In cpanse/tartare: Raw ground spectra recorded on Thermo Fisher Scientific mass spectrometers
library(BiocStyle)
Sample description
Pierce HeLa protein digest standard (#1862824, Thermo Scientific) in 3% ACN,
0.1% FA
Data recording
Q Exactive HF-X
file name: 20190710_003_PierceHeLaProteinDigestStd.raw
LC
200 ng sample were seperated on a linear gradient from 5 to 35% B in 20 min
using a binary mobile phase composed of A) 0.1% FA in water, B) 0.1% FA in ACN. The stationary phase was ...
MS
The MS acquision was started 18 min post sample injection and lastet only 4 min. Be aware that spray
voltage and source gas was turned on at 18.5 min (see Tunefiles section of the method).
This means the first 30 s were recorded
without ESI. In summary, 4 different experiments were recorded
- Full MS / dd-MS² (TopN)
- PRM
- Targeted-SIM
- DIA
Experiment 2 and 3 were executed in parrallel! All method details are listed below.
Method of Q Exactive HF-X
Overall method settings
Global Settings
Use lock masses best
Lock mass injection ―
Chrom. peak width (FWHM) 15 s
Time
Method duration 4.00 min
Customized Tolerances (+/-)
Lock Masses ―
Inclusion ―
Exclusion ―
Neutral Loss ―
Mass Tags ―
Dynamic Exclusion ―
Experiments
Full MS / dd-MS² (TopN)
General
Runtime 0 to 1 min
Polarity positive
In-source CID 0.0 eV
Default charge state 2
Inclusion ―
Exclusion ―
Tags ―
Full MS
Microscans 1
Resolution 120,000
AGC target 3e6
Maximum IT 100 ms
Number of scan ranges 1
Scan range 200 to 2000 m/z
Spectrum data type Centroid
dd-MS² / dd-SIM
Microscans 1
Resolution 30,000
AGC target 1e5
Maximum IT 50 ms
Loop count 5
MSX count 1
TopN 5
Isolation window 1.4 m/z
Isolation offset 0.0 m/z
Scan range 200 to 2000 m/z
Fixed first mass ―
(N)CE / stepped (N)CE nce: 30
Spectrum data type Centroid
dd Settings
Minimum AGC target 8.00e3
Intensity threshold 1.6e5
Apex trigger ―
Charge exclusion unassigned, 1, 5 - 8, >8
Multiple charge states all
Peptide match preferred
Exclude isotopes on
Dynamic exclusion 10.0 s
If idle .. do not pick others
PRM
General
Runtime 1 to 2 min
Polarity positive
In-source CID 0.0 eV
Dynamic RT off
Default charge state 2
Inclusion on
MS²
Microscans 1
Resolution 30,000
AGC target 2e5
Maximum IT 100 ms
Loop count 11
MSX count 1
MSX isochronous ITs on
Isolation window 4.0 m/z
Isolation offset 0.0 m/z
Fixed first mass ―
(N)CE / stepped (N)CE nce: 35
Spectrum data type Centroid
Full MS — SIM
General
Runtime 1 to 2 min
Polarity positive
In-source CID 0.0 eV
Full MS — SIM
Microscans 1
Resolution 120,000
AGC target 3e6
Maximum IT 200 ms
Number of scan ranges 2
Scan range 1 150 to 1000 m/z
Scan range 2 1000 to 2000 m/z
Spectrum data type Centroid
Targeted-SIM
General
Runtime 2 to 3 min
Polarity positive
In-source CID 0.0 eV
Inclusion on
SIM
Microscans 1
Resolution 240,000
AGC target 5e4
Maximum IT 200 ms
MSX count 5
Isolation window 4.0 m/z
Isolation offset 0.0 m/z
Scan range 150 to 2000 m/z
Spectrum data type Centroid
DIA
General
Runtime 3 to 4 min
Polarity positive
In-source CID 0.0 eV
Default charge state 2
DIA
Microscans 1
Resolution 30,000
AGC target 1e6
Maximum IT auto
Loop count 11
MSX count 1
MSX isochronous ITs on
Isolation window 20.0 m/z
Isolation offset 0.0 m/z
Fixed first mass 100.0 m/z
(N)CE / stepped (N)CE nce: 30
Spectrum data type Centroid
Setup
Tunefiles
General
Switch Count 2
Base Tunefile C:\Xcalibur\methods\nogas_novoltage_nano.mstune
Element 1
At 0.50 min
New Tunefile C:\Xcalibur\methods\default_nano.mstune
Element 2
At 34.50 min
New Tunefile C:\Xcalibur\methods\nogas_novoltage_nano.mstune
Contact Closure
General
Used True
Start in Closed False
Switch Count 2
Element 1
At 0.50 min
Switches to Open
Element 2
At 34.50 min
Switches to Closed
Syringe
General
Used False
Start in OFF True
Stop at end of run False
Switch Count 0
Pump setup
Syringe type Hamilton
Flow rate 3.000 µL/min
Inner diameter 2.303 mm
Volume 250 µL
Divert Valve A
General
Used False
Start in 1-2 True
Switch Count 0
Divert Valve B
General
Used False
Start in 1-2 True
Switch Count 0
Lock Masses
2 entries
Mass Polarity Start End Comment
[m/z] [min] [min]
371.10124 Positive
445.12002 Positive
Inclusion List
16 entries
Mass Formula Species CS Polarity Start End (N)CE MSX ID Comment
[m/z] [M] [z] [min] [min]
487.25671 2 Positive 1.00 3.00 28 LGGNEQVTR
(light)
644.82261 2 Positive 1.00 3.00 28 GAGSSEPVTGLDAK
(light)
683.82789 2 Positive 1.00 3.00 28 VEATFGVDESNAK
(light)
547.29804 2 Positive 1.00 3.00 28 YILAGVENSK
(light)
669.83806 2 Positive 1.00 3.00 28 TPVISGGPYEYR
(light)
683.85371 2 Positive 1.00 3.00 28 TPVITGAPYEYR
(light)
699.33842 2 Positive 1.00 3.00 28 DGLDAASYYAPVR
(light)
726.83571 2 Positive 1.00 3.00 28 ADVTPADFSEWSK
(light)
622.85351 2 Positive 1.00 3.00 28 GTFIIDPGGVIR
(light)
636.86916 2 Positive 1.00 3.00 28 GTFIIDPAAVIR
(light)
776.92975 2 Positive 1.00 3.00 28 LFLQFGAQGSPFLK
(light)
410.00000 2 Positive 3.00 4.00 28 window1
430.00000 2 Positive 3.00 4.00 28 window2
450.00000 2 Positive 3.00 4.00 28 window3
470.00000 2 Positive 3.00 4.00 28 window4
490.00000 2 Positive 3.00 4.00 28 window5
Exclusion List
(no entries)
Neutral Losses
(no entries)
Mass Tags
(no entries)
Fusion Lumos
file name: 20190716_004_PierceHeLa.raw
LC
200 ng sample were seperated on a linear gradient from 5 to 35% B in 20 min
using a binary mobile phase composed of A) 0.1% FA in water, B) 0.1% FA in ACN. The stationary phase was ...
