Sample Preparation Artifacts Research Articles

Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic mesophilic bacterial isolates in each sample. A high biodiversity of 70 and 110 species per sample was determined, of which 25–28% corresponded to yet unknown taxa. The isolates were dominated by Gram-positive members of the genera Staphylococcus, Corynebacterium, Streptococcus, or Janibacter, whilst Chryseobacterium and Acinetobacter were most abundant among the Gram-negative taxa. At the second time point, samples of the same farms were analyzed via both cultivation (1,500 individual colonies each) and high-throughput 16S rRNA gene amplicon sequencing. The latter revealed a threefold higher biodiversity at the genus level, as anaerobic or fastidious species were also detected. However, cultivation identified genera not captured by sequencing, indicating that both approaches are complementary. Using amplicon sequencing, the relative abundance of a few genera was distorted, which seems to be an artifact of sample preparation. Therefore, attention needs to be paid to the library preparation procedure with special emphasis on cell lysis and PCR.

STEM electron beam-induced current measurements of organic-inorganic perovskite solar cells

We describe a new approach for preparing organic-inorganic perovskite solar cells for electron beam-induced current (EBIC) measurements in plan-view geometry. This method substantially reduces sample preparation artefacts, provides good electrical contact and keeps the preparation steps as close as possible to those for real devices. Our EBIC images were acquired simultaneously with annular dark-field scanning transmission electron microscopy images using a home-made highly sensitive EBIC amplifier. High-angle annular dark-field images and energy dispersive X-ray maps were recorded from the same area immediately afterwards. This allowed the EBIC contrast to be correlated with regions containing N and a deficiency of O. The EBIC contrast was also found to be similar to secondary electron contrast recorded with a scanning electron microscope. By identifying the generation and absorption electron processes, we determine that EBIC cannot be separated from the secondary electron and absorbed currents. This means that careful analysis needs to be performed before conclusions can be made on the origin of the current measured across p-n or p-i-n junctions.

High porosity with tiny pore constrictions and unbending pathways characterize the 3D structure of intervessel pit membranes in angiosperm xylem.

Pit membranes between xylem vessels play a major role in angiosperm water transport. Yet, their three-dimensional (3D) structure as fibrous porous media remains unknown, largely due to technical challenges and sample preparation artefacts. Here, we applied a modelling approach based on thickness measurements of fresh and fully shrunken pit membranes of seven species. Pore constrictions were also investigated visually by perfusing fresh material with colloidal gold particles of known sizes. Based on a shrinkage model, fresh pit membranes showed tiny pore constrictions of ca. 20nm, but a very high porosity (i.e. pore volume fraction) of on average 0.81. Perfusion experiments showed similar pore constrictions in fresh samples, well below 50nm based on transmission electron microscopy. Drying caused a 50% shrinkage of pit membranes, resulting in much smaller pore constrictions. These findings suggest that pit membranes represent a mesoporous medium, with the pore space characterized by multiple constrictions. Constrictions are much smaller than previously assumed, but the pore volume is large and highly interconnected. Pores do not form highly tortuous, bent, or zigzagging pathways. These insights provide a novel view on pit membranes, which is essential to develop a mechanistic, 3D understanding of air-seeding through this porous medium.

MicroX-ray absorption near edge structure tomography reveals cell-specific changes of Zn ligands in leaves of turnip yellow mosaic virus infected plants

