Mass Spectrometry Proteotyping Research Articles

Universal Identification of Pathogenic Viruses by Liquid Chromatography Coupled with Tandem Mass Spectrometry Proteotyping

Accurate and rapid identification of viruses is crucial for an effective medical diagnosis when dealing with infections. Conventional methods, including DNA amplification techniques or lateral-flow assays, are constrained to a specific set of targets to search for. In this study, we introduce a novel tandem mass spectrometry proteotyping-based method that offers a universal approach for the identification of pathogenic viruses and other components, eliminating the need for a priori knowledge of the sample composition. Our protocol relies on a time and cost-efficient peptide sample preparation, followed by an analysis with liquid chromatography coupled to high-resolution tandem mass spectrometry. As a proof of concept, we first assessed our method on publicly available shotgun proteomics datasets obtained from virus preparations and fecal samples of infected individuals. Successful virus identification was achieved with 53 public datasets, spanning 23 distinct viral species. Furthermore, we illustrated the method's capability to discriminate closely related viruses within the same sample, using alphaviruses as an example. The clinical applicability of our method was demonstrated by the accurate detection of the vaccinia virus in spiked saliva, a matrix of paramount clinical significance due to its non-invasive and easily obtainable nature. This innovative approach represents a significant advancement in pathogen detection and paves the way for enhanced diagnostic capabilities.

GroEL-Proteotyping of Bacterial Communities Using Tandem Mass Spectrometry.

Profiling bacterial populations in mixed communities is a common task in microbiology. Sequencing of 16S small subunit ribosomal-RNA (16S rRNA) gene amplicons is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except a few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw dataset obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution.

Evaluation of the Limit of Detection of Bacteria by Tandem Mass Spectrometry Proteotyping and Phylopeptidomics.

Shotgun proteomics has proven to be an attractive alternative for identifying a pathogen and characterizing the antimicrobial resistance genes it produces. Because of its performance, proteotyping of microorganisms by tandem mass spectrometry is expected to become an essential tool in modern healthcare. Proteotyping microorganisms that have been isolated from the environment by culturomics is also a cornerstone for the development of new biotechnological applications. Phylopeptidomics is a new strategy that estimates the phylogenetic distances between the organisms present in the sample and calculates the ratio of their shared peptides, thus improving the quantification of their contributions to the biomass. Here, we established the limit of detection of tandem mass spectrometry proteotyping based on MS/MS data recorded for several bacteria. The limit of detection for Salmonella bongori with our experimental set-up is 4 × 104 colony-forming units from a sample volume of 1 mL. This limit of detection is directly related to the amount of protein per cell and therefore depends on the shape and size of the microorganism. We have demonstrated that identification of bacteria by phylopeptidomics is independent of their growth stage and that the limit of detection of the method is not degraded in presence of additional bacteria in the same proportion.

Deep Paleoproteotyping and Microtomography Revealed No Heart Defect nor Traces of Embalming in the Cardiac Relics of Blessed Pauline Jaricot.

Scientific examination of the heart of Blessed Pauline Jaricot-a French missionary figure-was carried out in 2022. As tandem mass spectrometry proteotyping has proven to be valuable to obtain the broad taxonomic repertoire of a given sample without any a priori information, we aimed at exploring the conditions of preservation of the relics and possible conditions of death. Metaproteomics and high-resolution microtomography imaging approaches were combined. A dataset comprising 6731 high-resolution MS/MS spectra was acquired and 968 of these spectra could be assigned to specific peptidic biomolecules. Based on the taxonomical information encompassed by the identified peptide sequences, 5 phyla were identified amongst eukaryota (94% of the biomass): Ascomycota (55%), with the species Aspergillus versicolor, Trichophyton mentagrophytes and Aspergillus glaucus, corresponding to expected cadaverous fungal flora; Chordata (42%), represented by a unique species, Homo sapiens; Streptophyta (3%); and Arthropoda (traces). Bacteria (6% of the biomass) were poorly represented. No trace of embalming substance could be retrieved, nor any pathogens. Imaging evidenced no heart defect nor embalming traces. No evidence that was inconsistent with natural and spontaneous conservation could be retrieved. This study prefigures the power of modern molecular techniques such as paleoproteotyping coupled to microtomography to gain insight into historical relics.

Identification and Characterization of Marine Microorganisms by Tandem Mass Spectrometry Proteotyping.

The vast majority of marine microorganisms and their functions are yet to be explored. The considerable diversity they encompass is an endless source of knowledge and wealth that can be valued on an industrial scale, emphasizing the need to develop rapid and efficient identification and characterization techniques. In this study, we identified 26 microbial isolates from coastal water of the NW Mediterranean Sea, using phylopeptidomics, a cutting-edge tandem mass spectrometry proteotyping technique. Taxonomical identification at the species level was successfully conducted for all isolates. The presence of strains belonging to the newly described Balneolaeota phylum, yet uncharacterized at the proteomics scale, was noted. The very first proteomics-based investigation of a representative of the Balneolaeota phylum, Balneola vulgaris, is proposed, demonstrating the use of our proteotyping workflow for the rapid identification and in-depth molecular characterization, in a single MS/MS analytical run. Tandem mass spectrometry proteotyping is a valuable asset for culturomic programs as the methodology is able to quickly classify the most atypical isolates.

