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

Abstract

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 Guideline on Validation of Bioanalytical Methods 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

“…46 The limit of detection (LOD) using the optimized MIP-SPE procedure on spiked serum samples was estimated to be 625 pM ProGRP isoform 1 using a signal-to-noise ratio (S/N) of 3. This detection limit is ca.…”

Water-Soluble Dried Blood Spot in Protein Analysis: A Proof-of-Concept Study

“…Stability of hCG stock solution and the prepared serum and urine samples has been evaluated in a previously published article. 21 The stability for hCG in whole blood stored on CMC was therefore the solely focus in the present work. Stability of the model analyte in whole blood on CMC was investigated using both high and low concentration of hCG in Figure 1.…”

“…Selected reaction monitoring (SRM) was selected as scan mode. The SRM method was based on the method by Lund et al 21 We monitored mainly for the signature peptide βT5 (VLQGVLPALPQVVCNYR) and the internal standard (VLQGVLPALPQVVCNY[R_ 13 …”

“…This was done as previously described by Lund et al: 21 A volume of 40 μL of stock solution (5 nmol/mL) was diluted with 940 μL of freshly prepared 200 mM ABC buffer. An amount of 8 μL of freshly prepared 200 mM DTT was then added and the solution was under vibration heated at 95°C for 20 min.…”

“…The SPE tips were made in house by punching out six discs of C8 3M Empore material (Teknolab AS, Kolbotn, Norway) with a diameter of approximately 1 mm using a Pasteur pipet as described elsewhere. 21 The discs were transferred from the Pasteur pipet and into the lower part of a 300 μL bevel point pipet tip (VWR, Hanover, Germany) using a metal wire. The SPE material was activated using 100 μL of MeCN followed by 100 μL of 20 mM FA.…”