As many metals are essential for plants, excess or deficiency of them or alteration of their uptake, translocation, sequestration or physiological use can have severe consequences for plant growth and fitness. Therefore, investigating the distribution and speciation of metals in tissues is essential to understand plant physiology. We present a method based on non-destructive Synchrotron X-ray microtomography combined with microspectroscopy for studying metal distribution and speciation in plant tissues. By using the Maia detector system and the high flux of the undulator beam at the P06 beamline of the PETRA III synchrotron (at DESY), it was possible to record micro X-ray Absorption Near Edge Structure (μXANES) for every voxel of a tomogram. The metal coordination in regions of interest within the tissue samples could be determined by comparing the XANES with spectra of relevant reference compounds. Metal distribution and coordination were measured in shock frozen hydrated plant leaves in a cryostream, avoiding sample preparation artefacts like liquid cell content redistribution that occurs with other preparation methods, unequal distribution of stains in staining assays, sample degradation by beam damage and thawing, etc. A spatial resolution of 5 μm was selected, which is sufficient to resolve all leaf tissues (epidermis, palisade mesophyll, spongy mesophyll, veins), larger cells and biomineralization hotspots.As an application example, we studied the effect of infection with Turnip Yellow Mosaic Virus (TYMV) on the Zn distribution and the Zn speciation in duplicates of Noccaea ochroleucum. This non-accumulator plant grown with 100 μM Zn had enough metal to allow collecting significant spectroscopic data. We found that the TYMV infected samples formed biomineralization crystallites, showing strong spectroscopic similarity to Zn silicate.

The Critical Importance of Rhodoliths in the Life Cycle Completion of Both Macro- and Microalgae, and as Holobionts for the Establishment and Maintenance of Marine Biodiversity

Rhodoliths are the main hard substrata for the attachment of benthic macroalgae in the NW Gulf of Mexico rubble habitats that are associated with salt domes, unique deep bank habitats at ~50-90m depth on the continental shelf offshore Louisiana and Texas. With the advent of additional sequencing technologies, methodologies for biodiversity assessments are now rapidly shifting to DNA metabarcoding, i.e. High Throughput Sequencing (HTS) of environmental DNA mixtures with standardized molecular markers, such as 16S V4, for rapid, cost-effective biodiversity measurement. We newly tested 16S V4 metabarcoding on endolithic portions of mesophtic rhodoliths exhibiting low phototroph colonization that revealed a hidden, cryptic algal diversity targeting spores, propagules, and unsuspected life history stages. We explored cryo-SEM as a potentially more informative method than regular SEM to minimize artefacts of sample preparation in the study of endolithic cell inclusions which brought to light a suite of microalgal stages. We were able to differentiate floridean starch from cellular inclusions. We associated the effect of anatomical growth pattern on presence or absence of cellular inclusions in biogenic rhodoliths. Analyses of combined 16S V4 metabarcodes and 16S Sanger sequences of two red algal orders, the Halymeniales and Bonnemaisoniales, increased the established record of diversity in the region. We view rhodoliths as marine biodiversity hotspots that may function as seedbanks, temporary reservoirs for life history stages of ecologically important eukaryotic microalgae, or as refugia for ecosystem resilience following environmental stress.

Undesired sample preparation artifacts can hamper the immuno-purification of lysine acetylation in proteomic studies.

Undesired sample preparation artifacts can hamper the immuno-purification of lysine acetylation in proteomic studies.

Validation of Remote Digital Frozen Sections for Cancer and Transplant Intraoperative Services

Introduction: Whole-slide imaging (WSI) technology can be used for primary diagnosis and consultation, including intraoperative (IO) frozen section (FS). We aimed to implement and validate a digital system for the FS evaluation of cancer and transplant specimens following recommendations of the College of American Pathologists. Materials and Methods: FS cases were routinely scanned at ×20 employing the “Navigo” scanner system. IO diagnoses using glass versus digital slides after a 3-week washout period were recorded. Intraobserver concordance was evaluated using accuracy rate and kappa statistics. Feasibility of WSI diagnoses was assessed by the way of sensitivity, specificity, as well as positive and negative predictive values. Participants also completed a survey denoting scan time, time spent viewing cases, preference for glass versus WSI, image quality, interface experience, and any problems encountered. Results: Of the 125 cases submitted, 121 (436 slides) were successfully scanned including 93 oncological and 28 donor-organ FS biopsies. Four cases were excluded because of failed digitalization due to scanning problems or sample preparation artifacts. Full agreement between glass and digital-slide diagnosis was obtained in 90 of 93 (97%, κ = 0.96) oncology and in 24 of 28 (86%, κ = 0.91) transplant cases. There were two major and one minor discrepancy for cancer cases (sensitivity 100%, specificity 96%) and two major and two minor disagreements for transplant cases (sensitivity 96%, specificity 75%). Average scan and viewing/reporting time were 12 and 3 min for cancer cases, compared to 18 and 5 min for transplant cases. A high diagnostic comfort level among pathologists emerged from the survey. Conclusions: These data demonstrate that the “Navigo” digital WSI system can reliably support an IO FS service involving complicated cancer and transplant cases.

