Funnel-freezing versus heat-stabilization for the visualization of metabolites by mass spectrometry imaging in a mouse stroke model.

Tissue preparation is the key to a successful matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) experiment. Rapid post-mortem changes contribute a significant challenge to the use of MSI approaches for the analysis of peptides and metabolites. In this technical note we aimed to compare the tissue fixation method ex-vivo heat-stabilization with in-situ funnel-freezing in a middle cerebral artery occlusion (MCAo) mouse model of stroke, which causes profound alterations in metabolite concentrations. The influence of the duration of the thaw-mounting of the tissue sections on metabolite stability was also determined. We demonstrate improved stability and biomolecule visualization when funnel-freezing was used to sacrifice the mouse compared with heat-stabilization. Results were further improved when funnel-freezing was combined with fast thaw-mounting of the brain sections.

Rapid Inactivation of Non-Endospore-Forming Bacterial Pathogens by Heat Stabilization is Compatible with Downstream Next-Generation Sequencing.

Introduction: Heat stabilization treatment preserves the in vivo state of biological samples by rapidly inactivating enzymes that cause degradation of proteins and nucleic acids. Historically, proteomics studies used this technique as an alternative to chemical fixation. More recently, microbiologists discovered that heat stabilization treatment rapidly inactivates pathogens present in tissue samples and preserves deoxyribonucleic acid (DNA) in the tissue. However, these recent studies did not investigate the inactivation of high-density bacterial suspensions and the quality of bacterial DNA. Methods and Results: High-density suspensions of Escherichia coli (>109 cfu/mL) were completely inactivated by heat stabilization treatment using the Denator Stabilizor T1 instrument at 72°C and 95°C for 45 seconds. Using the heat stabilization instrument, a panel of 30 species, 20 Gram-negative and 10 non-endospore-forming Gram-positive species, were fully inactivated by treatment (95°C for 45 seconds). DNA was isolated from bacterial suspensions of Gram-negative bacteria, including E. albertii, E. coli, Shigella dysenteriae, and S. flexneri, following inactivation via heat stabilization treatment and without treatment. DNA isolated following heat stabilization treatment was fully compatible with all downstream molecular applications tested, including next-generation sequencing, pulsed-field gel electrophoresis, multiplex polymerase chain reaction (PCR), and real-time PCR. Conclusions and Discussion: Heat stabilization treatment of Gram-negative and non-endospore-forming Gram-positive pathogens completely inactivates high-density bacterial suspensions. This treatment is compatible with downstream DNA molecular assays, including next-generation sequencing, pulsed-field gel electrophoresis, and PCR. Inactivation by heat stabilization is a rapid process that may increase safety by decreasing risks for laboratory-associated infections and risks associated with transportation of infectious materials.

Snap-heated freeze-free preservation and processing of the urine proteome using the combination of stabilizor-based technology and filter aided sample preparation.

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples.

qPCR based mRNA quality score show intact mRNA after heat stabilization.

Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

Effects of heat-treatment on the stability and composition of metabolomic extracts from the earthworm Eisenia fetida.

Environmental metabolomics studies employing earthworms as sentinels for soil contamination are numerous, but the instability of the metabolite extracts from these organisms has been minimally addressed. This study evaluated the efficacy of adding a heat-treatment step in two commonly used extraction protocols (Bligh and Dyer and D2O phosphate buffer) as a pre-analytical stabilization method. The resulting metabolic profiles of Eisenia fetida were assessed using principal component analysis and NMR spectral evaluations. The heated Bligh and Dyer extractions produced stabilized profiles with minimal variation of the extracted metabolomic profiles over time, providing a more suitable method for metabolomic analysis of earthworm extracts.

Impact of Wheat Bran Hydration Properties As Affected by Toasting and Degree of Milling on Optimal Dough Development in Bread Making.

The impact of the hydration capacity and hydration rate of wheat bran on optimal bread dough development and loaf volume was investigated using coarse bran, both native as well as after toasting, milling, presoaking, and combinations of the latter. It was found that toasting reduces bran's hydration rate, which, during mixing, results in a temporary excess of water in which dough development takes place inefficiently and hence requires additional time. This mechanism was further substantiated by the observation that delayed dough development can be counteracted by the presoaking of bran. Milling of bran increases its hydration rate and results in faster optimal dough development. Presoaking of nonmilled bran, however, did not result in faster dough development. Smaller bran particles do lead to faster dough development, probably due to increased proper contacts between flour particles. Optimal loaf volumes did not change upon milling and toasting.

MALDI Imaging Mass Spectrometry-A Mini Review of Methods and Recent Developments.

As the only imaging method available, Imaging Mass Spectrometry (IMS) can determine both the identity and the distribution of hundreds of molecules on tissue sections, all in one single run. IMS is becoming an established research technology, and due to recent technical and methodological improvements the interest in this technology is increasing steadily and within a wide range of scientific fields. Of the different IMS methods available, matrix-assisted laser desorption/ionization (MALDI) IMS is the most commonly employed. The course at IMSC 2012 in Kyoto covered the fundamental principles and techniques of MALDI-IMS, assuming no previous experience in IMS. This mini review summarizes the content of the one-day course and describes some of the most recent work performed within this research field.