Peptides In Biological Matrices Research Articles

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.

Application of peptide biomarkers in life analysis based on liquid chromatography-mass spectrometry technology.

Biomedicine is developing rapidly in the 21st century. Among them, the qualitative and quantitative analysis of peptide biomarkers is of considerable importance for the diagnosis and therapy of diseases and the quality evaluation of drugs and food. The identification and quantitative analysis of peptides have been going on for decades. Traditionally, immunoassays or biological assays are generally used to quantify peptides in biological matrices. However, the selectivity and sensitivity of these methods cannot meet the requirements of the application. The separation and analysis technique of liquid chromatography-mass spectrometry (LC-MS) supplies a reliable alternative. In contrast to immunoassays, LC-MS methods are capable of providing the analytical prowess necessary to satisfy the demands of peptide biomarker research in the life sciences arena. This review article provides a historical account of the in-roads made by LC-MS technology for the detection of peptide biomarkers in the past 10 years, with the focus on the qualification/quantification developments and their applications.

Follicular Fluid: A Powerful Tool for the Understanding and Diagnosis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) represents one of the leading causes of anovulatory infertility and affects 5% to 20% of women worldwide. Until today, both the subsequent etiology and pathophysiology of PCOS remain unclear, and patients with PCOS that undergo assisted reproductive techniques (ART) might present a poor to exaggerated response, low oocyte quality, ovarian hyperstimulation syndrome, as well as changes in the follicular fluid metabolites pattern. These abnormalities originate a decrease of Metaphase II (MII) oocytes and decreased rates for fertilization, cleavage, implantation, blastocyst conversion, poor egg to follicle ratio, and increased miscarriages. Focus on obtaining high-quality embryos has been taken into more consideration over the years. Nowadays, the use of metabolomic analysis in the quantification of proteins and peptides in biological matrices might predict, with more accuracy, the success in assisted reproductive technology. In this article, we review the use of human follicular fluid as the matrix in metabolomic analysis for diagnostic and ART predictor of success for PCOS patients.

Peptidomics: A Review of Clinical Applications and Methodologies.

Improvements in both liquid chromatography (LC) and mass spectrometry (MS) instrumentation have greatly enhanced proteomic and small molecule metabolomic analysis in recent years. Less focus has been on the improved capability to detect and quantify small bioactive peptides, even though the exact sequences of the peptide species produced can have important biological consequences. Endogenous bioactive peptide hormones, for example, are generated by the targeted and regulated cleavage of peptides from their prohormone sequence. This process may include organ specific variants, as proglucagon is converted to glucagon in the pancreas but glucagon-like peptide-1 (GLP-1) in the small intestine, with glucagon raising, whereas GLP-1, as an incretin, lowering blood glucose. Therefore, peptidomics workflows must preserve the structure of the processed peptide products to prevent the misidentification of ambiguous peptide species. The poor in vivo and in vitro stability of peptides in biological matrices is a major factor that needs to be considered when developing methods to study them. The bioinformatic analysis of peptidomics data sets requires the inclusion of specific post-translational modifications, which are critical for the function of many bioactive peptides. This review aims to discuss and contrast the various extraction, analytical, and bioinformatics approaches used for human peptidomics studies in a multitude of matrices.

Quantitation of Super Basic Peptides in Biological Matrices by a Generic Perfluoropentanoic Acid-Based Liquid Chromatography-Mass Spectrometry Method.

Peptides represent a promising modality for the design of novel therapeutics that can potentially modulate traditionally non-druggable targets. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are two large families that are being explored extensively as drug delivery vehicles, imaging reagents, or therapeutic treatments for various diseases. Many CPPs and AMPs are cationic among which a significant portion is extremely basic and hydrophilic (e.g., nona-arginine). Despite their attractive therapeutic potential, it remains challenging to directly analyze and quantify these super cationic peptides from biological matrices due to their poor chromatographic behavior and MS response. Herein, we describe a generic method that combines solid phase extraction and LC-MS/MS for analysis of these peptides. As demonstrated, using a dozen strongly basic peptides, low μM concentration of perfluoropentanoic acid (PFPeA) in the mobile phase enabled excellent compound chromatographic retention, thus avoiding co-elution with solvent front ion suppressants. PFPeA also had a charge reduction effect that allowed the selection of parent/ion fragment pairs in the higher m/z region to further reduce potential low molecular weight interferences. When the method was coupled to the optimized sample extraction process, we routinely achieved low digit ng/ml sensitivity for peptides in plasma/tissue. The method allowed an efficient evaluation of plasma stability of CPPs/AMPs without fluorescence derivatization or other tagging methods. Importantly, using the widely studied HIV-TAT CPP as an example, the method enabled us to directly assess its pharmacokinetics and tissue distribution in preclinical animal models.

