About GNPS analysis

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Dipesh Dhakal

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Apr 7, 2021, 4:45:39 PM4/7/21
to molecular_networ...@googlegroups.com
Recently, I used GNPS platform for analysis of extract from few of the cyanobacterial samples.
But from the analysis results, I can just get the networking for molecules with smaller molecular weights. The library search provided array of molecules related to fatty acid and terpenoids, whereas I expected molecules more related to cyanobacteria.
Did I missed anything?
 Is there any problem in my extraction method (its just regular methanol/ ACN/H2O extract)?
 Is there any problem in my HR-MS analysis (Data from LC-HR-MS and MS/MS was obtained using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with electrospray probe on Universal Ion Max API source. Acetonitrile (B)/water (A) containing 0.1% formic acid were used as mobile phases with a linear gradient program (10–90% solvent B over 15 min) to separate chemicals by the above reverse phase HPLC column at a flow rate of 0.3 mL/min. A pre-wash phase of 15 min with 10% solvent B was added at the beginning of each run, in which the elute was diverted to the waste by a diverting valve. MS1 were acquired under Full Scan mode of the Orbitrap, in which a mass range of m/z 150–2000 was covered and data were collected in the positive ion mode. Fragmentation was introduced by HCD technique with optimized collision energy ranging from 20 to 30. Other settings for the Orbitrap scan included resolution at 15,000 and AGC target at 5 × 105. Full scan mass spectra  were extracted from the raw files of the HPLC-MS/MS Experiment II using Xcalibur™ 2.1 (Thermo Scientific).
I followed standard protocol for data conversion and uploading the data file. 

Library Search File

Classical molecular networking

Best Regards,
Dipesh Dhakal,PhD
Post-doctoral Associate
Department of Medicinal Chemistry, University of Florida
1345 Center Dr. Gainesville, FL 32610



Vanessa Phelan

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Apr 8, 2021, 6:16:57 PM4/8/21
to GNPS Discussion Forum and Bug Reports
Hi Dipesh-
We can't provide advice for extraction or chromatographic parameters.

From the job status page of any of the links you've provided, you can see the chromatograms of your data in the GNPS Dashboard by clicking File Summaries > View LCMS.

Here is a link from the Dashboard for M134: https://gnps-lcms.ucsd.edu/?xic_mz=376.26&xic_formula=&xic_peptide=&xic_tolerance=0.5&xic_ppm_tolerance=10&xic_tolerance_unit=Da&xic_rt_window=&xic_norm=False&xic_file_grouping=MZ&xic_integration_type=AUC&show_ms2_markers=True&ms2marker_color=blue&ms2marker_size=5&ms2_identifier=MS2%3A3584&show_lcms_2nd_map=False&map_plot_zoom=%7B%7D&polarity_filtering=None&polarity_filtering2=None&tic_option=TIC&overlay_usi=None&overlay_mz=row+m%2Fz&overlay_rt=row+retention+time&overlay_color=&overlay_size=&overlay_hover=&overlay_filter_column=&overlay_filter_value=&feature_finding_type=Off&feature_finding_ppm=10&feature_finding_noise=10000&feature_finding_min_peak_rt=0.05&feature_finding_max_peak_rt=1.5&feature_finding_rt_tolerance=0.3&sychronization_session_id=796e2c88c13942979cd1f4e4776d28a9&chromatogram_options=%5B%5D&comment=&map_plot_color_scale=Hot_r&map_plot_quantization_level=Medium#%7B%22usi%22%3A%20%22mzspec%3AGNPS%3ATASK-a9edd5b8db3f4aecaa5d55a8eb2987e0-f.dipeshdhakal/M134.mzML%22%2C%20%22usi2%22%3A%20%22%22%7D

From the total ion chromatogram on the bottom right, it's clear you don't have many chromatographic peaks from your extracts. For the large peak, I pulled out the precursor m/z value and you can see based upon the red x position in the extracted ion chromatogram, the collection of the MS/MS data is at the bottom of the peak which means that your spectra will have low signal to noise. Low signal to noise in MS/MS data will lead to poor matches to the GNPS spectra libraries.

Based upon the mass resolving power of the data collection (15,000 on an orbitrap) and the precursor and fragment mass tolerance (2.0 Da and 0.5 Da) you're using for the library searches/classical molecular networking, the library matches may not be relevant to the samples. The m/z values from your data can have up to a 2.0 Da mass difference from the library spectra with some matches with ppm differences of >19000. 

Best wishes,
Vanessa

Pieter Dorrestein

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Apr 8, 2021, 11:34:21 PM4/8/21
to Vanessa Phelan, GNPS Discussion Forum and Bug Reports
Indeed, when the mass accuracy is considered most annotations are from plastics/plasticizers and the GNPS dashboard view that Vanessa provided shows that there is a large background of repeating units and/or little amount of sample itself. I suggest evaluating your extraction methods and tubes that you are using to eliminate as much material from plastics and background solvents and ensure that the extraction itself has a larger amount so that it becomes visible (at least it should be more than the background signals). Also you may want to expand your exclusion list when running the mass spec next time that should increase the likelihood to go deeper into the metabolome.

Good luck, P 

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