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1. Protocol for community‐created public MS/MS reference spectra within the Global Natural Products Social Molecular Networking infrastructure

   https://doi.org/10.1002/rcm.8725  

   Vargas, Fernando; Weldon, Kelly_C; Sikora, Nicole; Wang, Mingxun; Zhang, Zheng; Gentry, Emily_C; Panitchpakdi, Morgan_W; Caraballo‐Rodríguez, Andrés_Mauricio; Dorrestein, Pieter_C; Jarmusch, Alan_K (February 2020, Rapid Communications in Mass Spectrometry)

   RationaleA major hurdle in identifying chemicals in mass spectrometry experiments is the availability of tandem mass spectrometry (MS/MS) reference spectra in public databases. Currently, scientists purchase databases or use public databases such as Global Natural Products Social Molecular Networking (GNPS). The MSMS‐Chooser workflow is an open‐source protocol for the creation of MS/MS reference spectra directly in the GNPS infrastructure. MethodsAn MSMS‐Chooser Sample Template is provided and completed manually. The MSMS‐Chooser Submission File and Sequence Table for data acquisition were programmatically generated. Standards from the Mass Spectrometry Metabolite Library (MSMLS) suspended in a methanol–water (1:1) solution were analyzed. Flow injection on an LC/MS/MS system was used to generate negative and positive mode data using data‐dependent acquisition. The MS/MS spectra and Submission File were uploaded to MSMS‐Chooser workflow in GNPS for automatic selection of MS/MS spectra. ResultsData acquisition and processing required ~2 h and ~2 min, respectively, per 96‐well plate using MSMS‐Chooser. Analysis of the MSMLS, over 600 small molecules, using MSMS‐Chooser added 889 spectra (including multiple adducts) to the public library in GNPS. Manual validation of one plate indicated accurate selection of MS/MS scans (true positive rate of 0.96 and a true negative rate of 0.99). The MSMS‐Chooser output includes a table formatted for inclusion in the GNPS library as well as the ability to directly launch searches via MASST. ConclusionsMSMS‐Chooser enables rapid data acquisition, data analysis (selection of MS/MS spectra), and a formatted table for inspection and upload to GNPS. Open file‐format data (.mzML or.mzXML) from most mass spectrometry platforms containing MS/MS spectra can be processed using MSMS‐Chooser. MSMS‐Chooser democratizes the creation of MS/MS reference spectra in GNPS which will improve annotation and strengthen the tools which use the annotation information. 

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2. Effective discrimination of gas-phase peptide conformers using TIMS-ECD-ToF MS/MS

   https://doi.org/10.1039/d1ay01461g  

   Jeanne Dit Fouque, K.; Wellmann, M.; Leyva Bombuse, D.; Santos-Fernandez, M.; Cintron-Diaz, Y. L.; Gomez-Hernandez, M. E.; Kaplan, D.; Voinov, V. G.; Fernandez-Lima, F. (November 2021, Analytical Methods)

   In the present work, four, well-studied, model peptides ( e.g. , substance P, bradykinin, angiotensin I and AT-Hook 3) were used to correlate structural information provided by ion mobility and ECD/CID fragmentation in a TIMS-q-EMS-ToF MS/MS platform, incorporporating an electromagnetostatic cell (EMS). The structural heterogeneity of the model peptides was observed by (i) multi-component ion mobility profiles (high ion mobility resolving power, R ∼115–145), and (ii) fast online characteristic ECD fragmentation patterns per ion mobility band (∼0.2 min). Particularly, it was demonstrated that all investigated species were probably conformers, involving cis / trans -isomerizations at X-Pro peptide bond, following the same protonation schemes, in good agreement with previous ion mobility and single point mutation experiments. The comparison between ion mobility selected ECD spectra and traditional FT-ICR ECD MS/MS spectra showed comparable ECD fragmentation efficiencies but differences in the ratio of radical (˙)/prime (′) fragment species (H˙ transfer), which were associated with the differences in detection time after the electron capture event. The analysis of model peptides using online TIMS-q-EMSToF MS/MS provided complementary structural information on the intramolecular interactions that stabilize the different gas-phase conformations to those obtained by ion mobility or ECD alone. 

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3. Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics

   Zhichang Yang, Liangliang Sun (June 2022, Methods in molecular biology)

   Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms. 

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4. NanoSF-MS

   Mahmoud Elhusseiny Mostafa, James P. (September 2022, Journal of the American Society for Mass Spectrometry)

   Supercritical fluids are typically electrosprayed using an organic solvent makeup flow to facilitate continuous electrical connection and enhancement of electrospray stability. This results in sample dilution, loss in sensitivity, and potential phase separation. Premixing the supercritical fluid with organic solvent has shown substantial benefits to electrospray efficiency and increased analyte charge state. Presented here is a nanospray mass spectrometry system for supercritical fluids (nSF-MS). This split flow system used small i.d. capillaries, heated interface, inline frit, and submicron emitter tips to electrospray quaternary alkyl amines solvated in supercritical CO2 with a 10% methanol modifier. Analyte signal response was evaluated as a function of total system flow rate (0.5–1.5 mL/min) that is split to nanospray a supercritical fluid with linear flow rates between 0.07 and 0.42 cm/sec and pressure ranges (15–25 MPa). The nSF system showed mass-sensitive detection based on increased signal intensity for increasing capillary i.d. and analyte injection volume. These effects indicate efficient solvent evaporation for the analysis of quaternary amines. Carrier additives generally decreased signal intensity. Comparison of the nSF-MS system to the conventional SF makeup flow ESI showed 10-fold signal intensity enhancement across all the capillary i.d.s. The nSF-MS system likely achieves rapid solvent evaporation of the SF at the emitter point. The developed system combined the benefits of the nanoemitters, sCO2, and the low modifier percentage which gave rise to enhancement in MS detection sensitivity. 

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5. Direct sequencing of total S. cerevisiae tRNAs by LC-MS/MS

   https://doi.org/10.1261/rna.079656.123  

   Jones, Joshua D; Simcox, Kaley M; Kennedy, Robert T; Koutmou, Kristin S (May 2023, RNA)

   Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs. 

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