Sep 28, 2022

Public workspaceDiscovery proteomic (DIA) LC-MS/MS data acquisition and analysis V.2

Version 1 is forked from Discovery proteomic (DDA) LC-MS/MS data acquisition and analysis
DOI
dx.doi.org/10.17504/protocols.io.e6nvwk1z7vmk/v2
  • 1Lawrence Berkeley National Laboratory
Christopher J Petzold
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwk1z7vmk/v2
Protocol Citation: Yan Chen, Jennifer Gin, Christopher J Petzold 2022. Discovery proteomic (DIA) LC-MS/MS data acquisition and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwk1z7vmk/v2Version created by Christopher J Petzold
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 19, 2022
Last Modified: September 28, 2022
Protocol Integer ID: 70258
Keywords: Proteomics, Liquid chromatography, Mass spectrometry, DDA, Orbitrap
Disclaimer
This protocol is for research purposes only.
Abstract
This protocol details steps in discovery proteomic data-independent acquisition with a standard-flow UHPLC-Obitrap system and a subsequent DIA-NN library-free database search. The data acquisition method was adapted from González Fernández-Niño, S. M., et al. "Standard flow liquid chromatography for shotgun proteomics in bioenergy research."Frontiers in bioengineering and biotechnology, 3 (2015): 44.
Materials
MATERIALS
ReagentAcetonitrile LCMS qualityJT BakerCatalog #9829-02
ReagentLCMS grade waterVWR InternationalCatalog #BJLC365-2.5
ReagentIsopropanol VWR InternationalCatalog #BJ650447-4L
STEP MATERIALS
Analytical column: InfinityLab Poroshell 120 EC-C18 (Thikness2.1 mm ID ,Thikness100 mm length ,Thikness1.9 µm particle size , 120-Å pore size) (Agilent, Cat.#695675-902)
Guard column: InfinityLab Poroshell 120 EC-C18 guard column (Thikness2.1 mm ID ,Thikness5 mm length ,Thikness1.9 µm particle size , 160-Å pore size)(Agilent, Cat.#821725-940)
LC-MS system: Thermo Obitrap Exploris 480 (Thermo Fisher Scientific) coupled with an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA)
Protocol materials
ReagentAcetonitrile LCMS qualityJT BakerCatalog #9829-02
Materials
ReagentLCMS grade waterVWR InternationalCatalog #BJLC365-2.5
Materials
ReagentIsopropanol VWR InternationalCatalog #BJ650447-4L
Materials
ReagentPierce™ FlexMix™ Calibration SolutionThermo FisherCatalog #A39239
Step 11
Safety warnings
Wear proper PPE (gloves, safety goggle, and lab coat), and prepare solvents in a chemical fume hood.
Store organic solvents in a flammable storage cabinet when not in use.
Before start
Prepare the following solvents:
Needle wash solvents: Add Amount100 mL isopropanol into Amount900 mL water .
Solvent A: Add Concentration0.1 % volume formic acid into LC-MS grade water.
Solvent B: Add Concentration0.1 % volume formic acid into LC-MS grade acetonitrile.






Proteomics: HPLC and Mass Spectromtery
Proteomics: HPLC and Mass Spectromtery
Thaw peptide samples TemperatureOn ice , and transfer Amount30 µL of each sample to LC autosampler vials (Agilent, Cat.#5182-0567,#5182-0564) or 96-well plate (Bio-Rad, Cat.#HSP9655).
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is performed with a Thermo Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA).



Equipment
Obitrap Exploris 480
NAME
Mass spectrometer
TYPE
Thermo Fisher
BRAND
BRE725532
SKU
https://www.thermofisher.com/order/catalog/product/BRE725532#/BRE725532
LINK

Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems
LINK

Samples were loaded into a temperature controlled autosampler operating at Temperature4 °C . The separation on the UHPLC is achieved by using an Agilent InfinityLab Poroshell 120 EC-C18 (Thikness2.1 mm ID ,Thikness100 mm length ,Thikness1.9 µm particle size , 120-Å pore size) (Agilent, Cat.#695675-902) coupled with an Agilent InfinityLab Poroshell 120 EC-C18 guard column (Thikness2.1 mm ID ,Thikness5 mm length ,Thikness1.9 µm particle size , 160-Å pore size)(Agilent, Cat.#821725-940). The column is operated at Temperature60 °C .

