Increasing Proteome Depth While Maintaining Quantitative Precision in Short-Gradient Data-Independent Acquisition Proteomics.

The combination of short liquid chromatography (LC) gradients and data-independent acquisition (DIA) by mass spectrometry (MS) has proven its huge potential for high-throughput proteomics. However, the optimization of isolation window schemes resulting in a certain number of data points per peak (DPPP) is understudied, although it is one of the most important parameters for the outcome of this methodology. In this study, we show that substantially reducing the number of DPPP for short-gradient DIA massively increases protein identifications while maintaining quantitative precision. This is due to a large increase in the number of precursors identified, which keeps the number of data points per protein almost constant even at long cycle times. When proteins are inferred from its precursors, quantitative precision is maintained at low DPPP while greatly increasing proteomic depth. This strategy enabled us to quantify 6018 HeLa proteins (>80 000 precursor identifications) with coefficients of variation below 20% in 30 min using a Q Exactive HF, which corresponds to a throughput of 29 samples per day. This indicates that the potential of high-throughput DIA-MS has not been fully exploited yet. Data are available via ProteomeXchange with identifier PXD036451.

Perchlorate-specific proteomic stress responses of Debaryomyces hansenii could enable microbial survival in Martian brines.

If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganisms on the cellular level. Here, we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4 -induced stresses share many common metabolic features, for example, signalling pathways, elevated energy metabolism, or osmolyte biosynthesis. Nevertheless, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for putative life on Mars, which-given the environmental conditions-likely developed chaotropic defence strategies such as stabilized confirmations of biomacromolecules or the formation of cell clusters.

Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in Silico Peptide Mass Libraries.

Over the past decade, modern methods of MS (MS) have emerged that allow reliable, fast and cost-effective identification of pathogenic microorganisms. Although MALDI-TOF MS has already revolutionized the way microorganisms are identified, recent years have witnessed also substantial progress in the development of liquid chromatography (LC)-MS based proteomics for microbiological applications. For example, LC-tandem MS (LC-MS2) has been proposed for microbial characterization by means of multiple discriminative peptides that enable identification at the species, or sometimes at the strain level. However, such investigations can be laborious and time-consuming, especially if the experimental LC-MS2 data are tested against sequence databases covering a broad panel of different microbiological taxa. In this proof of concept study, we present an alternative bottom-up proteomics method for microbial identification. The proposed approach involves efficient extraction of proteins from cultivated microbial cells, digestion by trypsin and LC-MS measurements. Peptide masses are then extracted from MS1 data and systematically tested against an in silico library of all possible peptide mass data compiled in-house. The library has been computed from the UniProt Knowledgebase covering Swiss-Prot and TrEMBL databases and comprises more than 12,000 strain-specific in silico profiles, each containing tens of thousands of peptide mass entries. Identification analysis involves computation of score values derived from correlation coefficients between experimental and strain-specific in silico peptide mass profiles and compilation of score ranking lists. The taxonomic positions of the microbial samples are then determined by using the best-matching database entries. The suggested method is computationally efficient - less than 2 mins per sample - and has been successfully tested by a test set of 39 LC-MS1 peak lists obtained from 19 different microbial pathogens. The proposed method is rapid, simple and automatable and we foresee wide application potential for future microbiological applications.

Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction.

The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples.

Unbiased Antimicrobial Resistance Detection from Clinical Bacterial Isolates Using Proteomics.

Antimicrobial resistance (AMR) poses an increasing challenge for therapy and clinical management of bacterial infections. Currently, antimicrobial resistance detection relies on phenotypic assays, which are performed independently from species identification. Sequencing-based approaches are possible alternatives for AMR detection, although the analysis of proteins should be superior to gene or transcript sequencing for phenotype prediction as the actual resistance to antibiotics is almost exclusively mediated by proteins. In this proof-of-concept study, we present an unbiased proteomics workflow for detecting both bacterial species and AMR-related proteins in the absence of secondary antibiotic cultivation within <4 h from a primary culture. The workflow was designed to meet the needs in clinical microbiology. It introduces a new data analysis concept for bacterial proteomics, and a software (rawDIAtect) for the prediction and reporting of AMR from peptide identifications. The method was validated using a sample cohort of 7 bacterial species and 11 AMR determinants represented by 13 protein isoforms, which resulted in a sensitivity of 98% and a specificity of 100%.

A robust metabolomics approach for the evaluation of human embryos from in vitro fertilization.