Visualising the 3D microstructure of stained and native intervertebral discs using X-ray microtomography

Intervertebral disc degeneration (IVDD) is linked to low back pain. Microstructural changes during degeneration have previously been imaged using 2D sectioning techniques and 3D methods which are limited to small specimens and prone to inducing artefacts from sample preparation. This study explores micro computed X-ray tomography (microCT) methods with the aim of resolving IVD 3D microstructure whilst minimising sample preparation artefacts. Low X-ray absorption contrast in non-mineralised tissue can be enhanced using staining and phase contrast techniques. A step-wise approach, including comparing three stains, was used to develop microCT for bovine tail IVD using laboratory and synchrotron sources. Staining successfully contrasted collagenous structures; however not all regions were stained and the procedure induced macroscopic structural changes. Phase contrast microCT of chemically fixed yet unstained samples resolved the nucleus pulposus, annulus fibrosus and constituent lamellae, and finer structures including collagen bundles and cross-bridges. Using the same imaging methods native tissue scans were of slightly lower contrast but free from sample processing artefacts. In the future these methods may be used to characterise structural remodelling in soft (non-calcified) tissues and to conduct in situ studies of native loaded tissues and constructs to characterise their 3D mechanical properties.

Particle arrangements in clay slurries: The case against the honeycomb structure

The properties of clay slurries (porosity ~0.75) impact a wide range of materials such as commercial clay dispersions and sedimentary deposits. Their material behavior, and in particular the gelation of clay slurries, is thought to be governed by clay particle interactions. In the literature, such interactions are rarely directly probed, but rather inferred from structures observed by cryo-electron microscopy. For example, the honeycomb structure is a widely accepted textbook model that is used to rationalize the observed behavior of clay slurries. Using high-pressure freezing, cryo-electron microscopy, and cryo-synchrotron wide-angle X-ray scattering, this study shows that the honeycomb-structure is an artifact of sample preparation. When samples are high-pressure frozen, individual clay particles and aggregates of particles arrange in a random orientation rather than the closed-cell structure dominated by face-face and face-edge contacts observed in plunge frozen samples. These results substantially contribute to the understanding of the gelation mechanism and particle interactions in colloidal clay slurries, and provide valuable input parameters for meso-scale modeling efforts of clay dispersions and sedimentary deposits to upscale their mechanical properties to the macroscale.

Quantification of fenestrations in liver sinusoidal endothelial cells by atomic force microscopy

Liver sinusoidal endothelial cells present unique morphology characterized by the presence of transmembrane pores called fenestrations. The size and number of fenestrations in live cells change dynamically in response to variety of chemical and physical factors. Although scanning electron microscopy is a well-established method for investigation of fixed liver sinusoidal endothelial cells morphology, atomic force microscopy is the interesting alternative providing detailed 3D topographical information. Moreover, simple sample preparation, only by wet-fixation, minimizing sample preparation artifacts enable high-resolution atomic force microscopy-based measurements. In this work, we apply imaging methods based on atomic force microscopy, to describe characteristic features of glutaraldehyde-fixed primary murine liver sinusoidal endothelial cells, namely: mean fenestration diameter, porosity, and fenestrations frequency. We also investigate the effect of different tip apex radius on evaluation of single fenestration diameter.By quantitative description of fenestrations, we demonstrate that atomic force microscopy became a well competing tool for nondestructive quantitative investigation of the liver sinusoidal endothelial cell morphology.

Electron Microscopy of Fission Yeast.

