Quantitation of Thyroglobulin in Serum Using SISCAPA and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate measurement of thyrogloblulin (Tg) at low concentrations is essential for recurrence-monitoring in patients who have been treated for papillary and follicular thyroid cancers. The immunoassays commonly employed by clinical laboratories to measure Tg are known to suffer interferences from thyroglobulin autoantibodies (TgAb).We describe a semiautomated stable isotope standards and capture by antipeptide antibodies (SISCAPA®) LC-MS/MS method for the accurate and precise measurement of Tg using 400 uL of serum. Following trypsin digestion of serum proteins in a 96-well plate format, a Tg-specific peptide is captured and concentrated using a monoclonal antibody bound to protein G-coated paramagnetic beads. Eighteen microliters of concentrate are injected into the LC-MS/MS system. Quantitation is performed against a 6-point linear calibration curve prepared in a blank matrix. The assay calibration range is 0.1-10 ng/mL, the range of clinical interest for recurrence detection. Total imprecision in clinical production has been observed to be 13.8% and 6.54% for in-house prepared control materials having Tg concentrations of 0.24 ng/mL and 0.94 ng/mL, respectively. Limit of quantitation was determined to be 0.1 ng/mL.

Sportomics method to assess acute phase proteins in Olympic level athletes using dried blood spots and multiplex assay.

Sportomics is a subject-centered holistic method similar to metabolomics focusing on sports as the metabolic challenge. Dried blood spot is emerging as a technique due to its simplicity and reproducibility. In addition, mass spectrometry and integrative computational biology enhance our ability to understand exercise-induced modifications. We studied inflammatory blood proteins (Alpha-1-acid glycoprotein-A1AG1; Albumin; Cystatin C; C-reactive protein-CRP; Hemoglobin-HBA; Haptoglobin-HPT; Insulin-like growth factor 1; Lipopolysaccharide binding protein-LBP; Mannose-binding lectin-MBL2; Myeloperoxidase-PERM and Serum amyloid A1-SAA1), in 687 samples from 97 World-class and Olympic athletes across 16 sports in nine states. Data were analyzed with Spearman's rank-order correlation. Major correlations with CRP, LBP; MBL2; A1AG1, and SAA1 were found. The pairs CRP-SAA1 and CRP-LBP appeared with a robust positive correlation. Other pairs, LBP-SAA1; A1AG1-CRP; A1AG1-SAA1; A1AG1-MBL, and A1AG1-LBP, showed a broader correlation across the sports. The protein-protein interaction map revealed 1500 interactions with 44 core proteins, 30 of them linked to immune system processing. We propose that the inflammation follow-up in exercise can provide knowledge for internal cargo management in training, competition, recovery, doping control, and a deeper understanding of health and disease.

Development of an automated, interference-free, 2D-LC-MS/MS assay for quantification of a therapeutic mAb in human sera.

AIM: Hybrid LC-MS/MS assays are increasingly used to quantitate proteins in biological matrices. These assays involve analyte enrichment at the protein level. Although suitability has been demonstrated, they are limited by the lack of appropriate affinity reagents and may suffer from interferences caused by binding proteins or antibodies. RESULTS: An online stable isotope standards and capture by anti-peptide antibodies assay was developed, which involves tryptic digestion of a therapeutic monoclonal antibody in human serum to destroy interfering proteins followed by enrichment using high affinity peptide antibodies. The assay was validated and compared with a standard ligand-binding assay currently used for quantification. CONCLUSION: The data show that the stable isotope standards and capture by anti-peptide antibodies-2D-LC-MS/MS assay can be used as an alternative method for measurement of monoclonal antibodies in clinical samples.