Using collective expert judgements to evaluate quality measures of mass spectrometry images.

MOTIVATION: Imaging mass spectrometry (IMS) is a maturating technique of molecular imaging. Confidence in the reproducible quality of IMS data is essential for its integration into routine use. However, the predominant method for assessing quality is visual examination, a time consuming, unstandardized and non-scalable approach. So far, the problem of assessing the quality has only been marginally addressed and existing measures do not account for the spatial information of IMS data. Importantly, no approach exists for unbiased evaluation of potential quality measures. RESULTS: We propose a novel approach for evaluating potential measures by creating a gold-standard set using collective expert judgements upon which we evaluated image-based measures. To produce a gold standard, we engaged 80 IMS experts, each to rate the relative quality between 52 pairs of ion images from MALDI-TOF IMS datasets of rat brain coronal sections. Experts' optional feedback on their expertise, the task and the survey showed that (i) they had diverse backgrounds and sufficient expertise, (ii) the task was properly understood, and (iii) the survey was comprehensible. A moderate inter-rater agreement was achieved with Krippendorff's alpha of 0.5. A gold-standard set of 634 pairs of images with accompanying ratings was constructed and showed a high agreement of 0.85. Eight families of potential measures with a range of parameters and statistical descriptors, giving 143 in total, were evaluated. Both signal-to-noise and spatial chaos-based measures performed highly with a correlation of 0.7 to 0.9 with the gold standard ratings. Moreover, we showed that a composite measure with the linear coefficients (trained on the gold standard with regularized least squares optimization and lasso) showed a strong linear correlation of 0.94 and an accuracy of 0.98 in predicting which image in a pair was of higher quality. AVAILABILITY AND IMPLEMENTATION: The anonymized data collected from the survey and the Matlab source code for data processing can be found at: https://github.com/alexandrovteam/IMS_quality.

Uncovering effects of ex vivo protease activity during proteomics and peptidomics sample extraction in rat brain tissue by oxygen-18 labeling.

In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations.

Impact of temperature dependent sampling procedures in proteomics and peptidomics--a characterization of the liver and pancreas post mortem degradome.

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision.

The effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and prevent proteome degradation has been evaluated. Mouse brains were bisected immediately following excision, with one hemisphere being heat treated followed by snap freezing in liquid nitrogen while the other hemisphere was snap frozen immediately. Sections were cut by cryostatic microtome and analyzed by MALDI-MS imaging and minimal label 2-D DIGE, to monitor time-dependent relative changes in intensities of protein and peptide signals. Analysis by MALDI-MS imaging demonstrated that the relative intensities of markers varied across a time course (0-5 min) when the tissues were not stabilized by heat treatment. However, the same markers were seen to be stabilized when the tissues were heat treated before snap freezing. Intensity profiles for proteins indicative of both degradation and stabilization were generated when samples of treated and nontreated tissues were analyzed by 2-D DIGE, with protein extracted before and after a 10-min warming of samples. Thus, heat treatment of tissues at the time of excision is shown to prevent subsequent uncontrolled degradation of tissues at the proteomic level before any quantitative analysis, and to be compatible with downstream proteomic analysis.

Assessing the use of thermal treatment to preserve the intact proteomes of post-mortem heart and brain tissue.

Protein degradation that occurs in tissue during post-mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap-freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2-DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high-molecular-weight proteins and reduction in protein fragmentation.

Preserving the yeast proteome from sample degradation.

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95 degrees C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.

Sample handling of gastric tissue and O-glycan alterations in paired gastric cancer and non-tumorigenic tissues.

Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N,N'-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.

Preservation of Specific Protein Signaling States Using Heat Based Stabilizor System.

The ability to adequately measure the phosphorylation state of a protein has major biological as well as clinical relevance. Due to its variable nature, reversible protein phosphorylations are sensitive to changes in the tissue environment. StabilizorTM T1 is a system for rapid inactivation of enzymatic activity in biological samples. Enzyme inactivation is accomplished using thermal denaturation in a rapid, homogeneous, and reproducible fashion without the need of added chemical inhibitors. Using pCREB(Ser133) as a model system, the applicability of the Stabilizor system to preserve a rapidly lost phosphorylation is shown.

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.

Sample preservation through heat stabilization of proteins: principles and examples.

Due to post-sampling changes, caused by residual enzyme activity in the sample, levels of analytes can change from their in vivo levels so that they no longer accurately reflect conditions in the living system. The Stabilizor(™) system accomplishes elimination of enzyme activity through heat-induced denaturation of enzymes by permanently altering the 3D protein structure of the enzymes. Heat stabilization can be introduced in the workflow either directly after sampling, with the instrument just next to where the sample is taken, or prior to sample homogenization and extraction, when samples are heat denatured directly from a frozen state. Initially, heat stabilization was developed to enable mass spectrometric analysis of neuropeptides. Heat stabilization has since been further developed and applied to a range of samples and downstream protein analysis techniques such as western blot, 2D gels and phosphorylation analysis with LC-MS.

The impact of pre-analytical conditions on the serum proteome: heat-stabilization versus nitrogen storage.

