Jun 08, 2020

Public workspaceTargeted proteomic LC-MS/MS analysis

DOI
dx.doi.org/10.17504/protocols.io.bf9xjr7n
  • 1Lawrence Berkeley National Laboratory
  • LBNL omics
Yan Chen
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DOI: dx.doi.org/10.17504/protocols.io.bf9xjr7n
External link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211582
Protocol Citation: Yan Chen, Jennifer Gin, Christopher J Petzold 2020. Targeted proteomic LC-MS/MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bf9xjr7n
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 11, 2020
Last Modified: June 08, 2020
Protocol Integer ID: 36887
Keywords: LC-MS, Targeted proteomics, MRM
Abstract
This protocol details steps in targeted proteomic data acquisition with a standard-flow UHPLC-QQQ system. It was adapted from Chen, Y. et al. “A rapid methods development workflow for high-throughput quantitative proteomic applications.” PloS ONE 14,2 e0211582. 14 Feb. 2019, doi:10.1371/journal.pone.0211582.
Materials
MATERIALS
ReagentAcetonitrile LCMS qualityJT BakerCatalog #9829-02
ReagentPierce™ Formic AcidThermo FisherCatalog #TS-28905
ReagentLCMS grade waterVWR International (Avantor)Catalog #BJLC365-2.5
ReagentIsopropanol VWR International (Avantor)Catalog #BJ650447-4L
STEP MATERIALS
Thikness2.7 µm
Analytical column: Ascentis Express Peptide C18 column (Thikness2.7 µm particle size, 160-Å pore size, 5-cm length × Thikness2.1 mm internal diameter).
Guard column: Ascentis guard column (5-mm × Thikness2.1 mm ID with Thikness2.7 µm particle size, 160-Å pore size)
LC-MS system: Agilent 6460 QQQ mass spectrometer system coupled with an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA)

Protocol materials
ReagentAcetonitrile LCMS qualityJT BakerCatalog #9829-02
ReagentPierce™ Formic AcidThermo FisherCatalog #TS-28905
ReagentLCMS grade waterVWR International (Avantor)Catalog #BJLC365-2.5
ReagentIsopropanol VWR International (Avantor)Catalog #BJ650447-4L
ReagentAgilent Tune Mix: G1969-85000Catalog #G1969-85000
Safety warnings
Wear proper PPE (gloves, safety goggle, and lab coat), and prepare solvents in a chemical hood.
Store organic solvents in a flammable storage cabinet.
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 samplesTemperatureOn 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 an Agilent 6460 QQQ mass spectrometer system coupled to an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA).
Equipment
6460QQQ
NAME
Triple quadrupole mass spectrometer
TYPE
Agilent Technologies
BRAND
6460QQQ
SKU
https://www.agilent.com/en/products/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-instruments/triple-quadrupole-lc-ms
LINK

Equipment
1290 UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290UHPLC
SKU
www.agilent.com
LINK


Samples were loaded on the temperature controlled autosampler atTemperature4 °C . The Agilent 1290 Infinity II UHPLC is equipped with a Sigma–Aldrich Ascentis Peptides ES-C18 analytical column (Thikness2.1 mm ID ,Thikness50 mm length ,Thikness2.7 µm particle size , and 160-Å pore size (Sigma-Aldrich, Cat.# 53301-U)) coupled to aThikness2.1 mm ID ,Thikness5 mm length guard column (Sigma-Aldrich, Cat.# 53536-U) with the same particle and pore size. The column is operated at Temperature60 °C .

Twenty micrograms Amount20 µg L of peptides are loaded onto the column from each sample and separated using a linear mobile-phase gradient consisting of 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 10 minutes gradient of chromatographic separation is as follows:
Step%A%BTime (min)
19820.00
29820.50
365355.50
420806.25
520808.00
69828.50
798210.00
Chromatographic gradient table
Chromatographic gradient
Note
The gradient length depends on the application of interest and the number of target peptides for detection.


The eluted peptides were ionized via an Agilent Jet Stream ESI source operating in positive ion mode with the following source parameters:

Gas temperature250 °C
Gas flow13 liters/min
Nebulizer pressure35 psi
Sheath gas temperature250 °C
Sheath gas flow11 liters/min
Capillary voltage3500 V
Nozzle voltage0 V


SRM transitions were monitored through either scanning or scheduled MRM mode with the following criteria:
  • Minimum dwell time of a transition is 2 ms
  • Maximum number of concurrent transitions are 200
  • Total cycle time less than 1.5 sec.

