Peptides In Biological Samples Research Articles

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.

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography–tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.

Analytical Methods for the Determination of Anti-diuretic Peptides in Biological Samples: Challenges and Perspectives

Anti-diuretic peptides are widely used for the control of body water balance. With the growing concerns on their pharmacological characteristics and clinical safety, there is an increasing request of sensitive, selective and robust analytical assays for the determination of anti-diuretics in biological samples. This article provides a perspective review of various methodologies developed and validated in the past, including immunoassay (IA), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), with detections of radio, UV, fluorescence and mass spectrometry. Systematic description of each technique was given and emphasis was laid on the problems encountered with each assay. Despite the infrequent application of mass spectrometry in anti-diuretics quantitative analysis, it was highlighted here for its remarkable sensitivity and selectivity. The ultimate aim of this review is to demonstrate the analytical options of peptide determination and promote the quantification of peptide-like drugs and biomarkers in respect to fast-expanding peptide market and therapeutics.

Sensitivity enhancement of CE and CE‐MS for the analysis of peptides by a dynamic pH junction

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, β-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100 mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1 M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.

Fragmentation of peptides with intra-chain disulfide bonds in triple quadrupole mass spectrometry and its quantitative application to biological samples

A growing number of peptides are being used today in bioanalytical laboratories. Because of this, there is an increasing interest in the development of highly sensitive, specific and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays for the quantitative analysis of peptides in biological samples. Among the mass spectrometers previously used for peptide quantification, triple quadrupole mass spectrometers are generally not considered the instrument of choice. With this instrumentation, collision cascades or multiple fragmentations tend to generate multiple peaks that have weak intensities. This leads to a loss in detection sensitivity. However, in cases where immonium product ions were formed in abundance, it was found that peptide quantification succeeded. A common feature of these peptides is their intra-loop structure. To elucidate the usefulness of this feature in fragmentation, several peptide analytes with intra-chain disulfide bonds were investigated in this study, including a newly synthesized analog having a single amino acid substitution. The results presented here indicate that abrupt bond cleavage from the intra-loop structure of peptides could be one of the premises for intense immonium ion generation. In contrast, any preferential cleavage of peptide bonds (e.g., proline effect) that gives rise to a linearized sequence would break the intactness of the loop and prevent it from completely dissociating. In addition, the utilization of immonium product ions in LC/MS/MS was demonstrated for the determination of peptides with intra-chain disulfide bonds in biological fluids.

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, gammaGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 microm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 microM respectively.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B1 in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity ( R 2 > 0.99) in the concentration range 0.05–10 μg/mL with a limit of detection of 0.05 μg/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures.

Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5 ± 2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.

On-line coupling of size-exclusion chromatography and capillary zone electrophoresis via a reversed-phase C18 trapping column for the determination of peptides in biological samples.

Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart-cut of 200 micro L, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 micro L plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 micro g/ mL with an injection volume of 50 micro L).

Detection of oligomeric β-amyloid peptide in biological samples using a singlesite monoclonal antibody sandwich enzyme-linked immunoabsorbant assay

Apolipoprotein E (apoE) levels were compared in cerebrospinal fluid (CSF) taken on two occasions, with an average 15 months follow up, from groups of patients with Alzheimer's disease (AD; n=18), mild cognitive impairment (MCI; n=9) and other dementia disorders (ODD; n=9). In these groups, CSF apoE levels were between 2–3-fold higher than values for a group of 27 healthy age-matched controls. CSF apoE levels in the AD group were significantly increased at follow up, compared to levels obtained on the first sampling occasion. For the same cases it had been shown previously that CSF tau protein levels were increased at follow up [Blomberg, M., Jensen, M., Basun, H., Lannfelt, L. and Wahlund, L-O., Neurosci. Lett., 214 (1996) 163–166]. The AD, but not MCI, ODD or control groups, also showed statistically significant correlations between CSF apoE and tau protein levels at both the first (r=0.585, P<0.01) and follow up (r=0.695, P>0.001) samplings. It is concluded that CSF measures of both apoE and tau may reflect an intimate relationship between these two proteins in AD and could prove useful in monitoring the progression of this condition.

Fluorescence derivatization approaches for quantitative high-performance liquid chromatography of peptides

Modern high-performance liquid chromatography (HPLC) can provide high sensitivity and selectivity for the measurement of peptides which have been suitably derivatized for fluorescence detection. Several approaches to the chemical modification of peptides for high sensitivity fluo-rescence detection in HPLC have been developed and are routinely useful for low level determinations. Under optimal conditions, HPLC with fluorescence detection can provide sensitivity for peptides comparable with that of immunoassay techniques, and, in some cases, affords additional selectivity. An example of the utility of HPLC approaches for the high sensitivity determination of disulfide- and arginine-containing peptides in biological samples in support of pharmacokinetic studies is described.

Multidimensional liquid chromatography of opioid peptides: Fluorogenic labelling, retention prediction and separation optimization

The ultra-trace analysis of opioid peptides in biological samples can be achieved by multidimensional liquid chromatography with pre-column fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion. However, in order to take full advantage of the high sensitivity possible with detectors based on laser-induced fluorescence or chemiluminescence, each component of the analytical method must be carefully optimized. In this study, strategies are presented for the prediction of retention time and the optimization of separations of derivatized opioid peptides in multidimensional LC systems.

Use of octadecasilyl-silica for the extraction and purification of peptides in biological samples. Application to the identification of circulating metabolites of corticotropin-(1-24)-tetracosapeptide and somatostatin in vivo

Peptides can be adsorbed on octadecasilyl-silica from large volumes of aqueous solution and eluted with aqueous solvent mixtures containing methanol or acetonitrile. These properties may be used for the extraction and purification of peptide fragments in plasma samples collected from rats. After intravenous injection of Synacthen [corticotropin-(1-24)-tetracosapeptide], it was shown that within 2 min the main circulating products were intact peptide and its sulphoxide. In addition, a number of fragments indicative of cleavage at the N- and C-termini were present. Most of the products formed from Synacthen have low biological activity. Somatostatin was rapidly cleaved in vivo and in vitro to a single product, which probably retains biological activity. The absence of other circulating products suggests that somatostatin is only inactivated once it leaves the circulation.