Proteomics in the COVID-19 Battlefield: First Semester Check-Up.

Proteomics offers a wide collection of methodologies to study biological systems at the finest granularity. Faced with COVID‐19, the most worrying pandemic in a century, proteomics researchers have made significant progress in understanding how the causative virus hijacks the host's cellular machinery and multiplies exponentially, how the disease can be diagnosed, and how it develops, as well as its severity predicted. Numerous cellular targets of potential interest for the development of new antiviral drugs have been documented. Here, the most striking results obtained in the proteomics field over this first semester of the pandemic are presented. The molecular machinery of SARS‐CoV‐2 is much more complex than initially believed, as many post‐translational modifications can occur, leading to a myriad of proteoforms and a broad heterogeneity of viral particles. The interplay of protein–protein interactions, protein abundances, and post‐translational modifications has yet to be fully documented to provide a full picture of this intriguing but lethal biological threat. Proteomics has the potential to provide rapid detection of the SARS‐CoV‐2 virus by mass spectrometry proteotyping, and to further increase the knowledge of severe respiratory syndrome COVID‐19 and its long‐term health consequences.

Proteotyping Environmental Microorganisms by Phylopeptidomics: Case Study Screening Water from a Radioactive Material Storage Pool.

The microbial diversity encompassed by the environmental biosphere is largely unexplored, although it represents an extensive source of new knowledge and potentially of novel enzymatic catalysts for biotechnological applications. To determine the taxonomy of microorganisms, proteotyping by tandem mass spectrometry has proved its efficiency. Its latest extension, phylopeptidomics, adds a biomass quantitation perspective for mixtures of microorganisms. Here, we present an application of phylopeptidomics to rapidly and sensitively screen microorganisms sampled from an industrial environment, i.e., a pool where radioactive material is stored. The power of this methodology is demonstrated through the identification of both prokaryotes and eukaryotes, whether as pure isolates or present as mixtures or consortia. In this study, we established accurate taxonomical identification of environmental prokaryotes belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla, as well as eukaryotes from the Ascomycota phylum. The results presented illustrate the potential of tandem mass spectrometry proteotyping, in particular phylopeptidomics, to screen for and rapidly identify microorganisms.

High-throughput proteotyping of bacterial isolates by double barrel chromatography-tandem mass spectrometry based on microplate paramagnetic beads and phylopeptidomics

Tandem mass spectrometry-based proteotyping of microorganisms presents several advantages over whole-cell MALDI-TOF mass spectrometry: because a larger number of signals are recorded with better accuracy and precision, the approach allows for the identification of microorganisms at more resolved taxonomic levels, and can easily manage complex samples. Additionally, the use of SP3 paramagnetic beads for cell lysis and protein cleanup simplifies sample preparation for proteotyping. Based on these features, we have developed and tested a 96-well plate platform for high-throughput proteotyping of a large variety of bacteria. We evaluated the performance of the platform in terms of bacterial load and found no cross-contamination between wells. Likewise, phylopeptidomics analysis revealed no alteration in the relative quantifications of microorganisms. Finally, we applied this new format for rapid proteotyping of a large set of dental isolates using double-barrel chromatography coupled to tandem mass spectrometry, which maximizes the number of spectra per unit of time. The procedure allowed us to establish whether these isolates were pure strains or mixtures of strains and to identify the microorganisms at the most resolved taxonomic level. SignificanceThe rapid and accurate identification of microorganisms is a clinical priority in medical diagnostics; however, specimens containing mixtures of microorganisms are difficult to analyze and the procedure is time-consuming. Tandem mass spectrometry proteotyping allows the fast identification of complex mixtures of microorganisms, known or unknown, and can also establish the biomass ratio of each component. We describe here a new workflow for preparing microbial samples in a 96-well-plate format for tandem mass spectrometry proteotyping and document its advantages and limitations. We demonstrate that this new format coupled to a highly efficient double-barrel LC-MS/MS system allows proteotyping of 96 isolates in 55 h.

Rapid and Culture Free Identification of Francisella in Hare Carcasses by High-Resolution Tandem Mass Spectrometry Proteotyping.

Zoonotic pathogens that can be transmitted via food to humans have a high potential for large-scale emergencies, comprising severe effects on public health, critical infrastructures, and the economy. In this context, the development of laboratory methods to rapidly detect zoonotic bacteria in the food supply chain, including high-resolution mass spectrometry proteotyping are needed. In this work, an optimized sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteome profiling was established for Francisella isolates, and a cluster analysis, as well as a phylogenetic tree, was generated to shed light on evolutionary relationships. Furthermore, this method was applied to tissues of infected hare carcasses from Germany. Even though the non-informative data outnumbered by a manifold the information of the zoonotic pathogen in the resulting proteome profiles, the standardized evaluation of MS data within an established automated analysis pipeline identified Francisella (F.) tularensis and, thus, could be, in principle, an applicable method to monitor food supply chains.

Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry.

Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.