MS
The MS acquision was started directly after sample injection and lastet for 35 min.
In summary, 5 different experiments were recorded:
- Exp. 1 Orbitrap HCD - High Load (greater than 500 ng)
- Exp. 2 ETD Data-Dependent Decision Tree
- Exp. 3 Glycopeptide - Enriched
- Exp. 4 TMT Quan Pierce TKO Standard QC MS3 SPS
- Exp. 5 MS
Exp. #5 (MS) was executed the entire method duration! Method details are listed below.
Method Summary
Method Settings
Application Mode: Peptide
Method Duration (min): 35
Global Parameters
Ion Source
Use Ion Source Settings from Tune: True
FAIMS Mode: Not Installed
MS Global Settings
Advanced Peak Determination: False
Default Charge State: 1
Internal Mass Calibration: Off
Experiment#1 [Orbitrap HCD - High Load (greater than 500 ng)]
Start Time (min): 20
End Time (min): 21
Cycle Time (sec): 3
Master Scan:
MS OT
Detector Type: Orbitrap
Orbitrap Resolution: 60000
Mass Range: Normal
Use Quadrupole Isolation: True
Scan Range (m/z): 375-1500
RF Lens (%): 30
AGC Target: 4.0e5
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Polarity: Positive
Source Fragmentation: Disabled
Scan Description:
Filters:
MIPS
Monoisotopic Peak Determination: Peptide
Charge State
Include charge state(s): 2-7
Include undetermined charge states: False
Include charge states 25 and higher: False
Dynamic Exclusion
Exclude after n times: 1
Exclusion duration (s): 60
Mass Tolerance: ppm
Low: 10
High: 10
Exclude Isotopes: True
Perform dependent scan on single charge state per precursor only: False
Intensity
Filter Type: Intensity Threshold
Intensity Threshold: 5.0e4
Data Dependent
Data Dependent Mode: Cycle Time
Time between Master Scans (sec): 3
Scan Event Type 1:
Scan:
ddMS² OT HCD
Isolation Mode: Quadrupole
Isolation Window (m/z): 1.6
Isolation Offset: Off
Activation Type: HCD
Collision Energy Mode: Fixed
HCD Collision Energy (%): 30
Detector Type: Orbitrap
Scan Range Mode: Auto: m/z Normal
Orbitrap Resolution: 15000
First Mass (m/z): 110
AGC Target: 5.0e4
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 30
Microscans: 1
Data Type: Centroid
Use EASY-IC™: False
Scan Description:
Experiment#2 [ETD Data-Dependent Decision Tree]
Start Time (min): 21
End Time (min): 22
Cycle Time (sec): 3
Master Scan:
MS OT
Detector Type: Orbitrap
Orbitrap Resolution: 120000
Mass Range: Normal
Use Quadrupole Isolation: True
Scan Range (m/z): 350-1500
RF Lens (%): 30
AGC Target: 4.0e5
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Polarity: Positive
Source Fragmentation: Disabled
Scan Description:
Filters:
MIPS
Monoisotopic Peak Determination: Peptide
Dynamic Exclusion
Exclude after n times: 1
Exclusion duration (s): 60
Mass Tolerance: ppm
Low: 10
High: 10
Exclude Isotopes: True
Perform dependent scan on single charge state per precursor only: False
Exclude Within Cycle: False
Intensity
Filter Type: Intensity Threshold
Intensity Threshold: 1.0e4
Data Dependent
Data Dependent Mode: Cycle Time
Time between Master Scans (sec): 3
Scan Event Type 1:
Charge State
Include charge state(s): 3
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 350-650
OR
Charge State
Include charge state(s): 4
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 350-900
OR
Charge State
Include charge state(s): 5
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 350-950
OR
Charge State
Include charge state(s): 6-8
Include undetermined charge states: False
Include charge states 25 and higher: False
Sort by Charge
Precursor Priority: Highest Charge State
Sort by m/z
Precursor Priority: Lowest m/z
Scan:
ddMS² IT ETD
Scan Priority: 1
Isolation Mode: Quadrupole
Isolation Window (m/z): 2
Isolation Offset: Off
Activation Type: ETD
Use Calibrated Charge-Dependent ETD Parameters: True
ETD Supplemental Activation: False
Detector Type: Ion Trap
Scan Range Mode: Auto: m/z Normal
Ion Trap Scan Rate: Rapid
First Mass (m/z): 120
AGC Target: 1.0e4
Inject Ions for All Available Parallelizable Time: True
Maximum Injection Time (ms): 100
Microscans: 1
Data Type: Centroid
Scan Description:
Scan Event Type 2:
Charge State
Include charge state(s): 2
Include undetermined charge states: False
Include charge states 25 and higher: False
OR
Charge State
Include charge state(s): 3
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 650-1200
OR
Charge State
Include charge state(s): 4
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 900-1200
OR
Charge State
Include charge state(s): 5
Include undetermined charge states: False
Include charge states 25 and higher: False
Precursor Selection Range
Mass Range (m/z): 950-1200
Scan:
ddMS² IT CID
Scan Priority: 1
Isolation Mode: Quadrupole
Isolation Window (m/z): 2
Isolation Offset: Off
Activation Type: CID
Collision Energy Mode: Fixed