Electron microscopy (EM) can provide images of cells with a spatial resolution that significantly surpasses that available from light microscopy (LM), even with modern methods that give LM "super resolution." However, EM resolution comes with costs in time spent with sample preparation, expense of instrumentation, and concerns regarding sample preparation artifacts. It is therefore important to know the limitations of EM as well as its strengths. Here we describe the most reliable methods for the preservation of fission yeast cells currently available. We describe the properties of images obtained by transmission EM (TEM) and contrast them with images from scanning EM (SEM). We also show how one can make three-dimensional TEM images and discuss several approaches to address the problem of localizing specific proteins within cells. We give references to work by others who have pursued similar goals with different methods, and we discuss briefly the complex subject of image interpretation.

Core size determination and structural characterization of intravenous iron complexes by cryogenic transmission electron microscopy

Understanding physicochemical properties of intravenous (IV) iron drug products is essential to ensure the manufacturing process is consistent and streamlined. The history of physicochemical characterization of IV iron complex formulations stretches over several decades, with disparities in iron core size and particle morphology as the major source of debate. One of the main reasons for this controversy is room temperature sample preparation artifacts, which affect accurate determination of size, shape and agglomeration/aggregation of nanoscale iron particles. The present study is first to report the ultra-fine iron core structures of four IV iron complex formulations, sodium ferric gluconate, iron sucrose, low molecular weight iron dextran and ferumoxytol, using a cryogenic transmission electron microscopy (cryo-TEM) preservation technique, as opposed to the conventional room temperature (RT-TEM) technique. Our results show that room temperature preparation causes nanoparticle aggregation and deformation, while cryo-TEM preserves IV iron colloidal suspension in their native frozen-hydrated and undiluted state. In contrast to the current consensus in literature, all four IV iron colloids exhibit a similar morphology of their iron oxide cores with a spherical shape, narrow size distribution and an average size of 2nm. Moreover, out of the four tested formulations, ferumoxytol exhibits a cluster-like community of several iron carbohydrate particles which likely accounts for its large hydrodynamic size of 25nm, measured with dynamic light scattering. Our findings outline a suitable method for identifying colloidal nanoparticle core size in the native state, which is increasingly important for manufacturing and design control of complex drug formulations, such as IV iron drug products.

The Measurement of Reversible Redox Dependent Post-translational Modifications and Their Regulation of Mitochondrial and Skeletal Muscle Function.

Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar, and excitation-contraction (EC) coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.

Sample preparation artifacts in nuclear materials and mitigation strategies

Sample preparation artifacts in nuclear materials and mitigation strategies

Experimental evaluation of environmental scanning electron microscopes at high chamber pressure.

In environmental scanning electron microscopy (ESEM) high pressure applications have become increasingly important. Wet or biological samples can be investigated without time-consuming sample preparation and potential artefacts from this preparation can be neglected. Unfortunately, the signal-to-noise ratio strongly decreases with increasing chamber pressure. To evaluate the high pressure performance of ESEM and to compare different electron microscopes, information about spatial resolution and detector type is not enough. On the one hand, the scattering of the primary electron beam increases, which vanishes the contrast in images; and on the other hand, the secondary electrons (SE) signal amplification decreases. The stagnation gas thickness (effective distance the beam has to travel through the imaging gas) as well as the SE detection system depend on the microscope and for a complete and serious evaluation of an ESEM or low vacuum SEM it is necessary to specify these two parameters. A method is presented to determine the fraction of scattered and unscattered electrons and to calculate the stagnation gas thickness (θ). To evaluate the high pressure performance of the SE detection system, a method is presented that allows for an analysis of a single image and the calculation of the signal-to-noise ratio of this image. All investigations are performed on an FEI ESEM Quanta 600 (field emission gun) and an FEI ESEM Quanta 200 (thermionic gun). These methods and measurements should represent opportunities for evaluating the high pressure performance of an ESEM.

