Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

Immunocapture sample clean-up in determination of low abundant protein biomarkers - a feasibility study of peptide capture by anti-protein antibodies.

Immunocapture in mass spectrometry based targeted protein analysis using a bottom-up workflow is nowadays mainly performed by target protein extraction using anti-protein antibodies followed by tryptic digestion. Already available monoclonal antibodies (mAbs) which were developed against intact target proteins (anti-protein antibodies) can capture proteotypic epitope containing peptides after tryptic digestion of the sample. In the present paper considerations when developing a method for targeted protein quantitation through capture of epitope containing peptides are discussed and a method applying peptide capture by anti-protein antibodies is compared with conventional immunocapture MS. The model protein used for this purpose was progastrin releasing peptide (ProGRP), a validated low abundant biomarker for Small Cell Lung Cancer with reference values in serum in the pg mL-1 range. A set of mAbs which bind linear epitopes of ProGRP are available, and after a theoretical consideration, three mAbs (E146, E149 and M18) were evaluated for extraction of proteotypic epitope peptides from a complex sample. M18 was the best performing mAb for peptide capture by anti-protein antibodies, matching the LOD (54 pg mL-1) and LOQ (181 pg mL-1) of the existing conventional immunocapture LC-MS/MS method for determination of ProGRP. Peptide and protein capture using the same mAb were also compared with respect to sample clean-up, and the peptide capture workflow yielded cleaner extracts and therewith less complex chromatograms. Analysis of five patient samples demonstrated that peptide capture by anti-protein antibodies can be used for the determination of various levels of endogenously present ProGRP.

Exploration of the impact of stereochemistry on the identification of the novel translocator protein PET imaging agent [(18)F]GE-180.

INTRODUCTION: The tricyclic indole compound, [(18)F]GE-180 has been previously identified as a promising positron emission tomography (PET) imaging agent of the translocator protein (TSPO) with the potential to aid in the diagnosis, prognosis and therapy monitoring of degenerative neuroinflammatory conditions such as multiple sclerosis. [(18)F]GE-180 was first identified and evaluated as a racemate, but subsequent evaluations of the resolved enantiomers have shown that the S-enantiomer has a higher affinity for TSPO and an improved in vivo biodistribution performance, in terms of higher uptake in specific brain regions and good clearance (as described previously). Here we describe the additional biological evaluations carried out to confirm the improved performance of the S-enantiomer and including experiments which have demonstrated the stability of the chiral centre to chemical and biological factors. MATERIALS AND METHODS: GE-180 and the corresponding radiolabelling precursor were separated into single enantiomers using semi-preparative chiral supercritical fluid chromatography (SFC). A detailed comparison of the individual enantiomers and the racemate was carried out in a number of biological studies. TSPO binding affinity was assessed using a radioligand binding assay. Incubation with rat hepatic S9 fractions was used to monitor metabolic stability. In vivo biodistribution studies up to 60 min post injection (PI) in naïve rats were carried out to monitor uptake and clearance. Achiral and chiral in vivo metabolite detection methods were developed to assess the presence of metabolite/s in plasma and brain samples, with the chiral method also determining potential racemisation at the chiral centre. RESULTS: Evaluation of the chiral stability of the two enantiomers to metabolism by rat S9 fractions, showed no racemisation of enantiomers. There were notable differences in the biodistribution between the racemate and the R- and S-enantiomers. All compounds had similar initial brain uptake between 0.99 and 1.01% injected dose (id) at 2 min PI, with S-[(18)F]GE-180 showing significantly greater retention than the R-enantiomer at 10 and 30 min PI (P<0.05). S-[(18)F]GE-180 uptake to the TSPO-expressing olfactory bulbs was 0.45% id (SD ± 0.17) at 30 min PI in comparison to RS-[(18)F]GE-180 or R-[(18)F]GE-180 levels of 0.41% id ± 0.09 and 0.23% id ± 0.02 respectively, at the same timepoint (P > 0.05). The signal-to-noise ratio (ratio olfactory bulb to striata binding) were similar for both RS-[(18)F]GE-180 and S-[(18)F]GE-180 (3.2 and 3.4 respectively). Initial uptake to the lungs (an organ with high TSPO expression) was more than 3-fold greater with S-[(18)F]GE-180 than R-[(18)F]GE-180, and significantly higher at 10 and 30 min PI (P < 0.05). Furthermore lung uptake of S-[(18)F]GE-180 at 2 and 10 min PI was also significant when compared to the racemate (P < 0.05). The majority of the radioactivity in the rat brain following administration of RS-[(18)F]GE-180 or S-[(18)F]GE-180 was due to the presence of the parent compound (91% ± 1.5 and 94% ± 2.0 of total radioactivity at 60 min PI respectively). In contrast at 60 min PI for the plasma samples, the parent compounds accounted for only 28% ± 1.2 and 21% ± 4.6 of total radioactivity for RS-[(18)F]GE-180 and S-[(18)F]GE-180 respectively. Chiral assessment confirmed that the S-enantiomer was chirally stable in vivo, with no stereochemical conversion in brain and plasma samples up to 60 min PI. CONCLUSIONS: Developing racemic radiotracers, as for racemic therapeutics, is a considerable challenge due to differences of the enantiomers in pharmacokinetics, efficacy and potential toxicity. We have shown that the enantiomers of the promising racemic PET ligand [(18)F]GE-180 do not share identical performance, with S-[(18)F]GE-180 demonstrating higher TSPO affinity, higher brain uptake and better retention to the high TSPO-expressing lungs. Furthermore, S-[(18)F]GE-180 has also been shown to be enantiomerically stable in vivo, with no observed conversation of the eutomer to the distomer. As a single enantiomer, S-[(18)F]GE-180 retains the beneficial characteristics of the racemate and is a promising imaging agent for imaging neuroinflammation in vivo.