CID Collision Energy (%): 30
CID Activation Time (ms): 10
Activation Q: 0.25
Multistage Activation: False
Detector Type: Ion Trap
Scan Range Mode: Auto: m/z Normal
Ion Trap Scan Rate: Rapid
AGC Target: 1.0e4
Inject Ions for All Available Parallelizable Time: True
Maximum Injection Time (ms): 35
Microscans: 1
Data Type: Centroid
Scan Description:
Experiment#3 [Glycopeptide - Enriched]
Start Time (min): 22
End Time (min): 23
Cycle Time (sec): 3
Master Scan:
MS OT
Detector Type: Orbitrap
Orbitrap Resolution: 120000
Mass Range: Normal
Use Quadrupole Isolation: True
Scan Range (m/z): 350-1800
RF Lens (%): 30
AGC Target: 4.0e5
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Polarity: Positive
Source Fragmentation: Disabled
Scan Description:
Filters:
MIPS
Monoisotopic Peak Determination: Peptide
Charge State
Include charge state(s): 2-8
Include undetermined charge states: False
Include charge states 25 and higher: False
Dynamic Exclusion
Exclude after n times: 1
Exclusion duration (s): 60
Mass Tolerance: ppm
Low: 10
High: 10
Exclude Isotopes: True
Perform dependent scan on single charge state per precursor only: False
Intensity
Filter Type: Intensity Threshold
Intensity Threshold: 5.0e4
Data Dependent
Data Dependent Mode: Cycle Time
Time between Master Scans (sec): 3
Scan Event Type 1:
Sort by Charge
Precursor Priority: Highest Charge State
Sort by m/z
Precursor Priority: Lowest m/z
Scan:
ddMS² OT EThcD
Isolation Mode: Quadrupole
Isolation Window (m/z): 3
Isolation Offset: Off
Activation Type: ETD
Use Calibrated Charge-Dependent ETD Parameters: True
ETD Supplemental Activation: True
SA Collision Energy Type: EThcD
SA Collision Energy (%): 15
Detector Type: Orbitrap
Scan Range Mode: Auto: m/z Normal
Orbitrap Resolution: 60000
First Mass (m/z): 120
AGC Target: 5.0e4
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 118
Microscans: 1
Data Type: Centroid
Use EASY-IC™: False
Scan Description:
Experiment#4 [TMT Quan Pierce TKO Standard QC MS3 SPS]
Start Time (min): 23
End Time (min): 24
Cycle Time (sec): 2
Master Scan:
MS OT
Detector Type: Orbitrap
Orbitrap Resolution: 120000
Mass Range: Normal
Use Quadrupole Isolation: False
Scan Range (m/z): 375-1500
RF Lens (%): 30
AGC Target: 4.0e5
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Polarity: Positive
Source Fragmentation: Disabled
Scan Description:
Filters:
MIPS
Monoisotopic Peak Determination: Peptide
Charge State
Include charge state(s): 2-6
Include undetermined charge states: False
Include charge states 25 and higher: False
Dynamic Exclusion
Exclude after n times: 1
Exclusion duration (s): 60
Mass Tolerance: ppm
Low: 10
High: 10
Exclude Isotopes: True
Perform dependent scan on single charge state per precursor only: True
Exclude Within Cycle: True
Intensity
Filter Type: Intensity Threshold
Intensity Threshold: 5.0e3
Data Dependent
Data Dependent Mode: Cycle Time
Time between Master Scans (sec): 2
Scan Event Type 1:
Charge State
Include charge state(s): 2
Include undetermined charge states: False
Include charge states 25 and higher: False
Scan:
ddMS² IT CID
Scan Priority: 1
Isolation Mode: Quadrupole
Isolation Window (m/z): 1.2
Isolation Offset: Off
Activation Type: CID
Collision Energy Mode: Fixed
CID Collision Energy (%): 35
CID Activation Time (ms): 10
Activation Q: 0.25
Multistage Activation: False
Detector Type: Ion Trap
Scan Range Mode: Auto: m/z Normal
Ion Trap Scan Rate: Turbo
AGC Target: 1.0e4
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Scan Description:
Filters:
Precursor Selection Range
Selection Range Mode: Mass Range
Mass Range (m/z): 400-1200
Isobaric Tag Loss Exclusion
Reagent: TMT
Precursor Ion Exclusion
Exclusion mass width: m/z
Low: 50
High: 5
Data Dependent
Data Dependent Mode: Scans Per Outcome
Scan Event Type 1:
Scan:
ddMS³ OT HCD
MSⁿ Level: 3
Synchronous Precursor Selection: True
Number of SPS Precursors: 5
MS Isolation Window (m/z): 1.3
MS2 Isolation Window (m/z): 2
Isolation Offset: Off
Activation Type: HCD
HCD Collision Energy (%): 65
Detector Type: Orbitrap
Scan Range Mode: Define m/z range
Orbitrap Resolution: 50000
Scan Range (m/z): 100-500
AGC Target: 1.0e5
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 105
Microscans: 1
Data Type: Centroid
Use EASY-IC™: False
Scan Description:
Number of Dependent Scans: 5
Scan Event Type 2:
Charge State
Include charge state(s): 3
Include undetermined charge states: False
Include charge states 25 and higher: False
Scan:
ddMS² IT CID
Scan Priority: 1
Isolation Mode: Quadrupole
Isolation Window (m/z): 0.7
Isolation Offset: Off
Activation Type: CID
Collision Energy Mode: Fixed
CID Collision Energy (%): 35
CID Activation Time (ms): 10
Activation Q: 0.25
Multistage Activation: False
