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In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 μg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out. • An optimized comparison of cell and tissue sample preparation protocols for proteomics is provided. • FASP surpassed in-solution and in-gel in terms of identified peptides and proteins. • Detergents-based buffer (RIPA) led to more efficient protein extraction from tissue. • Choice of workflow depends on sample nature, lysis buffer, and equipment/resources. • Knowledge of intra and inter-day variances is key for reproducible results. [ABSTRACT FROM AUTHOR]

Pharmacological effect of a drug depends on its free fraction in plasma. By certain drugs, like immunosupresive mycophenolic acid, concentrations below therapeutic lead to rejection of the transplanted organ while concentrations above therapeutic lead to toxic effects, primarily on kidney and liver. For this reason constant monitoring of mycophenolic acid in plasma is necessary. Concerning frequency of sampling, availability and non-invasive way of sampling, saliva presents one of the best biological materials for analysis. On the other hand, numerous interfering endogen substances, impurities and high viscosity of saliva make this biological material difficult for purification. Mycophenolic acid has to be purified and concentrated from saliva to be consequently determined applying appropriate analytical method. In this paper solid phase extraction method for purification of saliva has been presented. The efficacy of solid phase extraction has been influenced by many different experimental parameters, so these parameters has to be strongly controlled, and protocol of extraction procedure has been proposed. In this way conditioning of polymeric sorbent has been done with methanol and purified water, samples have been applied together with 0.1 % formic acid, washing has been done with purified water and elution of mycophenolic acid has been performed with acetonitrile and 0.2% formic acid.

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C 60 + cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites. Subject category : Mass spectrometry. [ABSTRACT FROM AUTHOR]

Understanding ageing mechanisms of Li-ion batteries is essential for further optimizations. To determine performance loss causes, post-mortem analyses are commonly applied. For each type of post-mortem test, different sample preparation protocols are adopted. However, reports on the reliability of these protocols are rare. Herein, Li-ion pouch cells with LiNi 1/3 Mn 1/3 Co 1/3 O 2 – polyvinylidene fluoride positive electrode, graphite–carboxymethyl cellulose–styrene rubber negative electrode and LiPF 6 – carbonate solvents mixture electrolyte, are opened and electrodes are recovered following a specified protocol. Negative and positive symmetric cells are assembled and their impedances are recorded. A signal analysis is applied to reconstruct the Li-ion pouch cell impedance from the symmetric cells, then comparison against the pouch cell true impedance allows the evaluation of the sample preparation protocols . The results are endorsed by Transmission Electronic Microscopy (TEM) and Gas Chromatography – Mass Spectrometry (GC-MS) analyses. Carbonate solvents used to remove the salt impacts slightly the surface properties of both electrodes. Drying electrodes under vacuum at 25 °C produces an impedance increase, particularly very marked for the positive electrode. Drying at 50 °C under vacuum or/and exposition to the anhydrous room atmosphere is very detrimental. [ABSTRACT FROM AUTHOR]