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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details steps in discovery proteomic data-dependent acquisition with a standard-flow UHPLC-Obitrap 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."</span><span style = "font-style:italic;">Frontiers in bioengineering and biotechnology, </span><span>3 (2015): 44.</span></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 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).
?. [Proteomics: HPLC and Mass Spectromtery]
Samples were loaded into a temperature controlled autosampler operating at . 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 .
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  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:   ABCD1Step%A%BTime (minute)219820.03298101.543653511.554208012.065208013.57698214.08798215.0
20 µg
ABCD1Step%A%BTime (minute)219820.03298101.543653511.554208012.065208013.57698214.08798215.0
The gradient length depends on the application of interest and the depth of proteome coverage a study is pursuing.
?. [Proteomics: HPLC and Mass Spectromtery]
The eluted peptides were ionized via OptaMaxTM NG Electrospray Ion Source operating in positive ion mode with the following source parameters:Vaporizer temp 250 °CIon transfer tube temp300 °CPositive ion voltage3500 VShealth gas50Aux gas20
Vaporizer temp 250 °CIon transfer tube temp300 °CPositive ion voltage3500 VShealth gas50Aux gas20
?. [Proteomics: HPLC and Mass Spectromtery]
The mass spectrometer is operated in data dependent mode with cycles of a survey scan followed by 10 ddMS2 events. The survey scan and ddMS2 scan parameters are as follows:   AB1Survey scan obitrap resolution60K2Survey scan MS range300 to 1,200 m/z3Survey scan AGC target300%4Survey scan maximum ion injection time60 ms5Ion intensity threshold triggering MS2 analysis5.0e36Isolation window1.6 m/z7ddMS2 scan obitrap resolution15K8ddMS2 scan AGC target100%9ddMS2 scan maximum ion injection time50s
AB1Survey scan obitrap resolution60K2Survey scan MS range300 to 1,200 m/z3Survey scan AGC target300%4Survey scan maximum ion injection time60 ms5Ion intensity threshold triggering MS2 analysis5.0e36Isolation window1.6 m/z7ddMS2 scan obitrap resolution15K8ddMS2 scan AGC target100%9ddMS2 scan maximum ion injection time50s
?. [Proteomics: HPLC and Mass Spectromtery]
The MS raw data were acquired using Thermo Scientific Xcalibur version 4.3.73
?. [Proteomics: HPLC and Mass Spectromtery]
The acquired raw data were converted to .mgf files using RawConverter tool and searched against the protein database with Mascot search engine version 2.3.02 (Matrix Science).
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.
?. [Proteomics: HPLC and Mass Spectromtery]
The following Mascot search parameters are applied:   AB1EnzymeTrypsin2Maximum missed cleavages13Precursor ion tolerance20 ppm4Fragment ion tolerance0.1 Da5Fixed modificationsCarbamidomethyl (Cys)6Variable modificationsDeamination (Asn, Gln); Oxidation (Met)
AB1EnzymeTrypsin2Maximum missed cleavages13Precursor ion tolerance20 ppm4Fragment ion tolerance0.1 Da5Fixed modificationsCarbamidomethyl (Cys)6Variable modificationsDeamination (Asn, Gln); Oxidation (Met)
?. [Proteomics: HPLC and Mass Spectromtery]
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.
?. [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.
?. [LCMS QC and performance monitoring]
A weekly mass calibration is performed to maintain
?. [LCMS QC and performance monitoring]
The mass spectrometer is subjected to a system calibration at least quarterly (and more frequently, if transmission tune fails, or performance issues arise).
?. [LCMS QC and performance monitoring]
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.