NC100668, A NEW TRACER FOR IMAGING OF VENOUS THROMBOEMBOLISM: DISPOSITION AND METABOLISM IN RATS

ABSTRACT: The NC100668 consists of a 13-amino acid peptide, N-terminally blocked with an acetyl group containing an iodinated tyrosine and coupled to a Tc-chelator (NC100194) via the C-terminal glycine. Using the common three-letter abbreviations for amino acids, the structure of NC100668 is Acetyl-Asn-Gln-Glu-Gln-Val-Ser-ProTyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194, where NC100194 is represented by the chemical formula -NH-CH 2 -CH 2 -N(CH 2 -CH 2 -NH-C(CH 3 ) 2 -C(CH 3 )ϭN-OH) 2 . The detailed structure is shown in 99m Tc-NC100668 is being developed as a diagnostic radiopharmaceutical for imaging of venous thromboembolism, which is a major health problem with an estimated average annual incidence in the United States exceeding 1 per 1000 Diagnostic radiopharmaceuticals are radioactive substances that are administered to patients. Many of these agents are labeled with the ␥-emitter 99m Tc, which has a half-life of 6.02 h doi:10.1124/dmd.105.006239. ABBREVIATIONS: NC100668, Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194; NC100194, -NH-CH 2 -CH 2 -N(CH 2 -CH 2 -NH-C(CH 3 ) 2 -C(CH 3 )ϭN-OH) 2 ; Fmoc, 9-fluorenyloxycarbonyl; Boc, butyl oxy carbonyl; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate; DMF, dimethylformamide; TFA, trifluoroacetic acid; MS, mass spectrometry; HPLC, high-performance liquid chromatography; QWBA, quantitative whole-body autoradiography; SAC, self-absorption coefficient; % ID, percentage of injected dose; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry

Exploring peptide capture by anti-protein antibodies for LC-MS-based biomarker determination

Immunocapture LC-MS/MS is a promising technique to ensure high sensitivity and selectivity of low-abundant protein biomarkers. For this purpose, the use of monoclonal antibodies (mAb) is especially attractive as they are renewable reagents that can be standardized. In this article we investigated the possibility of using mAbs developed against intact proteins (anti-protein antibodies) to capture proteotypic epitope peptides. Three mAbs were tested, and all selectively extracted proteotypic epitope peptides from a complex sample. Compared to intact protein extraction, this concept which we call peptide capture by anti-protein antibodies provided cleaner extracts, which further improved the sensitivity. Analysis of three patient samples demonstrated that p can be used for the determination of different endogenous protein levels. </p

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