Peptides In Biological Samples Research Articles

Pharmacokinetics of collagen dipeptides (Gly-Pro and Pro-Hyp) and tripeptides (Gly-Pro-Hyp) in rats.

Although systemic exposure to peptides, such as Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro, has been reported following administration of collagen hydrolysates from fish scale and porcine skin in vivo, the individual peptide pharmacokinetics remain unknown. We administered the three peptides individually to rats via the intravenous (5mg/kg) and intragastric (100mg/kg) routes and then monitored systemic exposure and urinary excretion. The peptides in biological samples were analyzed via liquid chromatography/tandem mass spectrometry. Gly-Pro-Hyp tended to exhibit higher first-pass metabolism than Pro-Hyp; the absolute oral bioavailabilities of Gly-Pro-Hyp and Pro-Hyp were 4.4% and 19.3%, respectively. Gly-Pro levels were very low in the systemic circulation. Pro-Hyp biotransformed from Gly-Pro-Hyp behaved similarly to Pro-Hyp alone when administered orally. Flip-flop kinetics (elimination rate ≫ absorption rate) were evident, probably reflecting transporter-mediated slow absorption. A double-peak phenomenon was observed for Gly-Pro-Hyp and Pro-Hyp when administered orally, and 5.9%±2.6% and 1.9%±0.3% of each dose were excreted in urine after intravenous administration, respectively. Urinary recovery of Gly-Pro was limited to 0.4%±0.5% of the intravenous dose. This work represents the first individual pharmacokinetics of Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro in vivo.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Advances in sample preparation and HPLC-MS/MS methods for determining amyloid-β peptide in biological samples: a review.

Alzheimer's disease (AD), a neurological disorder, is a major public health concern and the most common form of dementia. Its typical symptoms include memory loss, confusion, changes in personality, and cognitive impairment, which result in patients gradually losing independence. Over the last decades, some studies have focused on searching for effective biomarkers as early diagnostic indicators of AD. Amyloid-β (Aβ) peptides have been consolidated as reliable AD biomarkers and have been incorporated into modern diagnostic research criteria. However, quantitative analysis of Aβ peptides in biological samples remains a challenge because both the sample and the physical-chemical properties of these peptides are complex. During clinical routine, Aβ peptides are measured in the cerebrospinal fluid by immunoassays, but the availability of a specific antibody is critical-in some cases, an antibody may not exist, or its specificity may be inadequate, leading to low sensitivity and false results. HPLC-MS/MS has been reported as a sensitive and selective method for determining different fragments of Aβ peptides in biological samples simultaneously. Developments in sample preparation techniques (preconcentration platforms) such as immunoprecipitation, 96-well plate SPME,onlineSPME, andfiber-in-tubeSPME have enabled not only effective enrichment of Aβ peptides present at trace levels in biological samples, but also efficient exclusion of interferents from the sample matrix (sample cleanup). This high extraction efficiency has provided MS platforms with higher sensitivity. Recently, methods affording LLOQ values as low as 5pgmL-1 have been reported. Such low LLOQ values are adequate for quantifying Aβ peptides in complex matrixes including cerebrospinal fluid (CSF) and plasma samples. This review summarizes the advances in mass spectrometry (MS)-based methods for quantifying Aβ peptides and covers the period 1992-2022. Important considerations regarding the development of the HPLC-MS/MS method such as the sample preparation step, optimization of the HPLC-MS/MS parameters, and matrix effects are described. Clinical applications, difficulties related to analysis of plasma samples, and future trends of these MS/MS-based methods are also discussed.

Towards molecularly imprinted polymers that respond to and capture phosphorylated tyrosine epitopes using fluorescent bis-urea and bis-imidazolium receptors.

Early detection of cancer is essential for successful treatment and improvement in patient prognosis. Deregulation of post-translational modifications (PTMs) of proteins, especially phosphorylation, is present in many types of cancer. Therefore, the development of materials for the rapid sensing of low abundant phosphorylated peptides in biological samples can be of great therapeutic value. In this work, we have synthesised fluorescent molecularly imprinted polymers (fMIPs) for the detection of the phosphorylated tyrosine epitope of ZAP70, a cancer biomarker. The polymers were grafted as nanometer-thin shells from functionalised submicron-sized silica particles using a reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Employing the combination of fluorescent urea and intrinsically cationic bis-imidazolium receptor cross-linkers, we have developed fluorescent sensory particles, showing an imprinting factor (IF) of 5.0. The imprinted polymer can successfully distinguish between phosphorylated and non-phosphorylated tripeptides, reaching lower micromolar sensitivity in organic solvents and specifically capture unprotected peptide complements in a neutral buffer. Additionally, we have shown the importance of assessing the influence of counterions present in the MIP system on the imprinting process and final material performance. The potential drawbacks of using epitopes with protective groups, which can co-imprint with targeted functionality, are also discussed.