Enhanced UHPLC-MS/MS determination of a therapeutic heptapeptide mimic for inflammatory-related diseases in rat plasma: application to a pharmacokinetic study.

The seven amino acid peptide, GQTYTSG (named as SP), a peptide mimic derived from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) has presented remarkable anti-inflammatory activities in previous experiments, indicating that it could be a novel therapeutic peptide candidate for different inflammation-related diseases, such as HCV infection and asthma. A heptapeptide mimic discovery study highlighted the need for the development of quantitative bioanalytical assays for measuring the levels of SP. Herein, a reliable and sensitive ultrahigh-performance liquid chromatography (UHPLC) with tandem mass spectrometry (MS/MS) assay was established and validated for the determination of SP in rat plasma. C-11, with two amino acid substitutions compared to SP (Glycine 1 and Glycine 7) and a disulfide, acted as an internal standard (IS). SP and C-11 were isolated from acidified plasma using protein precipitation and the extracts were analyzed by reversed-phase UHPLC-MS/MS detection. We used an SHIM-PACK GISS C18 (2.1 × 100 mm, 1.9 μm) column with water containing 0.2% acetic acid as the aqueous mobile phase and methanol as the organic mobile phase with a 0.3 mL min−1 flow rate. We used an AB SCIEX TripleQuad™ 5500 mass spectrometer equipped with a TurboIon Spray interface and operated it in positive-ion mode. Multiple reaction monitoring (MRM) was used for the quantification of the precursor to the product ion at m/z 713.3 → 432.2 for SP and m/z 803.2 → 539.1 for IS. The method was fully validated according to the US Food and Drug Administration (FDA) guideline (2018), and provided satisfactory accuracy, precision, and reproducibility for the quantification of SP in rat plasma. Excellent linearity was achieved (r > 0.9977) over a linear dynamic range of 0.1–200 ng mL−1 with a lower limit of quantification (LLOQ) of 0.1 ng mL−1. The validated assay was applied to gain the pharmacokinetic (PK) parameters and the concentration–time profile for SP after subcutaneous administration in rats.

Development of a sensitive, accurate and robust liquid chromatography/mass spectrometric method for profiling of angiotensin peptides in plasma and its application for atherosclerotic mice

Quantification of angiotensin (Ang) peptides in biological matrices is a challenge due to their low picomolar (pM) concentration and poor analytical performance of current methods. This work aimed to select an optimal strategy for liquid chromatography/mass spectrometry (LC/MS) quantification of major angiotensins in plasma of wild type and atherosclerotic mice.Optimal LC/MS set-up for Ang quantification was chosen, based on analytical performance, from: nanoflow/orbitrap, nanoflow/triple quadrupole and preconcentration nanoflow/triple quadrupole. The best LC/MS configuration (preconcentration nanoflow/triple quadrupole) was validated and used for measurement of angiotensins (Ang I, II, III, IV and (1–7)) in plasma of 6-month-old atherosclerotic apolipoprotein E/LDL receptor double knock-outs (ApoE/LDLR (--/--)) and wild type C57BL/6J (WT) mice.The method established for Ang quantification was selective, accurate and highly sensitive with LLOQ of 5pgmL−1. The peak area intra-day precisions for Ang II and Ang-(1–7) were in the range 3.0–5.1 and 3.5–5.8, respectively, with corresponding accuracy of 95.4–103.5% and 95.6–106.3%. Plasma angiotensin profile was substantially modified in ApoE/LDLR knock-out mice with increase in concentration of Ang II from 37.6±21.3pgmL−1 in WT to 200.2±47.6pgmL−1. Concentrations of Ang I, III and IV were also increased 3–10 fold in ApoE/LDLR (--/--) mice while that of Ang-(1–7) was unchanged.We conclude that the method developed could be effectively used for accurate, comprehensive profiling of angiotensin peptides in mouse plasma. We identified substantial changes in renin–angiotensin system in a genetic mouse model of atherosclerosis consistent with the overactivation of angiotensin converting enzyme (ACE) and the impairment of ACE2.

Improving the LC-MS bioanalysis of hydrophilic peptides utilizing vacuum-based sample preparation

Hydrophilic peptides present great challenges for LC-MS analysis. Although the LC-MS method provides specificity and sensitivity, sample preparation is critical for bioanalytical assay performance. A practical sample preparation approach for improving the LC-MS analysis of hydrophilic peptides was developed, using hydrophilic isoforms of the Amyloid-β peptides as an example. A typical sample preparation procedure for peptides from biological matrices employs immunoaffinity purification; however, the high content of organic solvent in the elution buffer may cause insufficient chromatographic retention of the analytes on a RPLC column. By utilizing lyophilization or speed-vacuum drying followed by reconstitution with an aqueous solvent, the subsequent LC-MS bioanalysis showed these polar peptides being retained on the RPLC column and also an improved MS response. This approach is applicable for analytical studies on hydrophilic peptides in biological matrices.

