Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs.

AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy.

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct.

Liquid Chromatography-High Resolution Mass Spectrometry for Peptide Drug Quality Control.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed using three peptide drugs: salmon calcitonin, bivalirudin, and exenatide as model systems to assess the suitability of this approach for monitoring peptide drug product quality. Calcitonin and its related impurities displayed linear responses over the range from 0.1 to 10 µM (R (2) values for calcitonin salmon, Glu(14)-calcitonin, and acetyl-calcitonin were 0.995, 0.996, and 0.993, respectively). Intra-assay precision in terms of relative standard deviation (%RSD) was less than 10% at all tested concentrations. The accuracy of the method was greater than 85% as measured by spiking 0.1, 0.3, and 1% of Glu(14)-calcitonin and acetyl-calcitonin into a stock calcitonin solution. Limits of detection for calcitonin, Glu(14)-calcitonin, and acetyl-calcitonin were 0.02, 0.03, and 0.04 µM, respectively, indicating that an impurity present at less than 0.1% (0.1 µM) of the drug product API concentration (107 µM) could be detected. Method validation studies analyzing bivalirudin and exenatide drug products exhibited similar results to calcitonin salmon in regard to high selectivity, sensitivity, precision, and linearity. Added benefits of using LC-HRMS-based methods are the ability to also determine amino acid composition, confirm peptide sequence, and quantify impurities, even when they are co-eluting, within a single experiment. LC-HRMS represents a promising approach for the quality control of peptides including the measurement of any peptide-related impurities. While the development work performed here is focus on peptide drug products, the principles could be adapted to peptide drug substance.

[Emulsion liquid membrane extraction with D2EHPA mobile carrier Hirudinaria manillensis hirudin in experimental study].

OBJECTIVE: To study the extraction system of hirudin emulsion liquid membrane with the Poecilobdella manillensis as raw material, di-(2-ethylhexyl) phosphate (D2EHPA) as carrier, Span 80 as emulsifier, octane and D2EHPA mixed to constitute membrane solution, diluted HCl solutions as internal aqueous phase. METHOD: Using the orthogonal experiment to optimize the extraction conditions of hirudin reference substance such as membrane phase, internal aqueous phase volume ratio (MIPVR), external aqueous phase pH, internal aqueous phase pH, mobile carrier concentration and so on, and then using hirudin crude extracts to do purifying experiment, and gaining experimental samples. RESULT: The optimal conditions of hirudin extraction were as follows: MIPVR 10: 3, internal aqueous phase pH 2.6, external aqueous phase pH 3.4, the mass fraction of carrier D2EHPA 2%. In the optimal extraction conditions, when the initial concentration of hirudin was one anti-thrombin activity units (ATU) x mL(-1), ATU recovery rate of the reference substance was 83.06%. In the purifying experiment of crude extracts, ATU recovery rate was 82.99%, and the specific activity of sample was 3 289.48 the ATU x mg(-1). Discontinuous polyacrylamide gel electrophoresis and spectral scanning, the results showed that the purity and reference substance were considerable. CONCLUSION: The method of preparation hirudin was relatively simple, the purity of the experimental samples and ATU recovery were both high.

[Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds].

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.

[Determination of biological activity of extract from hirudo by N-benzoyl-L-arginine ethyl ester].

As a potent anticoagulant, leech a traditional Chinese medicine, has become increasing topics. Hirudin, which is the primary effective component in leech, is a specific and efficient inhibitor of thrombin, mainly used in prevention and treatment of thrombus on the clinic practice. However, there is still no accurate and convenient method reported about the determination of it's biological activity. This paper reported a method for the determination of the biological activity the of extract from hirudo. The extra thrombin, which was not inhibited by hirudin in the extract from hirudo, reacted with N-benzoyl-L-arginine ethyl ester and was determined. The biological activity of the hirudo extract was determined, indirectly. The linear of calibration curve and accuracy were both perfect, the method was accurate and reliable.

[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

[Quality comparison of Hirudo before and after processed by French chalk].

OBJECTIVE: To evaluate the effects of processing methods of Hirudo. METHOD: Both water and alcohol extracts of Hirudo were studied according to Chinese Pharmacopeia (Edition 2005). The content of hypoxanthine in Hirudo was measured by high performance liquid chromatography (HPLC) and Hirudin was determined by thrombin. RESULT: The contents of water, water soluble extraction, ethanol soluble extraction and hirudin in crude Hirudo are higher than those in processed Hirudo. But the contents of hypoxanthine in processed Hirudo is higher than in crude Hirudo. CONCLUSION: Hirudo fried by French chalk can decrease the active components with high intensive drug property, accordingly the toxicity of Hirudo was decreased. As a result, the effects of Hirudo as invigorate the circulation of blood and stimulate the menstrual flow were abated.

Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin.

The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL(-1) to 20 mg mL(-1) in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 x 10(8) to 1.7 x 10(8) microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery.

Design of FRET-based GFP probes for detection of protease inhibitors.

