Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC–MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC–MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

Direct Magnetic Bead-Based Extraction of MicroRNA from Urine with Capillary Electrophoretic Analysis Using Fluorescence Detection and Universal Label

Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.

Ti-containing mesoporous silica packed microcolumn separation/preconcentration combined with inductively coupled plasma-mass spectrometry for the determination of trace Cr, Cu, Cd and Pb in environmental samples

Solid phase extraction with Ti-containing mesoporous silica as adsorbent combined with ICP-MS for trace metal analysis in environmental samples.