Detector Type: Ion Trap
Scan Range Mode: Auto: m/z Normal
Ion Trap Scan Rate: Turbo
AGC Target: 1.0e4
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Scan Description:
Filters:
Precursor Selection Range
Selection Range Mode: Mass Range
Mass Range (m/z): 400-1200
Isobaric Tag Loss Exclusion
Reagent: TMT
Precursor Ion Exclusion
Exclusion mass width: m/z
Low: 50
High: 5
Data Dependent
Data Dependent Mode: Scans Per Outcome
Scan Event Type 1:
Scan:
ddMS³ OT HCD
MSⁿ Level: 3
Synchronous Precursor Selection: True
Number of SPS Precursors: 10
MS Isolation Window (m/z): 0.7
MS2 Isolation Window (m/z): 2
Isolation Offset: Off
Activation Type: HCD
HCD Collision Energy (%): 65
Detector Type: Orbitrap
Scan Range Mode: Define m/z range
Orbitrap Resolution: 50000
Scan Range (m/z): 100-500
AGC Target: 1.0e5
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 105
Microscans: 1
Data Type: Centroid
Use EASY-IC™: False
Scan Description:
Number of Dependent Scans: 10
Scan Event Type 3:
Charge State
Include charge state(s): 4-6
Include undetermined charge states: False
Include charge states 25 and higher: False
Scan:
ddMS² IT CID
Scan Priority: 1
Isolation Mode: Quadrupole
Isolation Window (m/z): 0.5
Isolation Offset: Off
Activation Type: CID
Collision Energy Mode: Fixed
CID Collision Energy (%): 35
CID Activation Time (ms): 10
Activation Q: 0.25
Multistage Activation: False
Detector Type: Ion Trap
Scan Range Mode: Auto: m/z Normal
Ion Trap Scan Rate: Turbo
AGC Target: 1.0e4
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Scan Description:
Filters:
Precursor Selection Range
Selection Range Mode: Mass Range
Mass Range (m/z): 400-1200
Isobaric Tag Loss Exclusion
Reagent: TMT
Precursor Ion Exclusion
Exclusion mass width: m/z
Low: 50
High: 5
Data Dependent
Data Dependent Mode: Scans Per Outcome
Scan Event Type 1:
Scan:
ddMS³ OT HCD
MSⁿ Level: 3
Synchronous Precursor Selection: True
Number of SPS Precursors: 10
MS Isolation Window (m/z): 0.5
MS2 Isolation Window (m/z): 2
Isolation Offset: Off
Activation Type: HCD
HCD Collision Energy (%): 65
Detector Type: Orbitrap
Scan Range Mode: Define m/z range
Orbitrap Resolution: 50000
Scan Range (m/z): 100-500
AGC Target: 1.0e5
Inject Ions for All Available Parallelizable Time: False
Maximum Injection Time (ms): 105
Microscans: 1
Data Type: Centroid
Use EASY-IC™: False
Scan Description:
Number of Dependent Scans: 10
Experiment#5 [MS]
Start Time (min): 0
End Time (min): 35
Master Scan:
MS OT
Detector Type: Orbitrap
Orbitrap Resolution: 120000
Mass Range: Normal
Use Quadrupole Isolation: True
Scan Range (m/z): 350-2000
RF Lens (%): 30
AGC Target: 2.0e5
Maximum Injection Time (ms): 50
Microscans: 1
Data Type: Centroid
Polarity: Positive
Source Fragmentation: Disabled
Scan Description:
mzXML conversion
The mzXML files were produced by using
proteowizard software using the below-listed wrapper script.
"C:\Program Files (x86)\ProteoWizard\ProteoWizard 3.0.10200\msconvert" %1 --mzXML -z -o %~dp1\msconvert_mzXML
The software versions are:
|software release | Q Exactive HF-X | Fusion Lumos |
|:---------------------|:------------------------|:---------------------|
|ProteoWizard | 3.0.10200 (2016-11-17) | 3.0.18109-390173050 |
|ProteoWizard MSData | 3.0.10200 (2016-11-17) | |
|ProteoWizard Analysis | 3.0.10112 (2016-10-19) | |
|Build date: | Nov 17 2016 10:59:56 | |
Uploading to S3
cp@fgcz-148:~ > aws --profile AnnotationContributor s3 cp tartare s3://annotation-contributor/tartare --recursive --acl public-read
upload: tartare/20190710_003_PierceHeLaProteinDigestStd.mzXML to s3://annotation-contributor/tartare/20190710_003_PierceHeLaProteinDigestStd.mzXML
upload: tartare/20190710_003_PierceHeLaProteinDigestStd.raw to s3://annotation-contributor/tartare/20190710_003_PierceHeLaProteinDigestStd.raw
upload: tartare/20190716_004_PierceHeLa.mzXML to s3://annotation-contributor/tartare/20190716_004_PierceHeLa.mzXML
upload: tartare/20190716_004_PierceHeLa.raw to s3://annotation-contributor/tartare/20190716_004_PierceHeLa.raw
Loading r Biocpkg("tartare") data using Bioconductor r Biocpkg("ExperimentHub")
load metadata
(fl <- system.file("extdata", "metadata.csv", package='tartare'))
metadata <- read.csv(fl, stringsAsFactors=FALSE)
library(knitr)
kable(metadata[,c(1,5,15,16)])
query and load r Biocpkg("tartare") package data from aws s3
library(ExperimentHub)
eh <- ExperimentHub();
query(eh, "tartare")
#### code snippets to be continued
# load(query(eh, c("NestLink", "F255744.RData"))[[1]])
# dim(F255744)
# load(query(eh, c("NestLink", "WU160118.RData"))[[1]])
# dim(WU160118)
Session info
Here is the compiled output of sessionInfo():
sessionInfo()
References
cpanse/tartare documentation built on Nov. 4, 2019, 9:14 a.m.