Internal standard strategies for relative and absolute quantitation of peptides in biological matrices by liquid chromatography tandem mass spectrometry

The development of LC-MS/MS instruments and related applications improved the large-scale analyses of proteins and peptides in complex biological mixtures. The historical factor limiting these types of studies was the lack of sensitivity and reproducibility. However, the capacity of these analyses to detect proteins and peptides was significantly enhanced to a point where they are routinely performed in specialized laboratories in support to drug development programs as well as prognostic and diagnostic investigations. The analytical strategy used in peptidomic analyses needs to minimize the fluctuation in data measurements that might mask or reduce the precision of the determinations and consequently reduce the sensitivity of the assay. Inherently, it outlines the importance of careful standardization to reduce technical and instrumental variation. Therefore, this review will focus on the strengths and the limitations of the different experimental approaches used for the integration of internal standards in peptidomic studies. This review will examine a wide variety of methods, reagents, instrumentations and data analysis tools available to design peptidomic experiments. Moreover, this review will focus on the importance of precision and accuracy in order to adequately establish analysis threshold to detect peptide expression differences.

Quantifying tetrapeptide SS-20 in rat plasma using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Quantitative analysis of peptides in biological matrices remains a challenging task. This is due to the low dosage and the complexity of both the matrix and the analytical characteristics of peptides. SS-20 is a tetrapeptide compound developed for the treatment of Parkinson's disease. To investigate the pharmacokinetics of SS-20, a sensitive and rapid liquid chromatography coupled with mass spectrometry method was developed and validated. An aliquot of 50 μL plasma sample was extracted via solid phase extraction. The extracts were separated using a hydrophilic interaction liquid chromatography column, and were then detected with a triple quadrupole mass spectrometer using electrospray ionization in positive-ion mode and selected reaction monitoring. The use of a deuterium-labeled internal standard provided acceptable accuracy, precision, and matrix effect. The lower limit of quantification was 0.30 ng/mL. The linear range of the method was from 0.30 to 1000 ng/mL. The intraday and interday precisions were lower than 10.2% in terms of relative standard deviation, and the accuracy was within ±2.1% in terms of relative error. The validated LC–MS/MS method was successfully applied to a pharmacokinetic study of SS-20 following an intravenous or subcutaneous injection administration of 1.0 mg/kg to Sprague-Dawley rats.

Application of micro‐ and nano‐HPLC to the determination and characterization of bioactive and biomarker peptides

This article reviews the works published since 2001 (included) on the micro/nano-HPLC analysis of bioactive and biomarker peptides. The main achievements related to the improvement of the detection sensitivity, quantitation repeatability and reproducibility, and separation selectivity are highlighted. A wide attention is paid to the application of micro/nano-HPLC to the analysis of bioactive peptides in biological matrices. The uses of micro/nano-HPLC in peptidomics to discover new endogenous bioactive peptides and to develop quantitation procedures to compare the levels of peptides of interest in two different biological samples are also considered. Finally, the application of micro/nano-HPLC to the analysis of biomarker peptides for various diseases is also included in this review.

Simple and sensitive quantification of bioactive peptides in biological matrices using liquid chromatography/selected reaction monitoring mass spectrometry coupled with trichloroacetic acid clean‐up

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed for the quantification of bioactive peptides in biological fluids. The method employs protein precipitation with 4% trichloroacetic acid (TCA) and selected reaction monitoring (SRM) using an immonium ion as the product ion. This method was applied to determine the synthetic parathyroid hormone (PTH) analog (MW 1721) in rat plasma and human hepcidin-25 (MW 2789) in human serum. TCA clean-up showed a sufficient recovery for peptides with a MW of less than 3000, and would be useful as a simple and rapid method because of direct injection of the supernatant without evaporation or dilution. In addition, TCA clean-up allowed us not only to reduce sample preparation time, but also to select an immonium ion as a product ion of SRM, which led to detection more sensitive than SRM using other types of product ions. The lower limits of quantitation (LLOQs) of the PTH analog and the human hepcidin-25 were 0.2 ng/mL and 5 ng/mL, respectively. This method was fully validated with acceptable linearity, intra- and inter-assay precisions, and accuracy. Furthermore, this simple and rapid method is applicable to pharmacokinetic studies.