Quantitative Assessment of Serine-8 Phosphorylated β-Amyloid Using MALDI-TOF Mass Spectrometry

The study of the molecular mechanisms of the pathogenesis of Alzheimer's disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of β-amyloid (Aβ) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aβ) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aβ based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aβ was estimated in the total Aβ-pool with a detection limit of 1 fmol for pSer8-Aβ (1-16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples.

Infrared Photoactivation Boosts Reporter Ion Yield in Isobaric Tagging.

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.

Hemorphins-From Discovery to Functions and Pharmacology.

During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.

An electrochemical biosensor for alpha-fetoprotein detection in human serum based on peptides containing isomer D-Amino acids with enhanced stability and antifouling property

The development of antifouling biosensors capable of detecting biomarkers at low concentrations in complex bio-fluids with many interference components is of great importance in the diagnosis and treatment of diseases. Certain zwitterionic peptides composed of natural L-amino acids have been used for the construction of low fouling biosensors and demonstrated excellent antifouling performances, but they are prone to enzymatic degradation in biological media, such as serum that contains a variety of enzymes. In this work, a novel antifouling peptide with the sequence of cppPPEKEKEkek was designed, and three unnatural D-amino acids were set at both ends of the peptide to enhance its tolerance to enzymatic degradation. An electrochemical biosensor was constructed by coupling the antifouling peptide with a conducting polymer polyaniline (PANI) to achieve accurate detection of alpha-fetoprotein (AFP) in clinical samples. Owing to the presence of the designed peptide with partial D-amino acids (pD-peptide), the biosensing interface showed significantly high antifouling performance and enhanced stability in human serum. Meanwhile, the pD-peptide based biosensor exhibited high sensitivity toward the target AFP, with the linear range from 0.1 fg mL−1 to 1.0 ng mL−1 and the limit of detection of 0.03 fg mL−1 (S/N = 3). This strategy of enhancing the stability (tolerance to enzymolysis) of antifouling peptides in biological samples provided an effective way to develop antifouling biosensors for practical applications.

Oxytocin Peptide Detection with Carbon Electrodes and Fast Scan Cyclic Voltammetry

Fast scan cyclic voltammetry (FSCV) used in conjunction with carbon fiber-microelectrodes (CFMEs) is an electrochemical technique used to detect neurotransmitters with a sub-section temporal resolution. This allows for a sensitive and selective subsecond analysis of changes in the brain. Typically, this technique is used to detect catecholamines in the brain, but recently, it has branched out into purinergic signaling and peptide neurotransmitters. This technique is not only able to detect neurochemical changes at a quick timescale, but it is able to detect a wide range of molecules with a high spatiotemporal resolution. This results in having chemical selectivity that may not be available in other electrochemical techniques. The nature of CFMEs also allows for the selective targeting of specific brain regions to ensure that precise measurements can be made.The peptide oxytocin is important in understanding stress, depression and anxiety, and it is one of the main components in clarifying signals sent throughout the brain. Oxytocin plays vital roles for women during childbirth as well as for lactation. Oxytocin is typically analyzed by taking a blood sample and running assays that can take a while to obtain the results. Peptides, like oxytocin, can also be detected through methods like mass spectrometry and various forms of chromatography but are sometimes very time consuming and can be destructive. On the other hand, it has been known that there are some amino acids are redox active and can be measured with electrochemical techniques.The detection of tyrosine-containing peptides using electrochemical techniques, such as oxytocin, can be quite difficult. The short amount of time that these molecules are in the extracellular space and its tendency to foul electrodes when oxidized make it hard to collect accurate and consistent data. Also, there is a higher concentration of more electroactive molecules that would make it harder to be specific for these molecules. However, due to the nature of FSCV, it is possible to change the waveform to be able to prevent the fouling of tyrosine on the surface of the electrode and improve the detection of tyrosine-containing peptides in biological samples. Recently, a modified sawhorse waveform (MSW) has been formulated that is able to specifically detect tyrosine in the methionine-enkephalin (M-ENK) neuropeptide using two specific scan rates in the anodic sweep and a short holding period at the switching potential. The MSW waveform was applied at 10 Hz with the holding potential at -0.2 V, then increased to +0.6 V at 100 V/s, and further increased to +1.2 V at 400 V/s. This potential was held in place for 3 ms before sweeping back down to the folding potential of -0.2 V at 100 V/s. This results in high specificity toward tyrosine and eliminates the fouling on the electrode. In this study, we target the tyrosine residue on oxytocin with a MSW waveform and characterize it with carbon fiber microelectrodes and fast scan cyclic voltammetry. We show the first electrochemical detection of oxytocin with FSCV with high sensitivity and spatiotemporal resolution that is distinguished from tyrosine and other amino acids. This work could potentially help further understand oxytocin’s role in vivo and importance in behavioral and disease states.

