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.

Selective Fishing for Peptides with Antibody-Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS.

An online microfluidics-mass spectrometry platform was developed for determining proteotypic peptides from in-solution digested samples. Accelerated and selective sample cleanup was achieved by integrating proteotypic epitope peptide immunoextraction with nano liquid chromatography-tandem mass spectrometry (online IE-nanoLC-MS/MS). Ten individually prepared 180 µm inner diameter capillaries with ethylene glycol dimethacrylate- co-vinyl azlactone (EDMA- co-VDM) monoliths were immobilized with anti-protein antibodies that are used in routine immunoassays of the intact small cell lung cancer biomarker ProGRP. The resulting AB columns provided linearity correlation coefficients of 0.96-0.99 for protein amounts and concentrations of 10 pg to 5 ng and 0.5-250 ng/mL, respectively. The columns/platform gave relative peak area RSDs below 15%. The IE-nanoLC-MS/MS platform provided a limit of detection (LOD) of 520 pg/mL of ProGRP in human serum. The approach was applicable for other matrixes and proteins, i.e., primary glioblastoma cells and endogenous αV integrin chain. Thus, EDMA- co-VDM monoliths immobilized with antibodies are suited for automated peptide capture in microfluidic formats.

Evaluation of affinity-based serum clean-up in mass spectrometric analysis: Plastic vs monoclonal antibodies.

Mass spectrometric assays are now of great relevance for trace compound analysis in complex matrices such as serum and plasma samples. Especially in the quantification of low abundant protein-biomarkers, the choice of the sample preparation is crucial. In the present paper immunocapture and Molecular Imprinted Polymers (MIPs) have been applied in the determination of pro-gastrin-releasing peptide, a Small Cell Lung Cancer marker. These affinity-based techniques were compared in terms of matrix effect, limits of detection, repeatability and extraction specificity. In addition, protein precipitation was included for comparison as it is a typical sample preparation method of biological matrices. The results highlighted differences in the methods' performance and specificity, strongly affecting the outcome of the mass spectrometric determination. Plastic and monoclonal antibodies confirmed to be sensitive and specific sample preparations able to determine ProGRP at clinical relevant concentration, although only the use of monoclonal antibodies allowed the reliable quantification of ProGRP at reference levels (8pM). In addition better insight in the specificity of the three sample preparation techniques was gained. This might also be of interest for other biological applications.

Synthesis, biological evaluation and molecular modeling studies of the PPARß/δ antagonist CC618.

Herein, we describe the synthesis, biological evaluation and molecular docking of the selective PPARß/δ antagonist (4-methyl-2-(4-(trifluoromethyl)phenyl)-N-(2-(5-(trifluoromethyl)-pyridin-2-ylsulfonyl)ethyl)thiazole-5-carboxamide)), CC618. Results from in vitro luciferase reporter gene assays against the three known human PPAR subtypes revealed that CC618 selectively antagonizes agonist-induced PPARß/δ activity with an IC50 = 10.0 µM. As observed by LC-MS/MS analysis of tryptic digests, the treatment of PPARß/δ with CC618 leads to a covalent modification of Cys249, located centrally in the PPARß/δ ligand binding pocket, corresponding to the conversion of its thiol moiety to a 5-trifluoromethyl-2-pyridylthioether. Finally, molecular docking is employed to shed light on the mode of action of the antagonist and its structural consequences for the PPARß/δ ligand binding pocket.

Multiplexing determination of small cell lung cancer biomarkers and their isovariants in serum by immunocapture LC-MS/MS.

A multiplex method for the determination of the small cell lung cancer (SCLC) markers progastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) is presented, which involves coextraction by immunoaffinity (IA) beads and codetermination by selected reaction monitoring (SRM). The performance was compared with two IA SRM methods which were recently validated for individual marker determination. The multiplexing method reduces sample volume, handling time per sample, and reagent consumption and shows good linearity, recovery, quantitative measurements, and sensitivity with lower limit of detection (LLOD) values of 7.2 pM (=90 pg/mL) and 4.5 pM (=210 pg/mL) and lower limit of quantitation (LLOQ) values of 24 pM (=300 pg/mL) and 15 pM (=700 pg/mL), for total ProGRP and γ-NSE, respectively. The novel aspect of this approach is the multiplexing of ProGRP and NSE with the additional ability to perform fingerprinting by the selective determination of ProGRP isoform 1, ProGRP isoform 3, and total ProGRP, as well as the α- and the γ-subunit of NSE isoenzymes. Six serum samples from patients with SCLC were analyzed to demonstrate the methods feasibility to simultaneously differ between and individually quantify ProGRP, NSE, and their isoform and isoenzyme variants, respectively. Both the presence of and variation between all the isoforms and isoenzymes, as well as covarying results with the conventional immunometric assays for total ProGRP and γ-NSE, were seen in the analyses of patient serum samples.

New labdane diterpenes from Solidago canadensis.

The ethanol extract of roots of Solidago canadensis yielded eight labdane-type diterpenes. Five of those were new natural compounds (9,13,15,16-bisepoxy-labdane-7-ene-6,15-dione (3a), 13-epi-9,13,15,16-bisepoxy-labdane-7-ene-6,15-dione (3b), 15,16-epoxy-labdane-7,13-diene-6,16-dione (5), 15-ethoxy-9,13,15,16-bisepoxy-labdane-7-ene-6-one (6a) and 13-epi-15-ethoxy-9,13,15,16-bisepoxy-labdane-7-ene-6-one (6b). The known labdane diterpenes deoxysolidagenone (1), solidagenone (2) and 15,16-epoxy-labdane-7,13-diene-6,15-dione (4) were also isolated. Chemical structures were determined using 1D and 2D NMR techniques and MS analysis.