Non-targeted analysis using LC-MS/MS DDA acquisition

This tutorial walks you through the workflow for analyzing non-targeted liquid chromatography high resolution mass spectrometry (LC-MS/MS) data-dependent acquisition (DDA) data starting from input file generation, to processing the data in SmartPeak, to reviewing the data in SmartPeak, to reporting the results.

Objectives

1. Obtaining the SOP for the workflow.

2. Choosing a data set for demonstrating the workflow.

3. Creating an optimized SmartPeak input templates for running the workflow.

The Workflows include

1. Defining the accurate mass search database

2. Processing Unknowns based MS1 accurate mass and creating a transition library based on MS2 product ion scans

3. Processing Unknowns based MS1 accurate mass and creating a spectral database based on MS2 product ion scans

4. Processing Unknowns based on MS2 product ion scans using an existing spectral database

5. Reviewing the results

Notes

The algorithm parameters used in the following workflows have been highly tuned for feature detection using Time of Flight (Tof) technologies. Slight modifications to the algorithm parameters in the TargetedSpectraExtractor section is needed for Orbitrap technology and other acquisition method specific parameters.

Steps

The tutorial includes the following steps :

1. Setting up the input files

   The data set used can be found in LC-MS/MS-DDA for the transition library and spectral database generation, and spectral library matching workflows.

   The dataset includes a CHEMISTRY folder which contains HMDB (Human Metabolome Database) mapping files organized as follows:

   1. HMDB2StructMapping.tsv

   This tab seperated file contains mapping of HMDB IDs, IUPAC Name, Compound Summary (Synonyms), Canonical SMILES and InChl.

   1. HMDBMappingFile.tsv

   This tab seperated file contains the Monoisotopic Molecular Weight and the Chemical Formula mapped to their respective HMDB ID.

   1. negative_adducts.tsv

   This file contains negative ion modes including the charge.

   1. positive_adducts.tsv

   This file contains positive ion modes including the charge.

   The above files provided in the example are appropriate for applications involving human serum and other biosamples. For applications involving other organisms such as bacteria (e.g., E. coli), the use of organism specific databases are recommended to reduce the number of false positives. See FIA_MS_database_construction.ipynb for an example notebook demonstrating how to convert the metabolites in a genome-scale reconstruction to SmartPeak accurate mass mapping files.

   Furthermore, the parameters.csv file contains the following settings for this workflow:

   DDA_parameters.csv

   function	name	value	type
   FeatureFindingMetabo	report_chromatograms	TRUE	bool
   FeatureFindingMetabo	report_convex_hulls	TRUE	bool
   FeatureFindingMetabo	use_smoothed_intensities	FALSE	bool
   Pick3DFeatures	enable_elution	FALSE	bool
   Pick3DFeatures	max_traces	1000	int
   Pick3DFeatures	force_processing	FALSE	bool
   TargetedSpectraExtractor	AccurateMassSearchEngine:db:mapping	[‘CHEMISTRY/HMDBMappingFileGermicidinA.tsv’]	list
   TargetedSpectraExtractor	AccurateMassSearchEngine:db:struct	[‘CHEMISTRY/HMDB2StructMappingGermicidinA.tsv’]	list
   TargetedSpectraExtractor	AccurateMassSearchEngine:positive_adducts	CHEMISTRY/PositiveAdducts.tsv	string
   TargetedSpectraExtractor	AccurateMassSearchEngine:negative_adducts	CHEMISTRY/NegativeAdducts.tsv	string
   TargetedSpectraExtractor	relative_allowable_product_mass	50	float
   TargetedSpectraExtractor	AccurateMassSearchEngine:ionization_mode	auto	string
   TargetedSpectraExtractor	AccurateMassSearchEngine:mass_error_value	10	float
   TargetedSpectraExtractor	rt_window	60	float
   TargetedSpectraExtractor	mz_tolerance	0.001	float
   TargetedSpectraExtractor	AccurateMassSearchEngine:keep_unidentified_masses	TRUE	bool

2. Defining the workflow in SmartPeak

For LC-MS/MS-DDA transition library generation analysis, the following steps are saved into the workflow.csv file. Alternatively, steps can be replaced, added or deleted direclty from SmartPeakGUI. A detailed explanation of each command step can be found in Workflow Commands.