GE-145, a new low-osmolar dimeric radiographic contrast medium.

BACKGROUND: Contrast-induced nephrotoxicity is a significant risk when using radiographic contrast media clinically, especially in high risk patients. Consequently, there is a need for a new contrast agent with improved clinical safety with regards to nephrotoxicity. PURPOSE: To evaluate the physicochemical properties as well as the preclinical safety and biodistribution parameters of the newly developed radiographic contrast medium GE-145. MATERIAL AND METHODS: Standard methods for radiographic contrast media were used for evaluation of physicochemical properties. The acute toxicity in rats was studied at 8, 10, and 12.5 gI/kg, the clinical chemistry parameters were determined, and histology of the kidneys was performed. Biodistribution was studied in rats using ¹²³I-labeled GE-145. RESULTS: GE-145 is more hydrophilic than iodixanol and has a considerably lower osmolality. The viscosity is similar to that of iodixanol and the protein binding is low. The acute toxicity is similar to that of iodixanol and the biodistribution is similar to that of other radiographic contrast media, showing mainly renal excretion. Kidney histology showed a moderate reversible vacuolization, similar to that of iodixanol. CONCLUSION: GE-145 exhibits similar preclinical properties to other dimeric radiographic contrast media. In addition, the low osmolality enables an iso-osmolar formulation containing a significantly higher concentration of electrolytes than Visipaque.

Phase I trial of the positron-emitting Arg-Gly-Asp (RGD) peptide radioligand 18F-AH111585 in breast cancer patients.

UNLABELLED: The integrin alpha v beta3 receptor is upregulated on tumor cells and endothelium and plays important roles in angiogenesis and metastasis. Arg-Gly-Asp (RGD) peptide ligands have high affinity for these integrins and can be radiolabeled for PET imaging of angiogenesis or tumor development. We have assessed the safety, stability, and tumor distribution kinetics of a novel radiolabeled RGD-based integrin peptide-polymer conjugate, 18F-AH111585, and its feasibility to detect tumors in metastatic breast cancer patients using PET. METHODS: The biodistribution of 18F-AH111585 was assessed in 18 tumor lesions from 7 patients with metastatic breast cancer by PET, and the PET data were compared with CT results. The metabolic stability of 18F-AH111585 was assessed by chromatography of plasma samples. Regions of interest (ROIs) defined over tumor and normal tissues of the PET images were used to determine the kinetics of radioligand binding in tissues. RESULTS: The radiopharmaceutical and PET procedures were well tolerated in all patients. All 18 tumors detected by CT were visible on the 18F-AH111585 PET images, either as distinct increases in uptake compared with the surrounding normal tissue or, in the case of liver metastases, as regions of deficit uptake because of the high background activity in normal liver tissue. 18F-AH111585 was either homogeneously distributed in the tumors or appeared within the tumor rim, consistent with the pattern of viable peripheral tumor and central necrosis often seen in association with angiogenesis. Increased uptake compared with background (P = 0.002) was demonstrated in metastases in lung, pleura, bone, lymph node, and primary tumor. CONCLUSION: 18F-AH111585 designed to bind the alpha v beta3 integrin is safe, metabolically stable, and retained in tumor tissues and detects breast cancer lesions by PET in most anatomic sites.

NC100668, a new tracer tested for imaging of venous thromboembolism: pharmacokinetics and metabolism in humans.

