Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between ...single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP), and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg range using varying amounts of HeLa cell lysate (1–20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25 000 and 30 000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R 2 = 0.97 (SP3); R 2 = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-associated macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a “mixed” M1/M2 phenotype.
Filter Aided Sample Preparation (FASP) is a widely used protein processing technique in “bottom-up” proteomics. Its popularity reflects the key features of the method: its applicability to a variety ...of sample types and the high quality of the released peptides. Successful application of FASP requires optimized properties of sample lysate and its amount, use of ultrafiltration units with membranes having large molecular mass cut-offs and well selected conditions for protein digestion. In contrast to the majority of sample preparation methods, FASP allows digestion of proteins with a variety of enzymes and a straightforward monitoring of protein-to-peptide conversion. A unique feature of FASP is the possibility to cleave proteins in a consecutive way using several proteases and to separate peptide fractions. Understanding principles of the method gives guidance in applying FASP to different types of samples in optimization of conditions of the FASP-workflow.
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•Filter Aided Sample Preparation method allows efficient sample preparation for “bottom up” LC-MS proteomic analysis.•Molecular foundations of the method are described.•Technological constrains of FASP are discussed.•This is a guide to the FASP method providing hints for optimization of sample preparation workflows.
This work proposes for the first time, a metric tool that gives prominence to sample preparation. The developed metric (termed AGREEprep) was based on 10 categories of impact that were recalculated ...to 0–1 scale sub-scores, and then used to calculate the final assessment score. The criteria of assessment evaluated, among others, the choice and use of solvents, materials and reagents, waste generation, energy consumption, sample size, and throughput. Assessment was also based on the possibility to differentiate between criteria importance by assigning them weights. The assessment procedure was performed using an open access, intuitive software that produced an easy-to-read pictogram with information on the total performance and structure of threats. A compiled version of the open access software can be obtained from mostwiedzy.pl/AGREEprep. The applicability of AGREEprep was successfully demonstrated using six different methods as case studies.
•Sample preparation analytical greenness metric is developed.•Assessment is based on ten criteria that refer to different greenness aspects.•Assessment is performed with user friendly, intuitive, free to download software.•The result reflects sample prep performance in criteria and criteria weights.
Quantitative protein extraction and high-yield generation of peptides from biological samples are the prerequisite for successful bottom-up type proteomic analysis. Filter aided sample preparation ...(FASP) is a method for processing of SDS-solubilized cells in a proteomic reactor format. In FASP, disposable centrifugal ultrafiltration units allow for detergent depletion, protein digestion, and isolation of peptides released by proteases from undigested material. Consecutive protein digestion with two or three proteases enables generation of peptide fractions with minimal overlap and considerably increases the number of identifications and protein sequence coverage. FASP is useful for analysis of samples varying in size from a few micrograms to several milligrams of total protein.
The determination of contaminants of emerging concern (CECs) in environmental samples has become a challenging and critical issue. The present work focuses on miniaturized analytical strategies ...reported in the literature for the determination of CECs. The first part of the review provides brief overview of CECs whose monitoring in environmental samples is of particular significance, namely personal care products, pharmaceuticals, endocrine disruptors, UV-filters, newly registered pesticides, illicit drugs, disinfection by-products, surfactants, high technology rare earth elements, and engineered nanomaterials. Besides, an overview of downsized sample preparation approaches reported in the literature for the determination of CECs in environmental samples is provided. Particularly, analytical methodologies involving microextraction approaches used for the enrichment of CECs are discussed. Both solid phase- and liquid phase-based microextraction techniques are highlighted devoting special attention to recently reported approaches. Special emphasis is placed on newly developed materials used for extraction purposes in microextraction techniques. In addition, recent contributions involving miniaturized analytical flow techniques for the determination of CECs are discussed. Besides, the strengths, weaknesses, opportunities and threats of point of need and portable devices have been identified and critically compared with chromatographic methods coupled to mass chromatography. Finally, challenging aspects regarding miniaturized analytical methods for determination of CECs are critically discussed.
