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.

Robust biomarker quantification is essential for the accurate diagnosis of diseases and is of great value in cancer management. In this paper, an innovative diagnostic platform is presented which provides automated molecularly imprinted solid-phase extraction (MISPE) followed by liquid chromatography-mass spectrometry (LC-MS) for biomarker determination using ProGastrin Releasing Peptide (ProGRP), a highly sensitive biomarker for Small Cell Lung Cancer, as a model. Molecularly imprinted polymer microspheres were synthesized by precipitation polymerization and analytical optimization of the most promising material led to the development of an automated quantification method for ProGRP. The method enabled analysis of patient serum samples with elevated ProGRP levels. Particularly low sample volumes were permitted using the automated extraction within a method which was time-efficient, thereby demonstrating the potential of such a strategy in a clinical setting.

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Abstract:Garlic is one of the most widespread and ancient medicinal plants. Its health benefits are due to its chemical components, and among these is carbohydrate, whose characteristics have been so far little investigated. The aim of this study is to typify the various components of carbohydrate (starch, individual sugars, fructans, and total dietary fibre) in four commonly consumed "Italian local landraces": Bianco Piacentino, Rosso di Castelliri, Rosso di Sulmona, Rosso di Proceno, which are grown in two different geographical areas-Viterbo and Alvito-under the same agronomic conditions. This study will also evaluate how genotype and the cultivation area can affect the profile of the carbohydrate components of these landrace strains. Regarding unavailable carbohydrates, all of the varieties showed appreciable contents of fructans, the most representative component, which ranged from 45.8 to 54.4 g/100 g d.w. In contrast, total dietary fibre values varied from 9.1 to 13.1 g/100 g d.w. in Rosso di Castelliri and Bianco Piacentino, respectively, which are both grown in Viterbo. As for starch, only some traces were found, while the amount of total sugars ranged between 2.12 and 3.27 g/100 g d.w., with higher levels of sucrose. Our findings could provide important information that may be adopted to enhance and promote the quality of some local Italian garlic landraces through highlighting the influence that the cultivar and the environmental conditions can have on carbohydrates components.

Affinity-based solid-phase extraction (SPE) is an attractive low-cost sample preparation strategy for biomarker analysis. Molecularly imprinted polymers (MIPs) as affinity sorbents offer unique opportunities for affinity SPE, due to their low manufacturing cost and high robustness. A limitation is the prediction of their affinity; therefore, screening of analyte recovery and specificity within a large range of SPE conditions is important in order to ensure high-sensitivity detection and assay reproducibility. Here, a µ-SPE method for screening of the MIP-SPE materials using a commercial 384-well filter plate is presented. The method allows for rapid and automated screening using 10-30 µL of packed SPE sorbent per well and sample volumes in the range of 10-70 µL. This enables screening of many different SPE sorbents while simultaneously identifying optimal SPE conditions. In addition, the 384-well format also facilitates detection with a multitude of analytical platforms. Performance of the µ-MIP-SPE method was investigated using a series of MIPs designed to capture pro-gastrin-releasing peptide (ProGRP). Fractions coming from sample load, cartridge wash, and elution were collected and analyzed using mass spectrometry (MS). The top-performing MIPs were identified, together with proper SPE conditions.