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of in vitro fertilization (IVF). We established a metabolomic-based approach by applying Fourier transform infrared (FTIR) spectroscopy on 130 samples of 3-day embryo culture supernatants from 26 embryos that implanted and 104 embryos that failed. On examining the internal structure of the data by unsupervised multivariate analysis, we found that the supernatant spectra of nonimplanted embryos constituted a highly heterogeneous group. Whereas ∼40% of these supernatants were spectroscopically indistinguishable from those of successfully implanted embryos, ∼60% exhibited diverse, heterogeneous metabolic fingerprints. This observation proved to be the direct result of pregnancy's multifactorial nature, involving both intrinsic embryonic traits and external characteristics. Our data analysis strategy thus involved one-class modelling techniques employing soft independent modelling of class analogy that identified deviant fingerprints as unsuitable for implantation. From these findings, we could develop a noninvasive Fourier-transform-infrared-spectroscopy-based approach that represents a shift in the fundamental paradigm for data modelling applied in assisted-fertilization technologies.

Isolation Window Optimization of Data-Independent Acquisition Using Predicted Libraries for Deep and Accurate Proteome Profiling.

In silico spectral library prediction of all possible peptides from whole organisms has a great potential for improving proteome profiling by data-independent acquisition (DIA) and extending its scope of application. In combination with other recent improvements in the field of mass spectrometry (MS)-based proteomics, including sample preparation, peptide separation, and data analysis, we aimed to uncover the full potential of such an advanced DIA strategy by optimization of the data acquisition. The results demonstrate that the combination of high-quality in silico libraries, reproducible and high-resolution peptide separation using micropillar array columns, as well as neural network supported data analysis enables the use of long MS scan cycles without impairing the quantification performance. The study demonstrates that mean coefficient of variations of 4% were obtained even at only 1.5 data points per peak (full width at half-maximum) across different gradient lengths, which in turn improved proteome coverage up to more than 8000 proteins from HeLa cells using empirically corrected libraries and more than 7000 proteins using a whole human in silico predicted library. These data were obtained using a Q Exactive orbitrap mass spectrometer with moderate scanning speed (12 Hz) and perform very well in comparison to recent studies using more advanced MS instruments, which underline the high potential of this optimization strategy for various applications in clinical proteomics, microbiology, and molecular biology.

Cold-adapted Bacilli isolated from the Qinghai-Tibetan Plateau are able to promote plant growth in extreme environments.

Nearly 1400 Bacillus strains growing in the plant rhizosphere were sampled from different sites on the Qinghai-Tibetan Plateau. Forty-five of the isolates, selected due to their biocontrol activity, were genome-sequenced and their taxonomic identification revealed that they were representatives of the Bacillus subtilis species complex (20) and the Bacillus cereus group (9). Majority of the remaining strains were found closely related to Bacillus pumilus, but their average nucleotide identity based on BLAST and electronic DNA/DNA hybridization values excluded closer taxonomic identification. A total of 45 different gene clusters involved in synthesis of secondary metabolites were detected by mining the genomes of the 45 selected strains. Except eight mesophilic strains, the 37 remaining strains were found either cold-adapted or psychrophilic, able to propagate at 10°C and below (Bacillus wiedmannii NMSL88 and Bacillus sp. RJGP41). Pot experiments performed at 10°C with winter wheat seedlings revealed that cold-adapted representatives of B. pumilus, B. safensis and B. atrophaeus promoted growth of the seedlings under cold conditions, suggesting that these bacilli isolated from a cold environment are promising candidates for developing of bioformulations useful for application in sustainable agriculture under environmental conditions unfavourable for the mesophilic bacteria presently in use.

Sialylation Controls Prion Fate in Vivo.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.

FT-IR Hyperspectral Imaging and Artificial Neural Network Analysis for Identification of Pathogenic Bacteria.

Identification of microorganisms by Fourier transform infrared (FT-IR) spectroscopy is known as a promising alternative to conventional identification techniques in clinical, food, and environmental microbiology. In this study we demonstrate the application of FT-IR hyperspectral imaging for rapid, objective, and cost-effective diagnosis of pathogenic bacteria. The proposed method involves a relatively short cultivation step under standardized conditions, transfer of the microbial material onto suitable IR windows by a replica method, FT-IR hyperspectral imaging measurements, and image segmentation by machine learning classifiers, a hierarchy of specifically optimized artificial neural networks (ANN). For cultivation, aliquots of the initial microbial cell suspension were diluted to guarantee single-colony growth on solid agar plates. After a short incubation period when microbial microcolonies achieved diameters between 50 and 300 µm, microcolony imprints were produced by using a specifically developed stamping device which allowed spatially accurate transfer of the microcolonies' upper cell layers onto IR-transparent CaF2 windows. Dry microcolony imprints were subsequently characterized using a mid-IR microspectroscopic imaging system equipped with a focal plane array (FPA) detector. Spectral data analysis involved preprocessing, quality tests, and the application of supervised modular ANN classifiers for hyperspectral image segmentation. The resulting easily interpretable segmentation maps suggest a taxonomic resolution below the species level.

Genome Mining of the Lipopeptide Biosynthesis of Paenibacillus polymyxa E681 in Combination with Mass Spectrometry: Discovery of the Lipoheptapeptide Paenilipoheptin.