Enzymatic digestion as a tool for the LC–MS/MS quantification of large peptides in biological matrices: Measurement of chymotryptic fragments from the HIV-1 fusion inhibitor enfuvirtide and its metabolite M-20 in human plasma

The use of enzymatic digests of the peptide HIV-1 fusion inhibitor enfuvirtide as a tool for the absolute quantification of this polypeptide (MW 4492 Da) in human plasma by LC–MS/MS has been evaluated. Two different methods applying digestion of enfuvirtide with chymotrypsin after solid phase extraction (SPE) of the plasma samples have therefore been developed and validated. One method used a stable isotopically labeled analog of the complete peptide (d60-enfuvirtide) as internal standard (IS) and could use as much as four different chymotryptic fragments for the quantification of enfuvirtide in a range of 100–10,000 ng/ml. Intra- and inter-assay precisions and deviations from the nominal concentrations varied for the different fragments, but were below 9% when the four results were averaged. The other method used a stable isotopically labeled chymotryptic fragment of the peptide (d10-ASLW) as IS. Although this IS does not correct for variations in digestion recovery, it allows the selective quantification of enfuvirtide (100–10,000 ng/ml), besides the quantification of the sum of enfuvirtide and its de-amidated metabolite M-20 (120–12,000 ng/ml). Both methods were suitable for the absolute quantification of enfuvirtide and M-20 in plasma, but proper selection of the fragment(s) used for the quantification appeared crucial when the deuterated fragment was used as IS.

Profiling histidine‐containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.

On‐line coupling of size exclusion and capillary zone electrophoresis via a reversed‐phase C18 trapping column for the analysis of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved. The degradation of the peptides, caused by endogenous peptidases in the matrix, is sufficiently inhibited with imipramine HCl. The assay has a satisfactory linearity and intraday (9.70-16.3%) precision considering the complexity of this multidimensional separation system. The sensitivity of the method, with a concentration limit of quantification of 2.5 nug/mL, is comparable with other CZE assays for peptides and sufficient for the quantification of peptide drugs in biological matrices.

On-line coupling of SEC and RP–LC for the determination of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP–LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.70–4.9%) precision. The sensitivity of the method, with a concentration limit of quantitation (LOQ) of 2 μg/ml is comparable with other kinds of assays for peptides and sufficient for the quantitation of peptide drugs with higher therapeutic ranges in biological matrices. However, for the assay of low concentrations of peptides, such as endogenous components of a biological matrix, the sensitivity may need improvement. The LOQ cannot be improved by increasing the sample amount, because of interference of other endogenous components of the CSF. This indicates that a larger selectivity is desired. The LOQ may be improved by using more sensitive and selective detection methods such as mass spectrometry or fluorescence after post-column derivatization. Miniaturization of the system, combined with on-line trapping may also contribute to a better sensitivity.

Sensitive analysis of [ d-Pen 2,5]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [ d-Pen 2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C 18 solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm×50 μm I.D. capillary column with borate buffer (25 m M, pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.

Development of a capillary zone electrophoresis assay to examine the disposition of [ d-pen 2,5]enkephalin in rats

A novel capillary zone electrophoresis (CZE) assay method was developed to evaluate the systemic disposition of [ d-pen 2,5]enkephalin (DPDPE) in rats. DPDPE was recovered from serum samples (200 μl) by solid-phase extraction. Complete resolution of DPDPE and the internal standard ([ d-ser 2]leucine-enkephalin; DSLET) from other serum components was achieved within 15 min on a 50-μm I.D. capillary column with borate buffer (25 m M, pH 8.3). The peak-height ratio (DPDPE to DSLET) was linear through 100 μg/ml, with a detection limit of 250 ng/ml in serum, when absorbance of the column eluent was monitored at 210 nm. Serum samples obtained from rats after a 10 mg/kg intravenous bolus dose of DPDPE were analyzed with the present CZE method. The results suggest that CZE is a useful technique for quantitating therapeutic peptides in biological matrices.

High-performance liquid chromatographic analysis of arginine-containing peptides in biological fluids by means of selective post-column reaction with fluorescence detection

Highly sensitive and specific high-performance liquid chromatograhic methodology for the fluorescence detection of arginine-containing peptides in biological matrices is described. Following HPLC separation, a post-column reaction of the guanidino group of the arginine moiety with ninhydrin under basic conditions is utilized to generate a fluorescent peptide product which can be measured at high sensitivity. Careful optimization of the post-column reaction conditions, and the use of HPLC columns of reduced internal diameter, resulted in on-column detection limits as low as 50 fmol. Application to the determination of synthetic arginine-containing vasopressin antagonists in human plasma resulted in a quantitative response which is linear over the range 0.5–100 pmol/ml. The assay method is sufficiently sensitive, accurate, and precise for use in pharmacokinetic studies of these synthetic peptides. The methodology also has general applicability in the detection of naturally occurring arginine-containing peptides.