Quantitative analysis of peptides and proteins as biomarkers in biological samples by peptide adsorption-controlled (PAC)-LC/MS

In the pharmaceutical industry, various compounds such as antibodies, peptides, oligonucleotides, etc. are being evaluated and developed as new modality drugs as well as small molecule compounds. For the evaluation of these new modalities, the development of analytical methods is essential, but often difficult because the analytical techniques related to these modalities are not as mature as to small molecule compounds. Liquid chromatography/mass spectrometry (LC/MS) is a highly selective and sensitive method with a wide dynamic range, and has become an indispensable instrument for analysis of small molecule compounds. Recently, LC/MS has attracted attention as an alternative to ligand-biding assays for the quantification of peptides and proteins. So far, we have found that the adsorption capacity of a peptide changes abruptly and reversibly around a specific organic solvent content (critical point) in the solution, and based on this property we developed a peptide adsorption-controlled liquid chromatography (PAC-LC) for quantitative peptide analysis. By using PAC-LC, it has become possible to quantify low concentration proteins and peptides in biological samples. In this symposium, I would like to introduce examples of analysis for biomarker proteins and peptides in biological samples using PAC-LC.

Multiplexed monitoring of therapeutic antibodies for inflammatory diseases using Fab-selective proteolysis nSMOL coupled with LC-MS

Monoclonal antibodies have accelerated the availability of treatment options for many diseases in which the molecular mechanism has been elucidated in detail. Therefore, an assay that can universally analyze antibodies for clinical pharmacokinetics and cross-sectional studies would be indispensable.We have developed a universal antibody bioanalysis with a Fab-selective tryptic reaction, named nano-surface and molecular-orientation limited (nSMOL) proteolysis, that collects the specific antibody signature peptides in biological samples. Using the nSMOL method, we have fully validated the bioanalysis of many antibodies, Fc-fusion proteins, and their biosimilars.Inflammatory immune diseases often require long-term clinical management because of the remission and relapse observed. Accurate antibody monitoring in systemic circulation could contribute to the improvement of clinical outcomes. Because several biopharmaceuticals can be selected as practical treatment options, the assay development that quantitates many antibodies simultaneously would be applicable in many theraprutic monitoring.In this study, we have validated the LC-MS bioanalysis method for seven-mixed antibodies (Infliximab, Adalimumab, Ustekinumab, Golimumab, Eculizumab, Etanercept, and Abatacept) using the nSMOL normal reaction condition and two-mixed antibodies (Tocilizumab and Mepolizumab) using the acidified reduction acceleration condition, as reported in our previous papers. Moreover, this multiplexed assay has been verified using clinical patient samples. The nSMOL approach enables the quantitation of several immunosuppressive antibodies simultaneously in human serum, and nSMOL can potentially be applicable to the drug-drug interaction assays or therapeutic antibody monitoring of several inflammatory immune diseases to optimize administration.