In this study, tandem Green fluorescent protein (GFP) fusion proteins were designed to detect proteolytic activity of thrombin based on the principle of fluorescence resonance energy transfer (FRET). The thrombin-specific recognition sequence, LVPR, was strategically placed in between a cyan-emitting mutant of the green fluorescent protein and an enhanced yellow-emitting fluorescent protein to allow thrombin-specific cleavage with detectable changes of FRET signal. A 4.6-fold increase of fluorescence emission ratio was observed upon addition of thrombin. This FRET-based probe was further tested for dose-dependent effects of thrombin specific inhibitor, hirudin. Our result showed a nice correlation between fluorescence emission ratios and concentrations of hirudin with subnanomolar sensitivity. We propose that FRET-based GFP probes can be used for high-throughput screening of protease inhibitors.

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.

Determination of recombinant hirudin structural deviants by capillary zone electrophoresis augmented with buffer additives.

The polypeptide hirudin is a potent and specific thrombin inhibitor used in anticoagulant therapy and naturally occurring in medicinal leech. Using gene-technology methods, recombinant (r) hirudin can be produced on a large scale. Purity evaluation of the synthesized r-hirudin is essential to monitor co-expressed structural deviants and degradation products before therapeutic use. Although the well established RP-HPLC analysis appears to be the method of choice, in the case of r-hirudin baseline separation of the structural deviants is not necessarily achieved. Capillary zone electrophoresis augmented with buffer additives was used as a complementary technique to separate r-hirudin successfully from several similar species, in order to provide characterization information, as well as performing purity control and stability studies.

Existence of beta-methylnorleucine in recombinant hirudin produced by Escherichia coli.

A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l(-1)) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as beta-methylnorleucine (beta MeNle; (2S,3S)-2-amino-3-methylhexanoic acid). The modification pathway of beta MeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, beta MeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of beta MeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.

Determination of the cysteine and cystine content of proteins by amino acid analysis: application to the characterization of disulfide-coupled folding intermediates.

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.

Capillary electrophoresis of r-hirudin and a polyethylene glycol derivative of r-hirudin (PEG-hirudin).

UNLABELLED: Recombinant (r-) hirudins and PEG-hirudin are currently tested for anticoagulant therapy. For their concentration measurement, radioimmunoassay and HPLC methods are available. The separation of r- and PEG-hirudin is currently performed by HPLC. However, the sensitivity of the method is low. Capillary electrophoresis is a rapid, selective technique that requires low sample amounts. Our aim was the development of a capillary electrophoresis method to measure r- and PEG-hirudin. The results are as follows: In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-hirudin was separated from PEG-hirudin in a purified system using a fused silica capillary (50 cm long and 75-micron i.d. and reversed polarity). A neutral capillary with a 20 mM tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r- from PEG-hirudin. A linear correlation was found between the peak area and the concentration between 20 micrograms/mL and 10 mg/mL for hirudin (r2 = 0.99) and between 1.25 and 10 mg/mL for PEG-hirudin (r2 = 0.99). In human plasma mixtures, r- and PEG-hirudin were completely separated. The linear correlation between the peak area and the concentration was r2 = 0.99. CONCLUSION: In a fused silica capillary, r- and PEG-hirudin are separated in a purified system. Capillary electrophoresis which is performed in a neutral capillary, resolves r- from PEG-hirudin in a purified system, in plasma and in urine. The sensitivities of the methods are comparable. Capillary electrophoresis separates r- from PEG-hirudin and may be applied to biologic systems to measure the concentration and purity of r- and PEG-hirudin.

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers.

On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins.

Quantitative determination of hirudin in blood and body fluids.

Recent clinical studies using hirudin as anticoagulant have demonstrated that an efficient method to determine the current blood level of hirudin is imperative for exact dose finding and adjustment. Only the exact determination of the hirudin content in blood, performed within a few minutes, prevents overdosage involving side effects or, otherwise, a subtherapeutic dose regimen. Therefore, a method for rapid, sensitive, and reproducible measurement of hirudin in blood, plasma, and other body fluids has been developed. The method, which is based on coagulation measurement, is called ecarin clotting time (ECT). In this test, ecarin, a purified enzyme of the Echis carinatus snake venom, acts as a prothrombin activator. In contrast to the "solid phase" prothrombin activation by prothrombinase, the ecarin-induced prothrombin activation proceeds in an alternative way, i.e., without the need of cofactors, resulting in intermediates such as meizothrombin. Compared to thrombin, meizothrombin has a lower procoagulant activity, but it still binds hirudin, which leads to the inhibition of meizothrombin. Depending on the sample's concentration of hirudin, ecarin forms a residual, nonhirudin-bound amount of intermediates of the prothrombin-thrombin conversion that are able to concentration-dependently convert fibrinogen to fibrin. There is an excellent linear correlation between ECT prolongation and the hirudin content of the sample in a range from 50 to 5,000 ng/mL blood or plasma. This allows immediate measurement not only of the therapeutic blood level of hirudin, but also of its concentration in blood following under- or overdosage. The ECT method is nearly independent of variations in the sample's content of fibrinogen (from 60% to 100%) and prothrombin (from 20% to 100%.) Heparin is not able to catalyze the very low antithrombin inhibition of meizothrombin. Therefore, it is also possible to determine hirudin in blood containing varying amounts of heparin. Another advantage of the method is that it can be applied to different mechanical measuring systems used in coagulation diagnostics.