The MS raw data were acquired using Agilent MassHunter version B.08.02
Software
MassHunter
NAME
Agilent Technologies, Inc
DEVELOPER

Acquired SRM data were imported and analyzed by Skyline software version 20.1 (MacCoss Lab Software).

Software
Skyline
NAME
University of Washington
DEVELOPER

For a small sample set, peak integration is visually checked and validated by browsing replicate peak area and retention time tabs. For a large set of more than 100 samples, peak integration is completed by using skyline advanced peak picking models (e.g., mProphet).
CITATION
Reiter L, Rinner O, Picotti P, Hüttenhain R, Beck M, Brusniak MY, Hengartner MO, Aebersold R (2011). mProphet: automated data processing and statistical validation for large-scale SRM experiments.. Nature methods.
LINK
https://doi.org/10.1038/nmeth.1584

A peptide quantitative report was exported to a .csv file for further analysis and sharing. The skyline file with the data and SRM methods information was uploaded to a project folder on the PanoramaWeb server. The project information (data and SRM methods) is made public when a manuscript is accepted for journal publication.
CITATION
Sharma V, Eckels J, Taylor GK, Shulman NJ, Stergachis AB, Joyner SA, Yan P, Whiteaker JR, Halusa GN, Schilling B, Gibson BW, Colangelo CM, Paulovich AG, Carr SA, Jaffe JD, MacCoss MJ, MacLean B (2014). Panorama: a targeted proteomics knowledge base.. Journal of proteome research.
LINK
https://doi.org/10.1021/pr5006636

CITATION
Sharma V, Eckels J, Schilling B, Ludwig C, Jaffe JD, MacCoss MJ, MacLean B (2018). Panorama Public: A Public Repository for Quantitative Data Sets Processed in Skyline.. Molecular & cellular proteomics : MCP.
LINK
https://doi.org/10.1074/mcp.RA117.000543

UHPLC-QQQ performance monitoring and QC
UHPLC-QQQ performance monitoring and QC
The mass spectrometer is subjected to a full autotune at least quarterly (and more frequently, if necessary) to optimize ion transmission and update EM voltage.

ReagentAgilent Tune Mix: G1969-85000Catalog #G1969-85000

Agilent 6460 QQQ mass spectrometer is subjected to a check tune prior to analyzing a batch of samples for resolution and mass accuracy verification.
The UHPLC-QQQ performance is tracked daily by injecting 50 fmol BSA tryptic digest standard and monitoring eight BSA peptides using a scheduled MRM assay. Peptide retention time, peak shape, and peak intensity of these peptides are monitored to determine if maintenance is required.

The performance tracking is automated and monitored via PanoramaWeb server through an established AutoQC pipeline.


An example of autoQC instrument performance track on the peak area of eight BSA tryptic peptide measurements

Software
AutoQC loader
NAME
University of Washington
DEVELOPER

CITATION
Bereman MS, Beri J, Sharma V, Nathe C, Eckels J, MacLean B, MacCoss MJ (2016). An Automated Pipeline to Monitor System Performance in Liquid Chromatography-Tandem Mass Spectrometry Proteomic Experiments. Journal of proteome research.
LINK
https://doi.org/10.1021/acs.jproteome.6b00744


Citations
Step 10
Sharma V, Eckels J, Taylor GK, Shulman NJ, Stergachis AB, Joyner SA, Yan P, Whiteaker JR, Halusa GN, Schilling B, Gibson BW, Colangelo CM, Paulovich AG, Carr SA, Jaffe JD, MacCoss MJ, MacLean B. Panorama: a targeted proteomics knowledge base.
https://doi.org/10.1021/pr5006636
Step 10
Sharma V, Eckels J, Schilling B, Ludwig C, Jaffe JD, MacCoss MJ, MacLean B. Panorama Public: A Public Repository for Quantitative Data Sets Processed in Skyline.
https://doi.org/10.1074/mcp.RA117.000543
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
https://doi.org/10.1021/acs.jproteome.6b00744
Step 9
Reiter L, Rinner O, Picotti P, Hüttenhain R, Beck M, Brusniak MY, Hengartner MO, Aebersold R. mProphet: automated data processing and statistical validation for large-scale SRM experiments.
https://doi.org/10.1038/nmeth.1584