Optimization of reversed-phase solid-phase extraction for shotgun proteomics analysis

Reversed-phase solid-phase extraction (SPE) is the method of choice for the purification of proteomics samples. Even though the efficacy of SPE methods is sample type-dependent, the manufacturers' protocols are used in most studies. Using an optimized SPE method can lead to a substantial gain in identification and recovery. In this tutorial, we give a brief introduction to the most important parameters influencing SPE performance, and we present a short workflow (16 measurements) for optimizing the SPE procedure. This is complemented by method performance assessment instructions and a short troubleshooting guide to help users further understand and investigate their SPE methods

The adsorption of CO on potassium doped molybdenum carbide surface: An ab-initio study

We have studied the effect of K on the adsorption of CO on the β-Mo 2C (0 0 1) surface with a periodic supercell method using Density Functional Theory calculations, with the PBE functional the generalized gradient approximation. The most favorable sites for CO adsorption are three-fold carbon deficient Mo sites on both clean and K promoted surface. Adsorption is more favorable in the presence of K. The electronic configuration analysis shows that in all cases the CO molecule withdraws charge from the surface, the larger charge transfer occurs on the K promoted surface.Fil: Pistonesi, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; ArgentinaFil: Pronsato, Maria Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; ArgentinaFil: Bugyi, László. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; Argentina. Hungarian Academy of Sciences; HungríaFil: Juan, Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; Argentin

Comparison of solid-phase extraction methods for efficient purification of phosphopeptides with low sample amounts

Efficient phosphoproteomic analysis of small amounts of biological samples (e.g. tissue biopsies) requires carefully selected enrichment and purification steps prior to the nanoflow HPLC-MS/MS analysis. Solid-phase extraction (SPE) is one of the most commonly used approaches for sample preparation. Several stationary phases are available for peptide SPE purification, however, most of the published methods are not optimized to provide good recoveries of phosphorylated peptides. Our goal was to investigate the performance of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips, thus enhancing the efficiency of the phosphoproteomic analysis of small amounts of complex protein mixtures. Eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) stationary phase were evaluated. Two RP, one graphite, and the HLB self-packed centrifugal SPE tips provided excellent results for the purification of 1 µg tissue and cell line digests. Using these methods, the sample loss was significantly reduced compared to one of the commercial SPE methods, 22-58% more unique phosphopeptides were identified, and the recovery was higher by 132-155%