3D Imaging of Fuel Cell Electrode Structure Degraded under Cell Voltage Reversal Conditions Using Nanoscale X-Ray Computed Tomography

Polymer electrolyte fuel cells (PEFCs) have gained significant attention as highly efficient and clean power sources for fuel cell electric vehicles (FCEVs). One challenge faced by PEFCs is hydrogen starvation at the anode that can arise from either malfunction of the hydrogen supply system or hydrogen passage blockage by water flooding, ice formation in wintertime, or foreign impurities [1,2]. In particular, hydrogen starvation could be exacerbated when FCEVs are operated under transient conditions such as start-up and rapid load change. When the anode is starved of hydrogen, the anode requires an additional source of protons and its potential increases until the cell potential reverses and water electrolysis occurs, typically followed by carbon corrosion at higher potentials. These cell voltage reversal events eventually lead to catastrophic cell failure [2]. In order to develop highly robust FCEVs, it is important to understand the detailed morphological change in fuel cell electrodes under cell voltage reversal conditions. Over the last few years, nano-scale X-ray computed tomography (nano-CT) with the ability to image within samples in a non-invasive and non-destructive nature has been adopted to investigate the three-dimensional (3D) structure of fuel cell electrodes [3,4]. However, visualization of the 3D structure of degraded electrodes using nano-CT has been lacking, with a key challenge being the extraction of undisturbed samples from end-of-life (EOL) cells. Here we present the morphological analysis of fuel cell electrodes degraded under cell voltage reversal conditions by nano-CT using high precision laser micro-milling for sample preparation. The value in nano-CT imaging is the ability to image internal 3D structure without surface sample preparation artifacts. A standard membrane-electrode assembly (MEA) consisting of typical membrane (Nafion® NRE211CS (H+), DuPont) and electrodes (Pt/C catalyst (HISPEC4000®, Johnson Matthey) and ionomer binder (Nafion® D2021, DuPont)) was used throughout the study. An in-house 4 cm2 MEA was produced through a decal transfer process using a polyimide (Kapton® HN, DuPont) film. Under hydrogen (anode) and air (cathode) conditions, a single fuel cell was fully activated and then the beginning-of-life (BOL) polarizations were measured at 65oC and 100 % relative humidity. Cell voltage reversal tests were performed by operating the fuel cell at 0.2 A cm-2 under nitrogen (anode) and air (cathode) conditions until the cell voltage reached -2 V using a potentiostat. Cell polarization curves were measured after each reversal test. When the cell voltage at 1.2 A cm-2 was reduced to 34% of its BOL value, the cell status was considered to be at EOL. The EOL MEA sample was taken out of the cell for post-mortem analysis. A nano-scale resolution X-ray computed tomography microscope (UltraXRM-L200, Carl Zeiss X-ray Microscopy) was used for resolving the electrode structures as described in literature [3,4]. For sample preparation, a razor blade was used to cut out a small area of MEA (ca. 1.0 x 0.7 mm). The anode side of the MEA was ablated using a high-precision (~ 1 μm) laser mill (QuickLaze 50ST2, ESI®), leaving only the pillar-shaped anode specimen of approximately 65 μm in diameter on the membrane surface. Figure 1(a) shows the polarization curves of the MEA measured after cell voltage reversal tests. The cell performance decreases significantly as the voltage reversal tests were repeated, which may be attributed to combined degradation due to carbon corrosion at anode and an increased Ohmic resistance as characterized by electrochemical impedance spectroscopy and in-situ cyclic voltammetry. Figure 1(b) represents the nano-CT image of the EOL anode in the MEA. It is found that the thickness of the EOL anode is reduced by approximately 50%, from about 4 μm to 2 μm (EOL), implying a severe carbon corrosion at anode during the cell voltage reversal tests. The interface between the membrane and electrode appears mostly intact over the interface area, a feature difficult to observe from 2D SEM cross-sections, indicating that the increased Ohmic resistance could be mainly related with the structural collapse of anode, but not with a significant delamination at the interface. A variety of post-mortem analysis data will be presented to further elucidate the fundamental aspects of the cell voltage reversal degradation.