R Package Documentation
Browse R Packages
We want your feedback!
Note that we can't provide technical support on individual packages. You should contact the package authors for that.
library(BiocStyle)
Sample description
Pierce HeLa protein digest standard (#1862824, Thermo Scientific) in 3% ACN, 0.1% FA
Data recording
Q Exactive HF-X
file name: 20190710_003_PierceHeLaProteinDigestStd.raw
LC
200 ng sample were seperated on a linear gradient from 5 to 35% B in 20 min using a binary mobile phase composed of A) 0.1% FA in water, B) 0.1% FA in ACN. The stationary phase was ...
MS
The MS acquision was started 18 min post sample injection and lastet only 4 min. Be aware that spray voltage and source gas was turned on at 18.5 min (see Tunefiles section of the method). This means the first 30 s were recorded without ESI. In summary, 4 different experiments were recorded
- Full MS / dd-MS² (TopN)
- PRM
- Targeted-SIM
- DIA
Experiment 2 and 3 were executed in parrallel! All method details are listed below.
Method of Q Exactive HF-X
Overall method settings
Global Settings
Use lock masses best
Lock mass injection ―
Chrom. peak width (FWHM) 15 s
Time
Method duration 4.00 min
Customized Tolerances (+/-)
Lock Masses ―
Inclusion ―
Exclusion ―
Neutral Loss ―
Mass Tags ―
Dynamic Exclusion ―
Experiments
Full MS / dd-MS² (TopN)
General
Runtime 0 to 1 min
Polarity positive
In-source CID 0.0 eV
Default charge state 2
Inclusion ―
Exclusion ―
Tags ―
Full MS
Microscans 1
Resolution 120,000
AGC target 3e6
Maximum IT 100 ms
Number of scan ranges 1
Scan range 200 to 2000 m/z
Spectrum data type Centroid
dd-MS² / dd-SIM
Microscans 1
Resolution 30,000
AGC target 1e5
Maximum IT 50 ms
Loop count 5
MSX count 1
TopN 5
Isolation window 1.4 m/z
Isolation offset 0.0 m/z
Scan range 200 to 2000 m/z
Fixed first mass ―
(N)CE / stepped (N)CE nce: 30
Spectrum data type Centroid
dd Settings
Minimum AGC target 8.00e3
Intensity threshold 1.6e5
Apex trigger ―
Charge exclusion unassigned, 1, 5 - 8, >8
Multiple charge states all
Peptide match preferred
Exclude isotopes on
Dynamic exclusion 10.0 s
If idle .. do not pick others
PRM
General
Runtime 1 to 2 min
Polarity positive
In-source CID 0.0 eV
Dynamic RT off
Default charge state 2
Inclusion on
MS²
Microscans 1
Resolution 30,000
AGC target 2e5
Maximum IT 100 ms
Loop count 11
MSX count 1
MSX isochronous ITs on
Isolation window 4.0 m/z
Isolation offset 0.0 m/z
Fixed first mass ―
(N)CE / stepped (N)CE nce: 35
Spectrum data type Centroid
Full MS — SIM
General
Runtime 1 to 2 min
Polarity positive
In-source CID 0.0 eV
Full MS — SIM
Microscans 1
Resolution 120,000
AGC target 3e6
Maximum IT 200 ms
Number of scan ranges 2
Scan range 1 150 to 1000 m/z
Scan range 2 1000 to 2000 m/z
Spectrum data type Centroid
Targeted-SIM
General
Runtime 2 to 3 min
Polarity positive
In-source CID 0.0 eV
Inclusion on
SIM
Microscans 1
Resolution 240,000
AGC target 5e4
Maximum IT 200 ms
MSX count 5
Isolation window 4.0 m/z
Isolation offset 0.0 m/z
Scan range 150 to 2000 m/z
Spectrum data type Centroid
DIA
General
Runtime 3 to 4 min
Polarity positive
In-source CID 0.0 eV
Default charge state 2
DIA
Microscans 1
Resolution 30,000
AGC target 1e6
Maximum IT auto
Loop count 11
MSX count 1
MSX isochronous ITs on
Isolation window 20.0 m/z
Isolation offset 0.0 m/z
Fixed first mass 100.0 m/z
(N)CE / stepped (N)CE nce: 30
Spectrum data type Centroid
Setup
Tunefiles
General
Switch Count 2
Base Tunefile C:\Xcalibur\methods\nogas_novoltage_nano.mstune
Element 1
At 0.50 min
New Tunefile C:\Xcalibur\methods\default_nano.mstune
Element 2
At 34.50 min
New Tunefile C:\Xcalibur\methods\nogas_novoltage_nano.mstune
Contact Closure
General
Used True
Start in Closed False
Switch Count 2
Element 1
At 0.50 min
Switches to Open
Element 2
At 34.50 min
Switches to Closed
Syringe
General
Used False
Start in OFF True
Stop at end of run False
Switch Count 0
Pump setup
Syringe type Hamilton
Flow rate 3.000 µL/min
Inner diameter 2.303 mm
Volume 250 µL
Divert Valve A
General
Used False
Start in 1-2 True
Switch Count 0
Divert Valve B
General
Used False
Start in 1-2 True
Switch Count 0
Lock Masses
2 entries
Mass Polarity Start End Comment
[m/z] [min] [min]
371.10124 Positive
445.12002 Positive
Inclusion List
16 entries
Mass Formula Species CS Polarity Start End (N)CE MSX ID Comment
[m/z] [M] [z] [min] [min]
487.25671 2 Positive 1.00 3.00 28 LGGNEQVTR
(light)
644.82261 2 Positive 1.00 3.00 28 GAGSSEPVTGLDAK
(light)
683.82789 2 Positive 1.00 3.00 28 VEATFGVDESNAK
(light)
547.29804 2 Positive 1.00 3.00 28 YILAGVENSK
(light)
669.83806 2 Positive 1.00 3.00 28 TPVISGGPYEYR
(light)
683.85371 2 Positive 1.00 3.00 28 TPVITGAPYEYR
(light)
699.33842 2 Positive 1.00 3.00 28 DGLDAASYYAPVR
(light)
726.83571 2 Positive 1.00 3.00 28 ADVTPADFSEWSK
(light)
622.85351 2 Positive 1.00 3.00 28 GTFIIDPGGVIR
(light)
636.86916 2 Positive 1.00 3.00 28 GTFIIDPAAVIR
(light)
