Magnetic Synthetic Receptors for Selective Clean-Up in Protein Biomarker Quantification.

Biomarker analysis by mass spectrometry (MS) can allow for the rapid quantification of low abundant biomarkers. However, the complexity of human serum is a limiting factor in MS-based bioanalysis; therefore, novel biomarker enrichment strategies are of interest, particularly if the enrichment strategies are of low cost and are easy to use. One such strategy involves the use of molecularly imprinted polymers (MIPs) as synthetic receptors for biomarker enrichment. In the present study, a magnetic solid-phase extraction (mSPE) platform, based on magnetic MIP (mMIP) sorbents, is disclosed, for use in the MS-based quantification of proteins by the bottom-up approach. Progastrin releasing peptide (ProGRP), a low abundant and clinically sensitive biomarker for small cell lung cancer (SCLC), was used to exemplify the mSPE platform. Four different mMIPs were synthesized, and an mSPE method was developed and optimized for the extraction of low concentrations of tryptic peptides from human serum. The mSPE method enabled the selective extraction of the ProGRP signature peptide, the nonapeptide NLLGLIEAK, prior to quantification of the target via LC-MS/MS. Overall, the mSPE method demonstrated its potential as a low cost, rapid, and straightforward sample preparation method, with demonstrably strong binding, acceptable recoveries, and good compatibility with MS. mMIPs are a potential low-cost alternative to current clinical methods for biomarker analysis.

Paper-based immunocapture for targeted protein analysis.

A novel sampling concept for mass spectrometric bottom-up targeted protein analysis is here demonstrated with polymeric sampling spots integrated with instant immunocapture for analysis of dried matrix spots. The polymers 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) and pHEMA-Tosyl for covalent attachment of antibodies where investigated alongside with adsorption on non-treated filter paper. From performance characterization, the pHEMA-VDM had the best performance. The sampling spots demonstrated fast and easy sampling and preparation of human serum spiked with the biomarker human chorionic gonadotropin. The sampling spots enabled a detection limit of 1 ng/mL (26.4 pM) within a five point concentration curve from 1 ng/mL to 20 ng/mL (R2 = 0.97). The detection limit was demonstrated to be two times lower than previously demonstrated with standard DMPK-C sampling cards. A five point concentration curve from 100 ng/mL to 2000 ng/mL was also investigated (R2 = 0.998). Intra day precision was within 16% and 23% for concentration range 1 ng/mL to 20 ng/mL and 100 ng/mL to 2000 ng/mL, respectively. Inter day precision was within 20%. Accuracy was determined to 10% and 11% for 2.5 ng/mL and 20 ng/mL, respectively. The sampling spots were also demonstrated in a realistic setting where serum samples from two confirmed patients with testicular germ cell cancer were analyzed. These analyses confirmed an elevated hCG content in the sera of 418.5 ±â€¯4.2 ng/mL and 21 ±â€¯0.02 ng/mL hCG for patient one and two respectively.