The (99m)Tc-complex of NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a new tracer tested for nuclear medical imaging of venous thromboembolism. NC100668 is a 13-amino acid peptide with a Tc-binding chelator [NC100194; -NH-CH2-CH2-N(CH2-CH2-NH-C(CH3)2-C(CH3)=N-OH)2] linked to the C-terminal end. The present study was performed following injection of (99m)Tc-NC100668 in healthy human volunteers with five dose levels of NC100668 (20-2000 microg) and a constant radioactivity dose. The rate at which the radioactivity was cleared from blood was independent of gender and dose of NC100668; more than half of the 82% urinary clearance of radioactivity was obtained 2 h postinjection. The radioactivity in blood was reduced to 50% of initial values within 12 min; this was followed by a more gradual decrease with a half-life of 1.2 h and a terminal elimination half-life of 10.5 h. The plasma concentration of NC100668 decreased rapidly with an initial half-life of 5 to 10 min. The half-life after this initial phase could be estimated for only two of the subjects in the highest-dose group because the NC100668 concentration in the other samples at these time points was below the limit of detection of the liquid chromatography/mass spectrometry (LC/MS) method. LC/MS analyses of urine samples revealed the identity of two metabolites generated from the C-terminal end of the molecule; Gly-NC100194 was identified as the major metabolite and NC100194 as a minor metabolite. The estimated sum of these two metabolites is in the same magnitude as the recoveries of (99m)Tc in these samples, indicating that most of the (99m)Tc excreted in urine is bound to one of these metabolites.

Quantification of NC100692, a new tracer for 99mTc-imaging of angiogenesis, in human plasma using reversed-phase liquid chromatography coupled with electrospray ionization ion-trap mass spectrometry.

NC100692 is under development as a diagnostic radiopharmaceutical for targeting angiogenesis associated with diseases, such as cancer and endometriosis. NC100692 consists of a cyclic RGD-containing peptide with an ethylene glycol chain linked to the C-terminal amino acid and a (99m)Tc-binding chelator linked to the N-terminal amino acid. The present report describes a method for quantification of NC100692 in human citrated plasma. The method is based on solid-phase extraction followed by reversed-phase liquid chromatography using a gradient of water and acetonitrile with 0.1% formic acid. The chromatographic system was coupled on-line with an electrospray mass spectrometer. The analyses were performed by selective ion monitoring of the [M+2H](2+) and the [M+3H](3+) ions of NC100692 and the internal standard, which was identical to NC100692 except for containing twice the length of the ethyleneglycol chain. The limit of quantification of the method was 0.5 ng NC100692/ml plasma. The calibration curve ranged from 0.5 to 250 ng NC100692/ml plasma and was fitted to a quadratic equation with a weighing factor of 1/y and found to be highly reproducible. The total precision of the method, expressed as the relative standard error of the mean, was 11.1, 10.8 and 9.7% for the low, medium and high control samples, respectively. The accuracy of the method was 103.4, 111.1 and 107.5% for the low, medium and high control samples, respectively. NC100692 was stable in human plasma during at least 3 freeze/thaw cycles, during 48 h on dry ice and at least 8 weeks when stored in a -20 degrees C freezer.

Disposition of perfluorobutane in rats after intravenous injection of Sonazoid.

The new ultrasound contrast agent Sonazoid was injected IV in rats at doses of 0.8 and 8 muL perfluorobutane (PFB)-containing microbubbles/kg body weight. Samples were obtained from blood, liver, spleen, fat, kidney, muscle, heart, lung and brain from both males and females and the PFB gas was analyzed using validated gas chromatography mass spectrometry methods. No differences were observed between genders or doses for any of the pharmacokinetic parameters. For all tissues, the highest concentrations were observed at the first time point (i.e., 5 min postinjection) (51% of injected dose in liver; total recovery of 69%). The highest concentrations of PFB in tissue were observed in spleen > liver > lung > kidney >> other tissues. At 24 h after dosing, the total amount of PFB remaining in the tissues was 1.9%. These data fit well with the finding that after a Sonazoid dose of 8 microL microbubbles/kg to male rats, more than 50% of the injected PFB was recovered in exhaled air by 20 min after dosing. During the first 24 h after administration, more than 96% of the PFB dose was recovered in exhaled air.

Nc100668, a new tracer for imaging of venous thromboembolism: disposition and metabolism in rats.

