Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.

Evaluation of Sample Preparation Methods for Non-Target Screening of Organic Micropollutants in Urban Waters Using High-Resolution Mass Spectrometry

Non-target screening (NTS) has gained interest in recent years for environmental monitoring purposes because it enables the analysis of a large number of pollutants without predefined lists of molecules. However, sample preparation methods are diverse, and few have been systematically compared in terms of the amount and relevance of the information obtained by subsequent NTS analysis. The goal of this work was to compare a large number of sample extraction methods for the unknown screening of urban waters. Various phases were tested for the solid-phase extraction of micropollutants from these waters. The evaluation of the different phases was assessed by statistical analysis based on the number of detected molecules, their range, and physicochemical properties (molecular weight, standard recoveries, polarity, and optical properties). Though each cartridge provided its own advantages, a multilayer cartridge combining several phases gathered more information in one single extraction by benefiting from the specificity of each one of its layers.

Comparison of Sample Preparation Methods for Shotgun Proteomic Studies in Aquaculture Species

Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC–MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies; however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).

Ecofriendly and low-cost sample preparation methods for magnesium determination in beer

Ultrasound-assisted extraction and direct analysis were compared with total digestion for magnesium determination in beer samples by flame atomic absorption spectrometry. The method for total digestion used concentrated nitric acid under plate heating. In optimized instrumental conditions, validation of the analytical method was promoted, with good linear range (0.06 to 0.5 mg L�1), low limits of detection and quantification (0.04 and 0.12 �g g�1, respectively), good precision, relative standard deviation (RSD) less than 3.4%, and accuracy (recovery levels of 91.5 to 99.0%). The characteristic concentration (C0) was 9 �g L�1. The extraction procedure was performed in a 1:1 nitric acid solution for 55 min in an ultrasonic bath at 60 �C, while the direct analysis involved a dilution of the samples in a 2% v/v nitric acid solution. The different sample preparation methods were applied to 13 beer samples and at a 95% confidence level, no significant differences were observed. Thus, direct analysis proved to be more suitable for quality control routines of beer samples in the industry.