Jun 04, 2020
Version 1
Discovery proteomic (DDA) LC-MS/MS data acquisition and analysis V.1
- 1Lawrence Berkeley National Laboratory
- LBNL omics
External link: https://doi.org/10.3389/fbioe.2015.00044
Protocol Citation: Yan Chen, Jennifer Gin, Christopher J Petzold 2020. Discovery proteomic (DDA) LC-MS/MS data acquisition and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bgbqjsmw
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: May 13, 2020
Last Modified: June 04, 2020
Protocol Integer ID: 36944
Abstract
This protocol details steps in discovery proteomic data-dependent acquisition with a standard-flow UHPLC-QTOF system and a subsequent Mascot database search. It 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
Acetonitrile LCMS qualityJT BakerCatalog #9829-02
LCMS grade waterVWR International (Avantor)Catalog #BJLC365-2.5
Isopropanol VWR International (Avantor)Catalog #BJ650447-4L
STEP MATERIALS
2.1 mm ID
Analytical column: Agilent AdvanceBio Peptide Map column (2.1 mm ID ,250 mm length ,2.7 µm particle size , 120-Å pore size) (Agilent, Cat.#651750-902)
Guard column: Ascentis guard column (2.1 mm ID ,50 mm length ,2.7 µm particle size , 160-Å pore size)(Sigma-Aldrich, Cat.#53536-U)
LC-MS system: Agilent 6550 QTOF mass spectrometer system coupled with an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA)
Protocol materials
Acetonitrile LCMS qualityJT BakerCatalog #9829-02
LCMS grade waterVWR International (Avantor)Catalog #BJLC365-2.5
Isopropanol VWR International (Avantor)Catalog #BJ650447-4L
Agilent Tune Mix: G1969-85000Catalog #G1969-85000
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 100 mL isopropanol into 900 mL water .
Solvent A: Add 0.1 % volume formic acid into LC-MS grade water.
Solvent B: Add 0.1 % volume formic acid into LC-MS grade acetonitrile.
Proteomics: HPLC and Mass Spectromtery
Proteomics: HPLC and Mass Spectromtery
Thaw peptide samples On ice , and transfer 30 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 an Agilent 6550 QTOF mass spectrometer system coupled with an Agilent 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA).
Equipment
6550 iFunnel Q-TOF
NAME
Quadruple-time-of-flight (TOF) tandem Mass Spectrometer
TYPE
Agilent Technologies
BRAND
6550 QTOF
SKU
LINK
Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
LINK
Samples were loaded into a temperature controlled autosampler operating at 4 °C . The separation on the UHPLC is achieved by using an Agilent AdvanceBio Peptide Map column (2.1 mm ID ,250 mm length ,2.7 µm particle size , 120-Å pore size (Agilent, Cat.#651750-902)) coupled to a guard column (2.1 mm ID ,50 mm length ,2.7 µm particle size , and 160-Å pore size (Sigma-Aldrich, Cat.#53536-U)). The column is operated at 60 °C .
Twenty micrograms 20 µg of peptides are loaded onto the column from each sample and separated using a gradient separaration 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 30 minutes linear elution gradient of chromatographic separation is as follows:
| Step | %A | %B | Time (minute) | |
| 1 | 98 | 2 | 0.00 | |
| 2 | 98 | 2 | 1.00 | |
| 3 | 65 | 35 | 31.00 | |
| 4 | 20 | 80 | 33.00 | |
| 5 | 20 | 80 | 39.00 | |
| 6 | 98 | 2 | 41.00 | |
| 7 | 98 | 2 | 45.00 |
Chromatographic gradient table
Chromatographic gradient graph
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 an Agilent Jet Stream ESI source operating in positive ion mode with the following source parameters:
| Gas temperature | 250 °C | |
| Drying gas flow | 14 liters/min | |
| Nebulizer pressure | 35 psi | |
| Sheath gas temparature | 250 °C | |
| Sheath gas flow | 11 liters/min | |
| Capillary voltage | 3500 V | |
| Fragmentor voltage | 180 V | |
| OCT 1 RF Vpp | 750 V |
The mass spectrometer is operated in data dependent acquisition (DDA) auto MS/MS mode with the following parameters:
| Charge states | 2 to 5 | |
| Precursor scan MS range | 300 to 1,400 m/z | |
| Maximum precursor ions allowed for MS2 event | 20 | |
| Fragment ion scan MS range | 100 to 1,700 m/z | |
| Ion intensity threshold triggering MS2 analysis | 1500 counts | |
| Quadrupole resolution | Medium | |
| Precursor ion accumulation | 45,000 counts | |
| Maximum MS2 accumulation time | 333 ms | |
| Precursor ion exclusion window | 0.3 minutes |
DDA parameters
The MS raw data were acquired using Agilent MassHunter version B.06.01
Software
MassHunter
NAME
Agilent Technologies, Inc
DEVELOPER
The acquired data were explored in Agilent Qualitative Analysis software version B.06.01. MS data were converted to .mgf files using inbuilt MGF file export and searched against the protein database with Mascot search engine version 2.3.02 (Matrix Science).
Software
Qualitative Analysis
NAME
Agilent Technologies, Inc
DEVELOPER
Software
Mascot Server
NAME
Matrix Science
DEVELOPER
Note
The latest protein database of interest was downloaded from the Universal Protein Resource database (https://www.uniprot.org/). Heterologous proteins of interest and common contaminant protein fasta sequences were subsequently added to the downloaded protein database, which then was used in the Mascot search.
The following Mascot search parameters are applied:
| Enzyme | Trypsin | |
| Maximum missed cleavages | 1 | |
| Precursor ion tolerance | 50 ppm | |
| Fragment ion tolerance | 0.1 Da | |
| Fixed modifications | Carbamidomethyl (Cys) | |
| Variable modifications | Deamination (Asn, Gln); Oxidation (Met) |
Mascot search parameters
Mascot search results are refined by using Scaffold. Identified peptides are filtered by a 1% peptide-level false discovery rate. In addition, the false discovery rate at the protein level is calculated, and only the proteins with false discovery rate ≤1% are reported.
Software
Scaffold
NAME
Proteome Software, Inc
DEVELOPER
QTOF QC and performance monitoring
QTOF QC and performance monitoring
The QTOF mass spectrometer is subjected to TOF mass calibration (Check Tune) prior to analyzing samples to verify mass accuracy, intensity, and resolution of ions in 10 times diluted ESI low concentration tune mix purchased from Agilent.
Agilent Tune Mix: G1969-85000Catalog #G1969-85000
A weekly TOF Quick Tune is performed to optimize ion transmission.
The mass spectrometer is subjected to a Standard Tune at least quarterly (and more frequently, if transmission tune fails, or performance issues arise).
UHPLC-QTOF system performance is monitored at the beginning, middle, and end of large sample sets by running full LC-MS/MS data collection of BSA tryptic digest standard (100 fmol). The HPLC retention times, mass accuracy, ion intensity, and resolution of BSA peptides are tracked via the PanoramaWeb server through an established AutoQC pipeline.
Software
AutoQC loader
NAME
University of Washington
DEVELOPER
CITATION
Citations
Step 14
Bereman MS, Beri J, Sharma V, Nathe C, Eckels J, MacLean B, MacCoss MJ. An Automated Pipeline to Monitor System Performance in Liquid Chromatography-Tandem Mass Spectrometry Proteomic Experiments.