Co2FeAl based magnetic tunnel junctions with BaO and MgO/BaO barriers

We succeed to integrate BaO as a tunneling barrier into Co2FeAl based magnetic tunnel junctions (MTJs). By means of Auger electron spectroscopy it could be proven that the applied annealing temperatures during BaO deposition and afterwards do not cause any diffusion of Ba neither into the lower Heusler compound lead nor into the upper Fe counter electrode. Nevertheless, a negative tunnel magnetoresistance (TMR) ratio of -10% is found for Co2FeAl (24 nm) / BaO (5 nm) / Fe (7 nm) MTJs, which can be attributed to the preparation procedure and can be explained by the formation of Co- and Fe-oxides at the interfaces between the Heusler and the crystalline BaO barrier by comparing with theory. Although an amorphous structure of the BaO barrier seems to be confirmed by high-resolution transmission electron microscopy (TEM), it cannot entirely be ruled out that this is an artifact of TEM sample preparation due to the sensitivity of BaO to moisture. By replacing the BaO tunneling barrier with an MgO/BaO double layer barrier, the electric stability could effectively be increased by a factor of five. The resulting TMR effect is found to be about +20% at room temperature, although a fully antiparallel state has not been realized.

Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary Cell Walls: Evidence from Solid-State Nuclear Magnetic Resonance

The structural role of pectins in plant primary cell walls is not yet well understood because of the complex and disordered nature of the cell wall polymers. We recently introduced multidimensional solid-state nuclear magnetic resonance spectroscopy to characterize the spatial proximities of wall polysaccharides. The data showed extensive cross peaks between pectins and cellulose in the primary wall of Arabidopsis (Arabidopsis thaliana), indicating subnanometer contacts between the two polysaccharides. This result was unexpected because stable pectin-cellulose interactions are not predicted by in vitro binding assays and prevailing cell wall models. To investigate whether the spatial contacts that give rise to the cross peaks are artifacts of sample preparation, we now compare never-dried Arabidopsis primary walls with dehydrated and rehydrated samples. One-dimensional (13)C spectra, two-dimensional (13)C-(13)C correlation spectra, water-polysaccharide correlation spectra, and dynamics data all indicate that the structure, mobility, and intermolecular contacts of the polysaccharides are indistinguishable between never-dried and rehydrated walls. Moreover, a partially depectinated cell wall in which 40% of homogalacturonan is extracted retains cellulose-pectin cross peaks, indicating that the cellulose-pectin contacts are not due to molecular crowding. The cross peaks are observed both at -20 °C and at ambient temperature, thus ruling out freezing as a cause of spatial contacts. These results indicate that rhamnogalacturonan I and a portion of homogalacturonan have significant interactions with cellulose microfibrils in the native primary wall. This pectin-cellulose association may be formed during wall biosynthesis and may involve pectin entrapment in or between cellulose microfibrils, which cannot be mimicked by in vitro binding assays.

Improved molecular imaging in rodent brain with time-of-flight-secondary ion mass spectrometry using gas cluster ion beams and reactive vapor exposure.

Imaging mass spectrometry has shown to be a valuable method in medical research and can be performed using different instrumentation and sample preparation methods, each one with specific advantages and drawbacks. Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) has the advantage of high spatial resolution imaging but is often restricted to low mass molecular signals and can be very sensitive to sample preparation artifacts. In this report we demonstrate the advantages of using gas cluster ion beams (GCIBs) in combination with trifluoracetic acid (TFA) vapor exposure for the imaging of lipids in mouse brain sections. There is an optimum exposure to TFA that is beneficial for increasing high mass signal as well as producing signal from previously unobserved species in the mass spectrum. Cholesterol enrichment and crystallization on the sample surface is removed by TFA exposure uncovering a wider range of lipid species in the white matter regions of the tissue, greatly expanding the chemical coverage and the potential application of TOF-SIMS imaging in neurological studies. Ar4000(+) (40 keV) in combination with TFA treatment facilitates high resolution, high mass imaging closing the gap between TOF-SIMS and matrix-assisted laser desorption ionization (MALDI).

Methionine to isothreonine conversion as a source of false discovery identifications of genetically encoded variants in proteogenomics.

A lot of effort is currently put into proteogenomics of cancer. Studies detect non-synonymous cancer mutations at protein level by search of high-throughput LC-MS/MS data against customized genomic databases. In such studies, much attention is paid to potential false positive identifications. Here we describe one possible cause of such false identifications, an artifact of sample preparation which mimics methionine to threonine nucleic acid-encoded variant. The methionine to isothreonine conversion should be taken into consideration for correct interpretation of proteogenomic data.