CONTEXT: Biological material reflecting the in vivo composition of markers provides a high potential for biomarker discovery. OBJECTIVE: We compared the serum proteome following heat- and nitrogen-preservation, with and without subsequent storage at room temperature. MATERIALS AND METHODS: Serum samples were collected, treated and analysed by two-dimensional gel electrophoresis. Protein spots were identified and confirmed by two mass spectrometry approaches (MALDI & ESI) and subjected to Ingenuity Pathway Analysis. RESULTS: We revealed 24 differentially expressed proteins (p ≤ 0.05) between nitrogen and heat preservation, and 87 between nitrogen and heat preservation with subsequent storage for 120 h at room-temperature. Mass spectrometry identified 25 polypeptides. Pathway analysis resulted in networks maintaining Cellular Assembly and Organization, Movement and Maintenance. CONCLUSION: Heat-stabilization does not substantially change the short-term proteome composition of serum compared with nitrogen treatment. However, heat-stabilization alone seems insufficient for long-term sample preservation for serum samples. We identified transthyretin and apolipoprotein A-IV as sample quality markers.

Methodology and technology for stabilization of specific states of signal transduction proteins.

The ability to adequately measure the phosphorylation state of a protein has major biological as well as clinical relevance. Due to its variable nature, reversible protein phosphorylations are sensitive to changes in the tissue environment. Stabilizor T1 is a system for rapid inactivation of enzymatic activity in biological samples. Enzyme inactivation is accomplished using thermal denaturation in a rapid, homogeneous, and reproducible fashion without the need for added inhibitors. Using pCREB(Ser133) as a model system, the applicability of the Stabilizor system to preserve a rapidly lost phosphorylation is shown.

Biomarkers of disease and post-mortem changes - Heat stabilization, a necessary tool for measurement of protein regulation.

This review focuses on post sampling changes and how the Stabilizor system has been used to control this natural biological process and potential implications on cancer-specific biomarkers due to post sampling changes. Tissue sampling is a major traumatic event that can have drastic effects within a very short timeframe at the molecular level [1] resulting in loss of sample quality due to post-mortem changes. A heat-stabilization technology, using the Stabilizor system, has been developed to quickly and permanently abolish the enzymatic activity that causes these changes post-sampling and so preserve sample quality. The Stabilizor system has been shown to give better sample quality when analyzing a variety of tissues in various proteomic workflows. In this paper we discuss the impact of using heat-stabilized tissue in different proteomic applications. Based on our observations regarding the overlap between commonly changing proteins and proteins found to change post-mortem we also highlight a group of proteins of particular interest in cancer studies.

Heat stabilization of the tissue proteome: a new technology for improved proteomics.

After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

Sugar-mediated semidian oscillation of gene expression in the cassava storage root regulates starch synthesis.

Starch branching enzyme (SBE) activity in the cassava storage root exhibited a diurnal fluctuation, dictated by a transcriptional oscillation of the corresponding SBE genes. The peak of SBE activity coincided with the onset of sucrose accumulation in the storage, and we conclude that the oscillatory mechanism keeps the starch synthetic apparatus in the storage root sink in tune with the flux of sucrose from the photosynthetic source. When storage roots were uncoupled from the source, SBE expression could be effectively induced by exogenous sucrose. Turanose, a sucrose isomer that cannot be metabolized by plants, mimicked the effect of sucrose, demonstrating that downstream metabolism of sucrose was not necessary for signal transmission. Also glucose and glucose-1-P induced SBE expression. Interestingly, induction by sucrose, turanose and glucose but not glucose-1-P sustained an overt semidian (12-h) oscillation in SBE expression and was sensitive to the hexokinase (HXK) inhibitor glucosamine. These results suggest a pivotal regulatory role for HXK during starch synthesis. Abscisic acid (ABA) was another potent inducer of SBE expression. Induction by ABA was similar to that of glucose-1-P in that it bypassed the semidian oscillator. Both the sugar and ABA signaling cascades were disrupted by okadaic acid, a protein phosphatase inhibitor. Based on these findings, we propose a model for sugar signaling in regulation of starch synthesis in the cassava storage root.

Developmental regulation of a VEIDase caspase-like proteolytic activity in barley caryopsis.

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.

A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter.

SURE (sugar responsive) is a cis element in plant sugar signaling. The SURE element was reported first for potato, in which it confers sugar responsiveness to the patatin promoter. A SURE binding transcription factor has not been isolated. We have isolated a transcription factor cDNA from barley and purified the corresponding protein. The transcription factor, SUSIBA2 (sugar signaling in barley), belongs to the WRKY proteins and was shown to bind to SURE and W-box elements but not to the SP8a element in the iso1 promoter. Nuclear localization of SUSIBA2 was demonstrated in a transient assay system with a SUSIBA2:green fluorescent protein fusion protein. Exploiting the novel transcription factor oligodeoxynucleotide decoy strategy with transformed barley endosperm provided experimental evidence for the importance of the SURE elements in iso1 transcription. Antibodies against SUSIBA2 were produced, and the expression pattern for susiba2 was determined at the RNA and protein levels. It was found that susiba2 is expressed in endosperm but not in leaves. Transcription of susiba2 is sugar inducible, and ectopic susiba2 expression was obtained in sugar-treated leaves. Likewise, binding to SURE elements was observed for nuclear extracts from sugar-treated but not from control barley leaves. The temporal expression of susiba2 in barley endosperm followed that of iso1 and endogenous sucrose levels, with a peak at approximately 12 days after pollination. Our data indicate that SUSIBA2 binds to the SURE elements in the barley iso1 promoter as an activator. Furthermore, they show that SUSIBA2 is a regulatory transcription factor in starch synthesis and demonstrate the involvement of a WRKY protein in carbohydrate anabolism. Orthologs to SUSIBA2 were isolated from rice and wheat endosperm.