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•Microextraction, millifluidic and microfluidic approaches used for CECs determination are reviewed.•The occurrence and fate of CECs in the environment is described.•Challenging aspects of miniaturized analytical approaches are identified and discussed.•Relevant applications to the determination of CECs in environmental samples are discussed.
The ten principles of green sample preparation López-Lorente, Ángela I.; Pena-Pereira, Francisco; Pedersen-Bjergaard, Stig ...
TrAC, Trends in analytical chemistry (Regular ed.),
03/2022, Letnik:
148
Journal Article
Recenzirano
Odprti dostop
The ten principles of GSP are presented with the aim of establishing a road map toward the development of overall greener analytical methodologies. Paramount aspects for greening sample preparation ...and their interconnections are identified and discussed. These include the use of safe solvents/reagents and materials that are renewable, recycled and reusable, minimizing waste generation and energy demand, and enabling high sample throughput, miniaturization, procedure simplification/automation, and operator's safety. Further, the importance of applying green metrics for assessing the greenness of sample preparation methods is highlighted, next to the contribution of GSP in achieving the broader goal of sustainability. Green sample preparation is sample preparation. It is not a new subdiscipline of sample preparation but a guiding principle that promotes sustainable development through the adoption of environmentally benign sample preparation procedures.
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•The ten principles of green sample preparation are presented.•Sustainability issues on solvents, reagents and materials are considered.•Fast, miniaturized, automated, in situ and low-energy methods are preferred.•Post-sample preparation configuration for analysis is considered.•Green metrics and the impact on sustainable development are discussed.
Sample preparation is a key step in the analytical procedure and a critical component for achieving analytical greenness. AGREEprep is the first metric intended for evaluating the environmental ...impact of sample preparation methods. The approach consists of ten steps of assessment that correspond to the ten principles of green sample preparation and uses a user-friendly open-source software to calculate and visualize the results. Despite the simplicity of the approach, some assessment steps can be difficult to evaluate in a straightforward manner, either because essential data are not readily available or, in some cases, are poorly defined.
This walkthrough tutorial on AGREEprep serves as an in depth yet simple guide for new users that elucidates all aspects of the greenness assessment. It will prove beneficial to analysts wishing to assess the greenness of their own developed procedures or those found in the literature, which can be challenging taken that critical data is often not reported. Particular attention is given to the calculations involved in estimating the amount of waste generated and energetic requirements. The impact of weight values of criteria on the overall final score are discussed and using two hypothetical scenarios is related to the analytical goals of operators. Finally, several assessment examples are given in the supplementary information to familiarize assessors with all assessment steps.
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The recent discovery of alternative open reading frames creates a need for suitable analytical approaches to verify their translation and to characterize the corresponding gene products at the ...molecular level. As the analysis of small proteins within a background proteome by means of classical bottom‐up proteomics is challenging, method development for the analysis of small open reading frame encoded peptides (SEPs) have become a focal point for research. Here, we highlight bottom‐up and top‐down proteomics approaches established for the analysis of SEPs in both pro‐ and eukaryotes. Major steps of analysis, including sample preparation and (small) proteome isolation, separation and mass spectrometry, data interpretation and quality control, quantification, the analysis of post‐translational modifications, and exploration of functional aspects of the SEPs by means of proteomics technologies are described. These methods do not exclusively cover the analytics of SEPs but simultaneously include the low molecular weight proteome, and moreover, can also be used for the proteome‐wide analysis of proteolytic processing events.
In spite of the huge development of analytical instrumentation during last two decades, sample preparation is still nowadays considered the bottleneck of the whole analytical process. In this regard, ...efforts have been conducted towards the improvement of the selectivity during extraction and/or subsequent clean-up of sample extracts. Molecularly imprinted polymers (MIPs) are stable polymers with molecular recognition abilities, provided by the presence of a template during their synthesis and thus are excellent materials to provide selectivity to sample preparation. In the present review, the use of MIPs in solid-phase extraction and solid-phase microextraction as well as its recent incorporation to other extraction techniques such as matrix-solid phase dispersion and stir bar sorptive extraction, among others, is described. The advantages and drawbacks of each methodology as well as the future expected trends are discussed.
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