Immuno–MS Based Targeted Proteomics: Highly Specific, Sensitive, and Reproducible Human Chorionic Gonadotropin Determination for Clinical Diagnostics and Doping Analysis

The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in <i>Guideline on Validation of Bioanalytical Methods</i> was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method’s application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis

Antibody-Free Biomarker Determination: Exploring Molecularly Imprinted Polymers for Pro-Gastrin Releasing Peptide

Biomarker mass spectrometry assays are in high demand, and analysis of pro-gastrin releasing peptide (ProGRP) as a small cell lung cancer marker has been recently investigated by mass spectrometry after immunoextraction. In this article, we introduce an assay based on molecularly imprinted polymers (MIPs) targeting the proteotypic peptide of ProGRP as a possible alternative to current immuno-based assay. The MIPs were prepared by surface-initiated reversible addition–fragmentation chain transfer polymerization and were introduced as sorbents for the cleanup and enrichment of a ProGRP signature peptide from tryptically treated serum samples. The use of an appropriate solid-phase extraction protocol allowed specific extraction of the target peptide while depleting other peptides that arose from the sample digestion, hence resulting in reduced background. The selective extraction of a ProGRP signature peptide, after digestion of serum samples, translates into a time- and cost-effective method suited for bottom-up analysis wherever targeted peptide extraction from complex matrices is required

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