Development of high-throughput chemical extraction techniques and quantitative HPLC-MS/MS (SRM) assays for clinically relevant plasma proteins.

The clinical application of targeted plasma protein analysis by selective reaction monitoring of peptides using LC-MS/MS requires the development of robust, inexpensive protein extraction techniques with the potential for high-throughput applications. We present the development of a novel mixed-mode solid phase extraction (SPE) technique for the removal of high abundance and high molecular weight proteins from plasma. This technique, coupled with fused-core HPLC-MS/MS analysis is compared to a previously developed extraction method to study a range of proteins in plasma, including routinely measured biomarkers of growth hormone action. To further validate this technique, it was used for the quantification of insulin-like growth factor I (IGF-I) levels and compared to a state-of-the-art immunoassay on a fully automated analyzer. Clinical reference materials were applied for method development to allow for further interlaboratory comparisons. The LC-MS/MS approach quantified IGF-I in plasma with an accuracy that is within the guidelines for macromolecular assays in a regulated laboratory environment. Furthermore, IGF-I levels determined using the SPE and ACN methods with LC-MS/MS analysis correlated well with the immunoassay results. This demonstrates the applicability of mixed-mode SPE coupled with fused-core HPLC-MS/MS to quantify plasma proteins with results suitable for clinical applications.

Development and multicenter evaluation of the N latex CDT direct immunonephelometric assay for serum carbohydrate-deficient transferrin.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is a promising biomarker of alcohol abuse. We describe the development and multicenter evaluation of N Latex CDT (Dade Behring), an automated, particle-enhanced, homogeneous immunonephelometric assay for directly determining CDT. METHODS: N Latex CDT uses a monoclonal antibody that recognizes the structure of transferrin glycoforms lacking 1 or 2 complete N-glycans [i.e., disialo-, monosialo-, and asialotransferrins (CDT glycoforms)] in combination with a simultaneous assay for total transferrin. The Dade Behring BN II and BN ProSpec systems automatically calculate the CDT value as a percentage of total transferrin (%CDT). No preanalytical sample treatment is used. RESULTS: Total imprecision values for serum pools containing 1.8%-8.7% CDT were 3.4%-10.4% (mean, 6.8%). The mean (SD) %CDT for 561 serum samples from healthy control individuals was 1.76% (0.27%; range, 1.01%-2.85%). No marked sex or age differences were noted. The 97.5th percentile was at 2.35%. Transferrin genetic variants did not interfere with measurements. High transferrin concentrations did not falsely increase %CDT values, but increased %CDT values were noted for some samples with transferrin concentrations <1.1 g/L. N Latex CDT results correlated with those of a commercial CDT immunoassay involving column separation (r(2) = 0.862) and an HPLC candidate reference method (r(2) = 0.978). CONCLUSION: N Latex CDT is the first direct immunoassay for quantifying %CDT in serum. The specificity of N Latex CDT for identifying alcohol abuse may be higher than for immunoassays that use column separation, because transferrin genetic variants do not interfere with measurements.