in response to stressor factors that affect bacterial survival in the aquatic environment and bacterial survival and multiplication inside the host cell. Measurement of protein abundance among different hosts showed that 5 and 26 proteins were exclusively found in the human- and fish-adapted GBS strains, respectively; the proteins exclusively identified in fish isolates were mainly related to virulence factors. Furthermore, 215 and 269 proteins were up- and down-regulated, respectively, in the fish-adapted GBS strains in comparison to the human isolate. CONCLUSIONS: Our study showed that the core proteome of fish-adapted GBS strains is conserved and demonstrated high similarity of the proteins expressed by fish-adapted strains to the proteome of the human GBS strain. This high degree of proteome conservation of different STs suggests that, a monovalent vaccine may be effective against these variants.

The renin-angiotensin system: going beyond the classical paradigms.

Thirty years ago, a novel axis of the renin-angiotensin system (RAS) was unveiled by the discovery of angiotensin-(1-7) [ANG-(1-7)] generation in vivo. Later, angiotensin-converting enzyme 2 (ACE2) was shown to be the main mediator of this reaction, and Mas was found to be the receptor for the heptapeptide. The functional analysis of this novel axis of the RAS that followed its discovery revealed numerous protective actions in particular for cardiovascular diseases. In parallel, similar protective actions were also described for one of the two receptors of ANG II, the ANG II type 2 receptor (AT2R), in contrast to the other, the ANG II type 1 receptor (AT1R), which mediates deleterious actions of this peptide, e.g., in the setting of cardiovascular disease. Very recently, another branch of the RAS was discovered, based on angiotensin peptides in which the amino-terminal aspartate was replaced by alanine, the alatensins. Ala-ANG-(1-7) or alamandine was shown to interact with Mas-related G protein-coupled receptor D, and the first functional data indicated that this peptide also exerts protective effects in the cardiovascular system. This review summarizes the presentations given at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, in 2017, during the symposium entitled "The Renin-Angiotensin System: Going Beyond the Classical Paradigms," in which the signaling and physiological actions of ANG-(1-7), ACE2, AT2R, and alatensins were reported (with a focus on noncentral nervous system-related tissues) and the therapeutic opportunities based on these findings were discussed.

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

Peptide de novo sequencing of mixture tandem mass spectra.

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20-35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications.

High-performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: Observations and implications.

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS-based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.

SuperQuant: A Data Processing Approach to Increase Quantitative Proteome Coverage.

SuperQuant is a quantitative proteomics data processing approach that uses complementary fragment ions to identify multiple coisolated peptides in tandem mass spectra allowing for their quantification. This approach can be applied to any shotgun proteomics data set acquired with high mass accuracy for quantification at the MS(1) level. The SuperQuant approach was developed and implemented as a processing node within the Thermo Proteome Discoverer 2.x. The performance of the developed approach was tested using dimethyl-labeled HeLa lysate samples having a ratio between channels of 10(heavy):4(medium):1(light). Peptides were fragmented with collision-induced dissociation using isolation windows of 1, 2, and 4 Th while recording data both with high-resolution and low-resolution. The results obtained using SuperQuant were compared to those using the conventional ion trap-based approach (low mass accuracy MS(2) spectra), which is known to achieve high identification performance. Compared to the common high-resolution approach, the SuperQuant approach identifies up to 70% more peptide-spectrum matches (PSMs), 40% more peptides, and 20% more proteins at the 0.01 FDR level. It identifies more PSMs and peptides than the ion trap-based approach. Improvements in identifications resulted in up to 10% more PSMs, 15% more peptides, and 10% more proteins quantified on the same raw data. The developed approach does not affect the accuracy of the quantification and observed coefficients of variation between replicates of the same proteins were close to the values typical for other precursor ion-based quantification methods. The raw data is deposited to ProteomeXchange (PXD001907). The developed node is available for testing at https://github.com/caetera/SuperQuantNode.

Discovery and characterization of alamandine: a novel component of the renin-angiotensin system.

RATIONALE: The renin-angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1-7). OBJECTIVE: To characterize a novel component of the RAS, alamandine. METHODS AND RESULTS: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1-7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1-7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein-coupled receptor, member D. Binding of alamandine to Mas-related G-protein-coupled receptor, member D is blocked by D-Pro(7)-angiotensin-(1-7), the Mas-related G-protein-coupled receptor, member D ligand ß-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/ß-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. CONCLUSIONS: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera) mesocarp and exocarp owing to Lobesia botrana infection.

Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lys-acetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lys-acetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to pathogen infection. Topographical distribution of phosphorylation sites within primary sequences reveal preferential phosphorylation at both the N- and C termini, and a clear preference for C-terminal phosphorylation in response to pathogen infection suggesting induction of region-specific kinase(s). Lys-acetylation analysis confirmed the consensus X-K-Y-X motif previously detected in mammals and revealed the importance of this modification in plant defense. The importance of N-linked protein glycosylation in plant response to biotic stimulus was evident by an up-regulated glycopeptide belonging to the disease resistance response protein 206. This study represents a substantial step toward the understanding of protein and PTMs-mediated plant-pathogen interaction shedding light on the mechanisms underlying the grape infection.

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT 2 -Receptor Agonist.

Background: The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. Methods: The effect of Ang-(1-5) (1µM) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT 2 -receptor (AT 2 R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT 2 R-knockout mice. Binding of Ang-(1-5) to the AT 2 R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. Results: Key findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at Ser1177 eNOS and Tyr657 eNOS and thereby (2) increased NO release from HAEC and AT 2 R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT 2 R-KO or in PD123319-treated mice and which were more potent than effects of the established AT 2 R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT 2 R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT 2 R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. Conclusions: Ang-(1-5) is a potent, endogenous AT 2 R-agonist.

Deconvolution of mixture spectra and increased throughput of peptide identification by utilization of intensified complementary ions formed in tandem mass spectrometry.

A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high-resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial data set.