TITLE: Discovery proteomic (DIA) LC-MS/MS data acquisition and analysis
AUTHORS: Yan Chen, Jennifer Gin, Christopher J Petzold

[DESCRIPTION]
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.

[BEFORE_START]
Prepare the following solvents:
Needle wash solvents: Add 100 mLinto 900 mL.
Solvent A: Add 0.1 % volumeinto LC-MS grade water.
Solvent B: Add 0.1 % volumeinto LC-MS grade acetonitrile.







[STEPS]
SECTION: Proteomics: HPLC and Mass Spectromtery
1. Thaw peptide samples on ice , and transfer 30 µLof each sample to LC autosampler vials (Agilent, Cat.#5182-0567,#5182-0564)  or 96-well plate (Bio-Rad, Cat.#HSP9655).
SECTION: Proteomics: HPLC and Mass Spectromtery
2. 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).



 

 

SECTION: Proteomics: HPLC and Mass Spectromtery
3. Samples were loaded into a temperature controlled autosampler operating at 4 °C. The separation on the UHPLC is achieved by using an Agilent InfinityLab Poroshell 120 EC-C18 ( , , , 120-Å pore size) (Agilent, Cat.#695675-902) coupled with an Agilent InfinityLab Poroshell 120 EC-C18 guard column ( , , , 160-Å pore size)(Agilent, Cat.#821725-940). The column is operated at 60 °C. 

SECTION: Proteomics: HPLC and Mass Spectromtery
4. Twenty micrograms 20 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: 

 

     
 


SECTION: Proteomics: HPLC and Mass Spectromtery
5. The eluted peptides were ionized via OptaMaxTM NG Electrospray Ion Source operating in positive ion mode with the following source parameters:



 


SECTION: Proteomics: HPLC and Mass Spectromtery
6. 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: 


  
 


SECTION: Proteomics: HPLC and Mass Spectromtery
7. The MS raw data were acquired using Thermo Scientific Xcalibur version 4.3.73 


 


SECTION: Proteomics: HPLC and Mass Spectromtery
8. The acquired DIA raw data files were analyzed by an integrated software suite DIA-NN v 1.8.1.
 

SECTION: Proteomics: HPLC and Mass Spectromtery
9. DIA-NN configurations for Library-free search and peptide quantification: 
 

 

SECTION: Proteomics: HPLC and Mass Spectromtery
10. Protein quantity reported by DIA-NN was further processed and visualized using an jupyter notebook described in detail through an established protocol.
 

SECTION: LCMS QC and performance monitoring
11. 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.
  
SECTION: LCMS QC and performance monitoring
12. A weekly mass calibration is performed to maintain <3 ppm mass accuracy without correction from internal calibrant. 
SECTION: LCMS QC and performance monitoring
13. The mass spectrometer is subjected to a system calibration at least quarterly (and more frequently, if transmission tune fails, or performance issues arise).
SECTION: LCMS QC and performance monitoring
14. 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.