Paenibacillus polymyxa strains are qualified for agro-biotechnological uses such as plant growth promotion and for biocontrol strategies against deleterious phytopathogenic competitors in the soil depending on their attractive arsenal of bioactive compounds. Moreover, they are potent producers of antibiotics for medical applications. To identify new products of such organisms, genome mining strategies in combination with mass spectrometry are the methods of choice. Herein, we performed such studies with the Paenibacillus strain E681. Bioinformatic evaluation of its genome sequence revealed four gene clusters A-D encoding nonribosomal peptide synthetases (NRPSs). Accordingly, four lipopeptide families were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Clusters A and D codify the well known fusaricidins and polymyxins. A yet-unknown lipoheptapeptide was discovered and structurally characterized by de novo sequencing by using MALDI-LIFT-TOF/TOF MS. It was designated as paenilipoheptin. From structure predictions we infer that the production of this agent is encoded by gene cluster C. Gene cluster B encodes the synthesis of tridecaptins, a family of open-chain lipotridecapeptides. Strain E681 produces new subspecies of such compounds (tridecaptins E) showing variations both in their fatty-acid part as well as in their peptide part.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.

Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires disease, has a biphasic life cycle with a switch from a replicative to a transmissive phenotype. During the replicative phase, the bacteria grow within host cells in Legionella-containing vacuoles. During the transmissive phenotype and the postexponential (PE) growth phase, the pathogens express virulence factors, become flagellated, and leave the Legionella-containing vacuoles. Using (13)C labeling experiments, we now show that, under in vitro conditions, serine is mainly metabolized during the replicative phase for the biosynthesis of some amino acids and for energy generation. During the PE phase, these carbon fluxes are reduced, and glucose also serves as an additional carbon substrate to feed the biosynthesis of poly-3-hydroxybuyrate (PHB), an essential carbon source for transmissive L. pneumophila. Whole-cell FTIR analysis and comparative isotopologue profiling further reveal that a putative 3-ketothiolase (Lpp1788) and a PHB polymerase (Lpp0650), but not enzymes of the crotonyl-CoA pathway (Lpp0931-0933) are involved in PHB metabolism during the PE phase. However, the data also reflect that additional bypassing reactions for PHB synthesis exist in agreement with in vivo competition assays using Acanthamoeba castellannii or human macrophage-like U937 cells as host cells. The data suggest that substrate usage and PHB metabolism are coordinated during the life cycle of the pathogen.

Two-Dimensional Correlation Spectroscopy for Multimodal Analysis of FT-IR, Raman, and MALDI-TOF MS Hyperspectral Images with Hamster Brain Tissue.

Hyperspectral imaging (HSI) techniques are useful for obtaining very detailed structural and compositional information from biomedical, pharmaceutical, or clinical samples, among others. The informative value of these methods can be further increased through the application of different HSI techniques and joint analysis of the data. However, interpretation and understanding of multimodal HSI have been impeded by difficulties in registration of the different HSI data sets and by the lack of integrative analysis concepts. Here, we introduce two-dimensional correlation spectroscopy (2DCOS) as a novel technique for jointly analyzing HSI data which allows one to obtain deeper insights into the chemistry of complex samples by decrypting auto- and heterospectral correlations that may exist between features of the different HSI data. The general workflow of 2DCOS analysis is demonstrated by HSI examples acquired from cryo-sections of hamster brain tissue using Fourier-transform infrared (FT-IR) microspectroscopy, confocal Raman microspectroscopy (CRM), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Multimodal hyperspectral image analysis by 2DCOS opens up new opportunities for spectral band assignments and thus the interpretation of structure-spectra and composition-spectra relationships. We foresee wide application potential for describing complex samples in various fields ranging from biomedicine to industrial applications.

Rapid characterisation of Klebsiella oxytoca isolates from contaminated liquid hand soap using mass spectrometry, FTIR and Raman spectroscopy.

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

[On-site detection of bioterrorism-relevant agents : Rapid detection methods for viruses, bacteria and toxins - capabilities and limitations]. / Vor-Ort-Nachweis bioterroristisch relevanter Agenzien : Schnelle Nachweismethoden für Viren, Bakterien und Toxine ­ Potenziale und Grenzen.

In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

Spectropathology for the next generation: quo vadis?

Although the potential of vibrational spectroscopy for biomedical applications has been well demonstrated, translation into clinical practice has been relatively slow. This Editorial assesses the challenges facing the field and the potential way forward. While many technological challenges have been addressed to date, considerable effort is still required to gain acceptance of the techniques among the medical community, standardise protocols, extend to a clinically relevant scale, and ultimately assess the health economics underlying clinical deployment. National and international research networks can contribute much to technology development and standardisation. Ultimately, large-scale funding is required to engage in clinical trials and instrument development.

Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.

Infrared microspectroscopy detects protein misfolding cyclic amplification (PMCA)-induced conformational alterations in hamster scrapie progeny seeds.

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.