Twenty micrograms Amount20 µg of peptides are loaded onto the column from each sample and separated using a gradient separation with 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) operating at a flow rate of 0.4 ml/min. A 15 minute total acquisition time with a 10 minute linear elution gradient of chromatographic separation is as follows:

ABCD
Step%A%BTime (minute)
19820.0
290101.5
3653511.5
4208012.0
5208013.5
698214.0
798215.0
Table 1. Chromatographic gradient table

Figure 1. Chromatographic gradient diagram
Note
The gradient length depends on the application of interest and the depth of proteome coverage a study is pursuing.


45m
The eluted peptides were ionized via OptaMaxTM NG Electrospray Ion Source operating in positive ion mode with the following source parameters:



AB
Vaporizer temp 250 °C
Ion transfer tube temp300 °C
Positive ion voltage3500 V
Shealth gas50
Aux gas20
Table 2. Source conditions


The mass spectrometer is operated in data independent mode with a duty cycle of 3 survey scans and 45 MS2 scans . The survey scan and MS2 scan parameters are as follows:


Figure 2. Sequencing scheme of the DIA acquisition method
AB
Survey scan obitrap resolution60K
Survey scan MS range380 to 985 m/z
Survey scan AGC target300%
Survey scan maximum ion injection time45 ms
DIA precusor isolation window13.5 m/z
MS2 scan range145 to 1450 m/z
MS2 scan obitrap resolution15K
MS2 scan AGC target1000%
MS2 scan maximum ion injection time22 ms
Table 3. DIA survey scan and MS2 scan parameters


The MS raw data were acquired using Thermo Scientific Xcalibur version 4.3.73


Software
Thermo Fisher Scientific
NAME
Thermo Fisher Scientific
DEVELOPER


The acquired DIA raw data files were analyzed by an integrated software suite DIA-NN v 1.8.1.
CITATION
Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M (2020). DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.. Nature methods.
LINK
https://doi.org/10.1038/s41592-019-0638-x

DIA-NN configurations for Library-free search and peptide quantification:
AB
EnzymeTrypsin
Maximum missed cleavages1
Precusor and Fragment MS accuriciesAutomatically determined
Precusor length range 7-30
Precusor charge range 2-4
Fixed modificationsCarbamidomethyl (Cys)
Variable modificationsDeamination (Asn, Gln); Oxidation (Met)
Precusor and protein identificaiton FDR1%
Quantification strategyRobust LC
Spectral libraryGenerated from fasta files of latest proteomes at Uniprot
Main configurations for DIA-NN search in library-free mode

Note
Note: DIA-NN could also utilize experimentally generated spectral libraries to analyze LCMS raw data, such as DDA based spectral library, gas-phased fractionation (GPF)-DIA based spectral library, etc.

Protein quantity reported by DIA-NN was further processed and visualized using an jupyter notebook described in detail through an established protocol.
Protocol
Label-free quantification (LFQ) proteomic data analysis from DIA-NN output files
NAME
Label-free quantification (LFQ) proteomic data analysis from DIA-NN output files
CREATED BY
Christopher J Petzold

LCMS QC and performance monitoring
LCMS QC and performance monitoring
The Exploris mass spectrometer is subjected to mass calibration check prior to analyzing samples to verify mass accuracy, intensity, and resolution of ions using Pierce™ FlexMix™ Calibration Solution purchased from Thermo Fisher Scientific.
ReagentPierce™ FlexMix™ Calibration SolutionThermo FisherCatalog #A39239
A weekly mass calibration is performed to maintain <3 ppm mass accuracy without correction from internal calibrant.
The mass spectrometer is subjected to a system calibration at least quarterly (and more frequently, if transmission tune fails, or performance issues arise).
UHPLC-Obitrap system performance is monitored at the beginning, middle, and end of large sample sets by running full LC-MS/MS data collection of 20 ug E.coli cell lysate protein tryptic digest. The protein identification, mass accuracy, peak shape, and resolution of peptides are evaluated.








Citations
Step 8
Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.
https://doi.org/10.1038/s41592-019-0638-x