The 99mTc-complex of NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is being evaluated for nuclear medical imaging of venous thromboembolism. NC100668 is a 13-amino acid peptide with a Tc-binding chelator [NC100194; -NH-CH2-CH2-N(CH2-CH2-NH-C(CH3)2-C(CH3)=N-OH)2] linked to the C-terminal end. Following injection in rats of [Asn-U-14C]NC100668 (labeling of the N-terminal amino acid), approximately 70% of the radioactivity was recovered in urine within 3 days. Following injection of [Lys-U-14C]NC100668 (labeling close to the C-terminal amino acid), radioactivity was cleared more slowly, with only 8% recovered in urine and approximately 80% of the radioactivity present in the body after 3 days. The highest concentration of radioactivity in the body following injection of [Lys-U-14C]NC100668 was observed in the kidney inner cortex; this probably represents 14C-labeled Lys, which is reabsorbed in the kidney tubules and incorporated into protein metabolism. Metabolite profiling by high-performance liquid chromatography with radiochemical detection revealed that following injection of [Asn-U-14C]NC100668, there is a rapid appearance in blood of one peak containing radioactive metabolite(s) originating from the N-terminal part of the molecule. In urine samples, only this radioactive peak was observed with no intact NC100668 remaining; this very hydrophilic N-terminal metabolite was probably either the N-terminal amino acid or a very short peptide. Liquid chromatography-mass spectrometry analyses of rat urine samples obtained after injection of nonlabeled NC100668 confirmed the identity of two metabolites generated from the C-terminal end of the molecule; Gly-NC100194 was identified as the major of these metabolites and NC100194 as a minor metabolite present at approximately one-tenth the amount of Gly-NC100194. No other metabolites were identified.

Quantification of NC100668, a new tracer for imaging of venous thromboembolism, in human plasma using reversed-phase liquid chromatography coupled with electrospray ionization ion-trap mass spectrometry.

NC100668 is being developed as a new tracer for radiopharmaceutical imaging of venous thromboembolism. NC100668 consists of a targeting peptide of 13 amino acids with a (99m)Tc-binding chelator linked to the C-terminal amino acid. The present report describes a method for quantification of NC100668 in human citrated plasma. The method is based on solid-phase extraction followed by reversed-phase liquid chromatography using a gradient of water and acetonitrile with 0.1% formic acid. The chromatographic system was coupled on-line with an electrospray mass spectrometer. The analyses were performed by selective ion monitoring of the [M+2H]2+ and the [M+3H]3+ ions of NC100668 and an internal standard which was identical to NC100668 except for not being iodinated in the tyrosine residue. The limit of quantification of the method was 2ng NC100668/ml plasma. The calibration curve ranged from 2 to 250 ng NC100668/ml plasma and was fitted to a linear equation with a weighing factor of 1/y2 and found to be highly reproducible. The total precision of the method, expressed as the relative standard error of the mean, was 23.2, 8.8 and 14.7% for the low, medium and high control samples, respectively. The accuracy of the method was 108.5, 100.0 and 105.0% for the low, medium and high control samples, respectively. NC100668 was stable in human plasma during at least three freeze/thaw cycles, during 30 h on dry ice and up to 3 months when stored in a -20 degrees C freezer.

Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex - a family of cyclic peptide antibiotics from Bacillus subtilis.

Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity. A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules. Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity. Characterisation of the peptides with LC-MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da. No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before. We have named them Maltacines.

In vitro stability analyses as a model for metabolism of ferromagnetic particles (Clariscan), a contrast agent for magnetic resonance imaging.

Clariscan is a new contrast agent for magnetic resonance imaging. It is an aqueous suspension of ferromagnetic particles injected for blood pool and liver imaging. Previous experiments showed that particles made of 59Fe were taken up by the mononuclear phagocytic system and then solubilised. The present work aims at explaining a possible mechanism for the dissolution of these ferromagnetic particles in the body. The particles were diluted in 10-mM citrate or 10-mM acetate buffers at pH 4.5, 5.0 and 5.5 and incubated at 37 degrees C for up to 22 days, protected from light. The mixtures were analysed at different times during this incubation period using photon correlation spectroscopy, magnetic relaxation, visible spectroscopy and reactivity of the iron with the chelator, bathophenanthroline disulphonic acid. The data obtained with these techniques showed that the particles were almost completely solubilised within 4-7 days when incubated in 10 mM citrate, pH 4.5. Incubation in 10 mM citrate buffer, pH 5.0 revealed a slower solubilisation of the particles, as the changes observed after 72 h of incubation at pH 5.0 were 43-71% of the changes observed at pH 4.5. Incubation in 10 mM citrate, pH 5.5 revealed an even slower solubilisation of the particles, as the changes observed after 72 h of incubation at pH 5.5 were 12-34% of those observed at pH 4.5. Incubation of the particles in 10 mM acetate at pH 4.5, 5.0 and 5.5, as well as incubation of the particles in water pH adjusted to pH 5.1, resulted in only minor or no solubilisation of the particles. The results indicate that the low pH of endosomes and lysosomes, as well as endogenous iron-complexing substances, may be important for the solubilisation of these ferromagnetic particles following i.v. injection of Clariscan.