TITLE: Targeted proteomic LC-MS/MS analysis
AUTHORS: Yan Chen, Jennifer Gin, Christopher Petzold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Proteomics: HPLC and Mass Spectromtery]
Thaw peptide samples, and transfer of each sample to LC autosampler vials (Agilent, Cat.# 5182-0567, 5182-0564) or 96-well plate (Bio-Rad, Cat.# HSP9655).
on ice
30 µl
?. [Proteomics: HPLC and Mass Spectromtery]
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).
?. [Proteomics: HPLC and Mass Spectromtery]
Samples were loaded on the temperature controlled autosampler at. The Agilent 1290 Infinity II UHPLC is equipped with a Sigma–Aldrich Ascentis Peptides ES-C18  analytical column (,,, and 160-Å pore size (Sigma-Aldrich, Cat.# 53301-U)) coupled to a,guard column (Sigma-Aldrich, Cat.# 53536-U) with the same particle and pore size. The column is operated at .
4 °C
60 °C
?. [Proteomics: HPLC and Mass Spectromtery]
Twenty micrograms  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:   ABCD1Step%A%BTime (min)219820.00329820.504365355.505420806.256520808.00769828.508798210.00
20 µg
ABCD1Step%A%BTime (min)219820.00329820.504365355.505420806.256520808.00769828.508798210.00
The gradient length depends on the application of interest and the number of target peptides for detection.
?. [Proteomics: HPLC and Mass Spectromtery]
The eluted peptides were ionized via an Agilent Jet Stream ESI source operating in positive ion mode with the following source parameters:  AB1Gas temperature250 °C2Gas flow13 liters/min3Nebulizer pressure35 psi4Sheath gas temperature250 °C5Sheath gas flow11 liters/min6Capillary voltage3500 V7Nozzle voltage0 V
AB1Gas temperature250 °C2Gas flow13 liters/min3Nebulizer pressure35 psi4Sheath gas temperature250 °C5Sheath gas flow11 liters/min6Capillary voltage3500 V7Nozzle voltage0 V
?. [Proteomics: HPLC and Mass Spectromtery]
The MS raw data were acquired using Agilent MassHunter version B.08.02
?. [Proteomics: HPLC and Mass Spectromtery]
SRM transitions were monitored through either scanning or scheduled MRM mode with the following criteria:Minimum dwell time of a transition is 2 msMaximum number of concurrent transitions are 200Total cycle time less than 1.5 sec.
?. [Proteomics: HPLC and Mass Spectromtery]
Acquired SRM data were imported and analyzed by Skyline software version 20.1 (MacCoss Lab Software).
?. [Proteomics: HPLC and Mass Spectromtery]
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).
?. [Proteomics: HPLC and Mass Spectromtery]
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.
?. [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.
?. [UHPLC-QQQ performance monitoring and QC]
Agilent 6460 QQQ mass spectrometer is subjected to a check tune prior to analyzing a batch of samples for resolution and mass accuracy verification.
?. [UHPLC-QQQ performance monitoring and QC]
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.
?. [UHPLC-QQQ performance monitoring and QC]
The performance tracking is automated and monitored via PanoramaWeb server through an established AutoQC pipeline.