Facile preparation of molybdenum (VI) oxide – Modified graphene oxide nanocomposite for specific enrichment of phosphopeptides

To promote the development of phosphoproteome analysis in highly selective efficient tracing phosphorylated proteins or peptides, views of researches should not confined with intrinsic materials and their modification. New materials are supposed to be explored for phosphoproteome analysis. In this work, we first introduced Molybdenum (VI) oxide (MoO3) into phosphoproteome, loading on the graphene oxide (GO) nanosheets forming MoO3/GO nanocomposites by a simple two-step strategy. The GO nanosheets offered MoO3 a perfect stable platform for separation and concentration and MoO3 exhibited wonderful property in enriching phosphopeptides with highly selectivity and sensitivity on GO nanosheets. Specifically, the as-synthesized MoO3/GO nanocomposites exhibited excellent specificity (β-casein: BSA=1:1000), high detection sensitivity (1 fmol/mL) and well recovery (91.13%) in enriching phosphopeptides by metal oxide affinity chromatography (MOAC). Moreover, the as-synthesized MoO3/GO nanocomposites provided effective enrichment of phosphopeptides from nonfat milk (a total of twelve phosphopeptides signals) and human serum (a total of four endogenous phosphopeptides signals), displaying great biological compatibility, which demonstrated that the MoO3/GO nanocomposites is a promising candidate in selectively identifying and determining low-abundance phosphorylated peptides in biological sample.

Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection

The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C18 column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10–50 pg mL−1 in plasma for most of the peptides and 100 pg mL−1 for the remaining. For urine, LOD was 20–400 pg mL−1 for most of the peptides and 1–4 ng mL−1 for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge.

Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

PK study of octreotide based on LC-MS/MS combining protein precipitation and impurity extraction technique.

To establish a robust methodology for quantitative analysis of therapeutic peptide in biological samples. Octreotide was chosen as a model therapeutic peptide, and oxidized-octreotide was synthesized as internal standard. Protein precipitation combining liquid-liquid extraction technique was adopted to enhance the recovery and reduce the endogenous interferences effectively. A LC-MS/MS method for the quantification of octreotide in plasma has been optimized and validated according to FDA guidelines. Linearity, selectivity, accuracy, precision, stability, matrix effect and recovery were within bioanalytical method validation acceptance criteria as FDA guidelines. The methodology was then successfully applied into the studies for octreotide. This robust methodology would be useful for the PK studies for octreotide and other therapeutic peptides.

4:15 PM CORTICAL THICKNESS IN INDIVIDUALS WITH NONCLINICAL AND CLINICAL PSYCHOSIS

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

Synthesis of C8 -Functionalized Magnetic Graphene with a Polydopamine Coating for the Enrichment of Low-Abundance Peptides.

For the first time, C8 -functionalized magnetic graphene (magG) has been designed and synthesized with a polydopamine (PDA) coating. The magG prepared by a solvothermal reaction was encapsulated in a layer of PDA through the oxidative polymerization of dopamine in alkaline buffer, and C8 groups were grafted onto magG/PDA composites through a silanization method. The as-prepared material integrates the merits of graphene, magnetic microspheres, PDA, and C8 chains; thus possessing an ultrahigh specific area, strong magnetic responsiveness, excellent solubility, and an extraordinary enrichment capability. The C8 -functionalized magnetic composites were employed in the enrichment and identification of low-concentration standard peptides, peptides in standard protein digest solutions, and endogenous peptides in human urine. The enriched peptides were eluted and analyzed by MALDI-TOF MS. The MS results indicated that the C8 -functionalized material exhibited the distinguished ability to enrich hydrophobic peptides mainly through hydrophobic-hydrophobic interactions. Moreover, thanks to the enrichment approach based on magG@PDA@C8 , the limit of detection of the standard peptide decreased to as low as 50 pM. The experimental results demonstrate that the magG@PDA@C8 composite is a promising candidate for the enrichment of low-abundance peptides in biological samples.

Estimation of angiotensin peptides in biological samples by LC–MS method

The low abundance of angiotensin peptides in biological tissues such as the kidney cortex, adipose tissue, urine and plasma makes their detection and quantification a challenge. A few available methods used to quantify these peptides involve lengthy processes of sample preparation and are hardly quantitative. Here, we report a mass spectrometry approach for quantifying angiotensin peptides [Ang II, Ang-(1-7)] in the kidney cortex, epididymal white adipose tissue (eWAT), urine and plasma of male mice. Tissue homogenates, urine and plasma samples were solid-phase extracted with C18 Sep-Pak cartridges and eluted off proteinaceous compounds. These extracted peptide samples were separated on C18 column with a linear acetonitrile gradient and detected by Q-ToF mass analyzer in ESI+-MS ion mode based on their retention time, accurate mass measurement of peptides, the isotope pattern of doubly charged molecular ion, and quantitation of peak area (or ion count) when referencing to the angiotensin peptide standards. The lower limit of quantitation for each angiotensin peptide was 10 pgmg-1 with the percent recovery at 100.6%. The intra-batch precision for Ang-(1-7) and Ang II were 24.0 and 12.7%, accuracy 84.0-123.0% and 100.2-116.0% respectively. Using this method, we determined the levels of Ang II and Ang-(1-7) in the kidney cortex, eWAT, urine and plasma. Quantification of angiotensin peptides could help target subtle therapeutics changes against pathophysiological conditions such as obesity, kidney disease and hypertension.