workflow_LCMS_DDA_transition.csv

workflow_step
LOAD_RAW_DATA
PICK_3D_FEATURES
SEARCH_SPECTRUM_MS1
MERGE_FEATURES_MS1
EXTRACT_SPECTRA_NON_TARGETED
SEARCH_SPECTRUM_MS2
MERGE_FEATURES_MS2
CONSTRUCT_TRANSITIONS_LIST
STORE_FEATURES

The workflow process’s data dependent acquisition (DDA) data generated by liquid chromatography mass spectrometry instrumentation.

The workflow first picks, annotates, and merges features found in the MS1 scans for each sample, second picks, annotates, and merges features found in the MS2 scans for each sample, and third constructs a transition list for downstream quantification using e.g., data independent acquisition (DIA) or single reaction monitoring (SRM) methods.

The transition library generated for each sample can be found in the output features directory. A snippet of the table is shown below.

DDA_transition_library.csv

PrecursorMz	ProductMz	PrecursorCharge	ProductCharge	LibraryIntensity	NormalizedRetentionTime	ProteinId	TransitionGroupId	TransitionId
235.0747741	134.9568506	0	NA	476	329.3195788	HMDB:HMDB0000001	HMDB:HMDB0000001	HMDB:HMDB0000001_scan=440_134.957_370.346
235.0747741	217.1050819	0	NA	464	329.3195788	HMDB:HMDB0000001	HMDB:HMDB0000001	HMDB:HMDB0000001_scan=440_217.105_370.346

Statistics on each of the individual scans analyzed can be viewed at view | scans.

The features found can be viewed at view | features (table) in long form.

Or at view | features (matrix) in compact form.

The TIC for each injection can be viewed at view | chromatograms.

Clicking on the chromatogram reveals the MS1 spectra acquired at the selected time-point.

Clicking on the spectra computes and displays the MS1 XIC for the selected m/z.

Clicking on the MS1 XIC chromatogram revealse the MS2 spectra acquired at the selected time-point.

1. Defining the workflow in SmartPeak

For LC-MS/MS-DDA spectral database generation analysis, the following steps are saved into the workflow.csv file. Alternatively, steps can be replaced, added or deleted direclty from SmartPeakGUI. A detailed explanation of each command step can be found in Workflow Commands.

workflow_LCMS_DDA_spectral.csv

workflow_step
LOAD_RAW_DATA
PICK_3D_FEATURES
SEARCH_SPECTRUM_MS1
MERGE_FEATURES_MS1
EXTRACT_SPECTRA_NON_TARGETED
STORE_MSP
STORE_FEATURES

The workflow process’s data dependent acquisition (DDA) data generated by liquid chromatography mass spectrometry instrumentation.

The workflow first picks, annotates, and merges features found in the MS1 scans for each sample, second picks features found in the MS2 scans for each sample, and third constructs a spectral library for downstream spectral annotation.

The spectral database in MSP format generated for each sample can be found in the output features directory. A snippet of the table is shown below.

DDA_spectral_library.csv

Name: HMDB:HMDB0000001

Retention Time: 370.346

base peak intensity: 476.0

total ion current: 940.0

Num Peaks: 2

134.957:476 217.105:464

1. Defining the workflow in SmartPeak

For LC-MS/MS-DDA spectral database matching analysis, the following steps are saved into the workflow.csv file. Alternatively, steps can be replaced, added or deleted direclty from SmartPeakGUI. A detailed explanation of each command step can be found in Workflow Commands.

workflow_LCMS_DDA_spectra.csv

workflow_step
LOAD_RAW_DATA
PICK_3D_FEATURES
EXTRACT_SPECTRA_NON_TARGETED
MATCH_SPECTRA
STORE_FEATURES

The workflow process’s data dependent acquisition (DDA) data generated by liquid chromatography mass spectrometry instrumentation.

The workflow first picks features found in the MS1 scans for each sample, second picks features found in the MS2 scans for each sample, and third annotates the MS1 features by matching the spectra of the MS2 scans with a spectral library.