776.92975 2 Positive 1.00 3.00 28 LFLQFGAQGSPFLK
(light)
410.00000 2 Positive 3.00 4.00 28 window1
430.00000 2 Positive 3.00 4.00 28 window2
450.00000 2 Positive 3.00 4.00 28 window3
470.00000 2 Positive 3.00 4.00 28 window4
490.00000 2 Positive 3.00 4.00 28 window5
Exclusion List
(no entries)
Neutral Losses
(no entries)
Mass Tags
(no entries)
Fusion Lumos
file name: 20190716_004_PierceHeLa.raw
LC
200 ng sample were seperated on a linear gradient from 5 to 35% B in 20 min using a binary mobile phase composed of A) 0.1% FA in water, B) 0.1% FA in ACN. The stationary phase was ...
MS
The MS acquision was started directly after sample injection and lastet for 35 min. In summary, 5 different experiments were recorded:
- Exp. 1 Orbitrap HCD - High Load (greater than 500 ng)
- Exp. 2 ETD Data-Dependent Decision Tree
- Exp. 3 Glycopeptide - Enriched
- Exp. 4 TMT Quan Pierce TKO Standard QC MS3 SPS
- Exp. 5 MS
Exp. #5 (MS) was executed the entire method duration! Method details are listed below.
Method Summary Method Settings Application Mode: Peptide Method Duration (min): 35 Global Parameters Ion Source Use Ion Source Settings from Tune: True FAIMS Mode: Not Installed MS Global Settings Advanced Peak Determination: False Default Charge State: 1 Internal Mass Calibration: Off Experiment#1 [Orbitrap HCD - High Load (greater than 500 ng)] Start Time (min): 20 End Time (min): 21 Cycle Time (sec): 3 Master Scan: MS OT Detector Type: Orbitrap Orbitrap Resolution: 60000 Mass Range: Normal Use Quadrupole Isolation: True Scan Range (m/z): 375-1500 RF Lens (%): 30 AGC Target: 4.0e5 Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Polarity: Positive Source Fragmentation: Disabled Scan Description: Filters: MIPS Monoisotopic Peak Determination: Peptide Charge State Include charge state(s): 2-7 Include undetermined charge states: False Include charge states 25 and higher: False Dynamic Exclusion Exclude after n times: 1 Exclusion duration (s): 60 Mass Tolerance: ppm Low: 10 High: 10 Exclude Isotopes: True Perform dependent scan on single charge state per precursor only: False Intensity Filter Type: Intensity Threshold Intensity Threshold: 5.0e4 Data Dependent Data Dependent Mode: Cycle Time Time between Master Scans (sec): 3 Scan Event Type 1: Scan: ddMS² OT HCD Isolation Mode: Quadrupole Isolation Window (m/z): 1.6 Isolation Offset: Off Activation Type: HCD Collision Energy Mode: Fixed HCD Collision Energy (%): 30 Detector Type: Orbitrap Scan Range Mode: Auto: m/z Normal Orbitrap Resolution: 15000 First Mass (m/z): 110 AGC Target: 5.0e4 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 30 Microscans: 1 Data Type: Centroid Use EASY-IC™: False Scan Description: Experiment#2 [ETD Data-Dependent Decision Tree] Start Time (min): 21 End Time (min): 22 Cycle Time (sec): 3 Master Scan: MS OT Detector Type: Orbitrap Orbitrap Resolution: 120000 Mass Range: Normal Use Quadrupole Isolation: True Scan Range (m/z): 350-1500 RF Lens (%): 30 AGC Target: 4.0e5 Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Polarity: Positive Source Fragmentation: Disabled Scan Description: Filters: MIPS Monoisotopic Peak Determination: Peptide Dynamic Exclusion Exclude after n times: 1 Exclusion duration (s): 60 Mass Tolerance: ppm Low: 10 High: 10 Exclude Isotopes: True Perform dependent scan on single charge state per precursor only: False Exclude Within Cycle: False Intensity Filter Type: Intensity Threshold Intensity Threshold: 1.0e4 Data Dependent Data Dependent Mode: Cycle Time Time between Master Scans (sec): 3 Scan Event Type 1: Charge State Include charge state(s): 3 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 350-650 OR Charge State Include charge state(s): 4 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 350-900 OR Charge State Include charge state(s): 5 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 350-950 OR Charge State Include charge state(s): 6-8 Include undetermined charge states: False Include charge states 25 and higher: False Sort by Charge Precursor Priority: Highest Charge State Sort by m/z Precursor Priority: Lowest m/z Scan: ddMS² IT ETD Scan Priority: 1 Isolation Mode: Quadrupole Isolation Window (m/z): 2 Isolation Offset: Off Activation Type: ETD Use Calibrated Charge-Dependent ETD Parameters: True ETD Supplemental Activation: False Detector Type: Ion Trap Scan Range Mode: Auto: m/z Normal Ion Trap Scan Rate: Rapid First Mass (m/z): 120 AGC Target: 1.0e4 Inject Ions for All Available Parallelizable Time: True Maximum Injection Time (ms): 100 Microscans: 1 Data Type: Centroid Scan Description: Scan Event Type 2: Charge State Include charge state(s): 2 Include undetermined charge states: False Include charge states 25 and higher: False OR Charge State Include charge state(s): 3 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 650-1200 OR Charge State Include charge state(s): 4 