Mesoporous Materials in Peptidome Analysis

Biologically active peptides, such as peptide hormones and cytokines, play pivotal roles in human health and disease development, as these peptides are important regulators for diverse functions, which range from the endocrine to the inflammatory and the nervous systems. Endogenous peptides can result from the proteolytic cleavage of proteins, and are indicative of protease activity, degradation, and degeneration. The overall concentrations of peptides in biological samples reflect certain biological events and may provide useful information for clinical diagnosis. Therefore, the comprehensive analysis of peptides in complex biological mixtures, termed peptidomics, can contribute to a better understanding of the biochemical functions of endogenous peptides, as well as to the development of diagnostic and prognostic biomarkers. To profile the peptidome in complicated samples, such as human serum, typical procedures involve sample collection, extraction, separation, detection, and data mining (Scheme 1). Of the five key steps, the selective extraction of peptides has been identified as an important obstacle to overcome for progress in peptidome research, because of the complexity and high dynamic range of biomolecules present in the samples from which the peptides must be extracted. For example, the detection of low-abundance peptides in human serum is very challenging because they are overwhelmed by larger and more abundant proteins, salts, and lipids. To confront this challenge, Zou s group has recently developed a highly efficient and selective method to extract peptides from human serum by using ordered mesoporous carbon (OMC). The 4.8 nm pore size of the OMC material enables a molecular-weight cut-off of approximately 10 kDa, which is sufficient to remove the majority of larger interfering serum proteins. The use of OMC greatly enhances the overall extraction efficiency of peptides, which includes peptides in the low mass range (< 2 kDa) that are traditionally difficult to extract. When combined with twodimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS), the researchers were able to identify 3402 unique endogenous peptides from 20 mL of serum. The significantly greater number of serum peptides identified by using this technique demonstrates the advantageous efficiency of mesoporous-based extraction over other sample treatment techniques. Mesoporous materials, such as OMC, have the desirable properties of an ideal extraction material, which include a high surface area, large pore volume, chemical inertness, and good mechanical stability. In recent years, various mesoporous materials have been synthesized to possess a welldefined pore-size distribution and controllable surface functionality. The narrow size distribution of the ordered mesopores facilitates size-selective extraction of endogenous peptides and size exclusion of larger proteins. For example, the highly ordered mesoporous-silica particles in MCM-41, have a pore size of 2 nm and a molecular-weight cut-off of Scheme 1. Typical steps involved in profiling peptidomes from complicated samples. The use of new mesoporous materials improves the efficiency and selectivity of peptide extraction.

Metabolism of bradykinin in aorta of hypertensive rats.

Alterations in the formation and metabolism of bradykinin (Bk) are hypothesized to play a role in the pathophysiology of hypertension, atherosclerosis and vascular complications of diabetes. However, despite its prominent role in cardiovascular regulation, studies on bradykinin have been limited by various difficulties in accurate measurements of this peptide in biological samples. In this study, using the LC-ESI-MS method we estimated the conversion of exogenous Bk to its main metabolites - Bk-(1-5) and Bk-(1-7) - in endothelial cell culture and in fragments of aorta of normotensive (WKY) and hypertensive rats (SHR). The effects of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors were more pronounced in SHR: perindoprilat inhibited Bk-(1-5) formation by 49 % and 76 % in WKY and SHR rats, respectively, and tiorphan tended to decrease formation of Bk-(1-5) in both groups of animals. The degradation of bradykinin and generation of both metabolites were significantly higher in the aorta of SHR rats than in WKY controls. Our results show that even in relatively early hypertension (in 4-month old SHR rats) inactivation of Bk by aorta wall is enhanced.