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 900-1200 OR Charge State Include charge state(s): 5 Include undetermined charge states: False Include charge states 25 and higher: False Precursor Selection Range Mass Range (m/z): 950-1200 Scan: ddMS² IT CID Scan Priority: 1 Isolation Mode: Quadrupole Isolation Window (m/z): 2 Isolation Offset: Off Activation Type: CID Collision Energy Mode: Fixed CID Collision Energy (%): 30 CID Activation Time (ms): 10 Activation Q: 0.25 Multistage Activation: False Detector Type: Ion Trap Scan Range Mode: Auto: m/z Normal Ion Trap Scan Rate: Rapid AGC Target: 1.0e4 Inject Ions for All Available Parallelizable Time: True Maximum Injection Time (ms): 35 Microscans: 1 Data Type: Centroid Scan Description: Experiment#3 [Glycopeptide - Enriched] Start Time (min): 22 End Time (min): 23 Cycle Time (sec): 3 Master Scan: MS OT Detector Type: Orbitrap Orbitrap Resolution: 120000 Mass Range: Normal Use Quadrupole Isolation: True Scan Range (m/z): 350-1800 RF Lens (%): 30 AGC Target: 4.0e5 Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Polarity: Positive Source Fragmentation: Disabled Scan Description: Filters: MIPS Monoisotopic Peak Determination: Peptide Charge State Include charge state(s): 2-8 Include undetermined charge states: False Include charge states 25 and higher: False Dynamic Exclusion Exclude after n times: 1 Exclusion duration (s): 60 Mass Tolerance: ppm Low: 10 High: 10 Exclude Isotopes: True Perform dependent scan on single charge state per precursor only: False Intensity Filter Type: Intensity Threshold Intensity Threshold: 5.0e4 Data Dependent Data Dependent Mode: Cycle Time Time between Master Scans (sec): 3 Scan Event Type 1: Sort by Charge Precursor Priority: Highest Charge State Sort by m/z Precursor Priority: Lowest m/z Scan: ddMS² OT EThcD Isolation Mode: Quadrupole Isolation Window (m/z): 3 Isolation Offset: Off Activation Type: ETD Use Calibrated Charge-Dependent ETD Parameters: True ETD Supplemental Activation: True SA Collision Energy Type: EThcD SA Collision Energy (%): 15 Detector Type: Orbitrap Scan Range Mode: Auto: m/z Normal Orbitrap Resolution: 60000 First Mass (m/z): 120 AGC Target: 5.0e4 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 118 Microscans: 1 Data Type: Centroid Use EASY-IC™: False Scan Description: Experiment#4 [TMT Quan Pierce TKO Standard QC MS3 SPS] Start Time (min): 23 End Time (min): 24 Cycle Time (sec): 2 Master Scan: MS OT Detector Type: Orbitrap Orbitrap Resolution: 120000 Mass Range: Normal Use Quadrupole Isolation: False Scan Range (m/z): 375-1500 RF Lens (%): 30 AGC Target: 4.0e5 Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Polarity: Positive Source Fragmentation: Disabled Scan Description: Filters: MIPS Monoisotopic Peak Determination: Peptide Charge State Include charge state(s): 2-6 Include undetermined charge states: False Include charge states 25 and higher: False Dynamic Exclusion Exclude after n times: 1 Exclusion duration (s): 60 Mass Tolerance: ppm Low: 10 High: 10 Exclude Isotopes: True Perform dependent scan on single charge state per precursor only: True Exclude Within Cycle: True Intensity Filter Type: Intensity Threshold Intensity Threshold: 5.0e3 Data Dependent Data Dependent Mode: Cycle Time Time between Master Scans (sec): 2 Scan Event Type 1: Charge State Include charge state(s): 2 Include undetermined charge states: False Include charge states 25 and higher: False Scan: ddMS² IT CID Scan Priority: 1 Isolation Mode: Quadrupole Isolation Window (m/z): 1.2 Isolation Offset: Off Activation Type: CID Collision Energy Mode: Fixed CID Collision Energy (%): 35 CID Activation Time (ms): 10 Activation Q: 0.25 Multistage Activation: False Detector Type: Ion Trap Scan Range Mode: Auto: m/z Normal Ion Trap Scan Rate: Turbo AGC Target: 1.0e4 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Scan Description: Filters: Precursor Selection Range Selection Range Mode: Mass Range Mass Range (m/z): 400-1200 Isobaric Tag Loss Exclusion Reagent: TMT Precursor Ion Exclusion Exclusion mass width: m/z Low: 50 High: 5 Data Dependent Data Dependent Mode: Scans Per Outcome Scan Event Type 1: Scan: ddMS³ OT HCD MSⁿ Level: 3 Synchronous Precursor Selection: True Number of SPS Precursors: 5 MS Isolation Window (m/z): 1.3 MS2 Isolation Window (m/z): 2 Isolation Offset: Off Activation Type: HCD HCD Collision Energy (%): 65 Detector Type: Orbitrap Scan Range Mode: Define m/z range Orbitrap Resolution: 50000 Scan Range (m/z): 100-500 AGC Target: 1.0e5 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 105 Microscans: 1 Data Type: Centroid Use EASY-IC™: False Scan Description: Number of Dependent Scans: 5 Scan Event Type 2: Charge State Include charge state(s): 3 Include undetermined charge states: False Include charge states 25 and higher: False Scan: ddMS² IT CID Scan Priority: 1 Isolation Mode: Quadrupole Isolation Window (m/z): 0.7 Isolation Offset: Off Activation Type: CID Collision Energy Mode: Fixed CID Collision Energy (%): 35 CID Activation Time (ms): 10 Activation Q: 0.25 Multistage Activation: False Detector Type: Ion Trap Scan Range Mode: Auto: m/z Normal Ion Trap Scan Rate: Turbo AGC Target: 1.0e4 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Scan Description: Filters: Precursor Selection Range Selection Range Mode: Mass Range Mass Range (m/z): 400-1200 Isobaric Tag Loss Exclusion Reagent: TMT Precursor Ion Exclusion Exclusion mass width: m/z Low: 50 High: 5 Data Dependent Data Dependent Mode: Scans Per Outcome Scan Event Type 1: Scan: ddMS³ OT HCD MSⁿ Level: 3 Synchronous Precursor Selection: True Number of SPS Precursors: 10 MS Isolation Window (m/z): 0.7 MS2 Isolation Window (m/z): 2 Isolation Offset: Off Activation Type: HCD HCD Collision Energy (%): 65 Detector Type: Orbitrap Scan Range Mode: Define m/z range Orbitrap Resolution: 50000 Scan Range (m/z): 100-500 AGC Target: 1.0e5 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 105 Microscans: 1 Data Type: Centroid Use EASY-IC™: False Scan Description: Number of Dependent Scans: 10 Scan Event Type 3: Charge State Include charge state(s): 4-6 Include undetermined charge states: False Include charge states 25 and higher: False Scan: ddMS² IT CID Scan Priority: 1 Isolation Mode: Quadrupole Isolation Window (m/z): 0.5 Isolation Offset: Off Activation Type: CID Collision Energy Mode: Fixed CID Collision Energy (%): 35 CID Activation Time (ms): 10 Activation Q: 0.25 Multistage Activation: False Detector Type: Ion Trap Scan Range Mode: Auto: m/z Normal Ion Trap Scan Rate: Turbo AGC Target: 1.0e4 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Scan Description: Filters: Precursor Selection Range Selection Range Mode: Mass Range Mass Range (m/z): 400-1200 Isobaric Tag Loss Exclusion Reagent: TMT Precursor Ion Exclusion Exclusion mass width: m/z Low: 50 High: 5 Data Dependent Data Dependent Mode: Scans Per Outcome Scan Event Type 1: Scan: ddMS³ OT HCD MSⁿ Level: 3 Synchronous Precursor Selection: True Number of SPS Precursors: 10 MS Isolation Window (m/z): 0.5 MS2 Isolation Window (m/z): 2 Isolation Offset: Off Activation Type: HCD HCD Collision Energy (%): 65 Detector Type: Orbitrap Scan Range Mode: Define m/z range Orbitrap Resolution: 50000 Scan Range (m/z): 100-500 AGC Target: 1.0e5 Inject Ions for All Available Parallelizable Time: False Maximum Injection Time (ms): 105 Microscans: 1 Data Type: Centroid Use EASY-IC™: False Scan Description: Number of Dependent Scans: 10 Experiment#5 [MS] Start Time (min): 0 End Time (min): 35 Master Scan: MS OT Detector Type: Orbitrap Orbitrap Resolution: 120000 Mass Range: Normal Use Quadrupole Isolation: True Scan Range (m/z): 350-2000 RF Lens (%): 30 AGC Target: 2.0e5 Maximum Injection Time (ms): 50 Microscans: 1 Data Type: Centroid Polarity: Positive Source Fragmentation: Disabled Scan Description:
mzXML conversion
The mzXML files were produced by using proteowizard software using the below-listed wrapper script.
"C:\Program Files (x86)\ProteoWizard\ProteoWizard 3.0.10200\msconvert" %1 --mzXML -z -o %~dp1\msconvert_mzXML
The software versions are:
|software release | Q Exactive HF-X | Fusion Lumos | |:---------------------|:------------------------|:---------------------| |ProteoWizard | 3.0.10200 (2016-11-17) | 3.0.18109-390173050 | |ProteoWizard MSData | 3.0.10200 (2016-11-17) | | |ProteoWizard Analysis | 3.0.10112 (2016-10-19) | | |Build date: | Nov 17 2016 10:59:56 | |
Uploading to S3
cp@fgcz-148:~ > aws --profile AnnotationContributor s3 cp tartare s3://annotation-contributor/tartare --recursive --acl public-read upload: tartare/20190710_003_PierceHeLaProteinDigestStd.mzXML to s3://annotation-contributor/tartare/20190710_003_PierceHeLaProteinDigestStd.mzXML upload: tartare/20190710_003_PierceHeLaProteinDigestStd.raw to s3://annotation-contributor/tartare/20190710_003_PierceHeLaProteinDigestStd.raw upload: tartare/20190716_004_PierceHeLa.mzXML to s3://annotation-contributor/tartare/20190716_004_PierceHeLa.mzXML upload: tartare/20190716_004_PierceHeLa.raw to s3://annotation-contributor/tartare/20190716_004_PierceHeLa.raw
Loading r Biocpkg("tartare") data using Bioconductor r Biocpkg("ExperimentHub")
load metadata
(fl <- system.file("extdata", "metadata.csv", package='tartare')) metadata <- read.csv(fl, stringsAsFactors=FALSE)
library(knitr) kable(metadata[,c(1,5,15,16)])
query and load r Biocpkg("tartare") package data from aws s3
library(ExperimentHub) eh <- ExperimentHub(); query(eh, "tartare") #### code snippets to be continued # load(query(eh, c("NestLink", "F255744.RData"))[[1]]) # dim(F255744) # load(query(eh, c("NestLink", "WU160118.RData"))[[1]]) # dim(WU160118)
Session info
Here is the compiled output of sessionInfo():
sessionInfo()
References
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