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![Page 1: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/1.jpg)
Application of Data Independent Acquisition Techniques Optimized for
Improved Precursor Selectivity
Jarrett D. Egertson, Ph.D.MacCoss Lab
Department of Genome SciencesUniversity of Washington
6/8/2013
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Acquisition Methods
Targeted DiscoveryData Dependent
Acquisition (DDA)
Peptide Identification
Data IndependentAcquisition (DIA)
Selected Reaction Monitoring (SRM)
Peptide Quantitation
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LC–MS/MS: Data Dependent Acquisition
m/z
MS Scan
MS Scan
1 234 5
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Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z500 900
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Data Independent Acquisition (DIA)
Scan 1
20 20 m/z-wide windows = 400 m/z
m/z500 900
![Page 6: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/6.jpg)
Data Independent Acquisition (DIA)
Scan 1
20 20 m/z-wide windows = 400 m/z
m/z500 900
Scan 2
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Data Independent Acquisition (DIA)
Scan 1
20 20 m/z-wide windows = 400 m/z
m/z500 900
Scan 2Scan 3Scan 4Scan 5Scan 6Scan 7
Scan 20
…
Scan 21
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Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z500 900
Tim
e
~2 secondsMS Scan
~30 seconds
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Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z500 900
Tim
e
![Page 10: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/10.jpg)
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z500 900
Tim
e
LGLVGGSTIDIK++ (586.85)
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LGLVGGSTIDIK++ (586.85)
VGGSTIDIK+
GGSTIDIK+
GSTIDIK+
LVGGSTIDIK+
STIDIK+
TIDIK+
IDIK+
(1002.58)
(889.50)
(790.43)
(676.39)(589.36)(488.31)(375.22)
Data Independent Acquisition (DIA)
![Page 12: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/12.jpg)
LGLVGGSTIDIK++ (586.85)
VGGSTIDIK+
GGSTIDIK+
GSTIDIK+
LVGGSTIDIK+
STIDIK+
TIDIK+
IDIK+
(1002.58)
(889.50)
(790.43)
(676.39)(589.36)(488.31)(375.22)
Data Independent Acquisition (DIA)
![Page 13: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/13.jpg)
48 49 50 51 52Retention Time
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Inte
nsi
ty x
10-6
LGLVGGSTIDIK++ (586.85)
VGGSTIDIK+
GGSTIDIK+
GSTIDIK+
LVGGSTIDIK+
STIDIK+
TIDIK+
IDIK+
(1002.58)
(889.50)
(790.43)
(676.39)(589.36)(488.31)(375.22)
Data Independent Acquisition (DIA)
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MS/MS
1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
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MS MS/MS
1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
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MS MS/MS
1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
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Theoretical Benefits of DIA
• Comprehensive Sampling– Reproducibility
• Improved Quantitation
500 – 900 m/z
MS MS/MS
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Isolation Window Width
Vs. Vs.2 m/z 10 m/z 20 m/z
DDA DIA
Lower precursor selectivity• More peptides co-
fragmented• More complex MS/MS
spectra• More interference
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Precursor Selectivity
2 m/zANFQGAITNR
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Precursor Selectivity
10 m/zANFQGAITNR
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Precursor Selectivity
20 m/zANFQGAITNR
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Precursor SelectivityIn
ten
sity
4e7
Retention Time (min)25 26
10 m/zANFQGAITNR
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Precursor SelectivityIn
ten
sity
4e7
10 m/z
Retention Time (min)
Inte
nsi
ty
4e7
25 26
20 m/z
ANFQGAITNR
X
X
X
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Precursor Selectivity
SLQDIIAILGMDELSEEDKLTVSR+++(892.47 m/z)
SLQDIIAILGMDELSEEDKLTVSR+++(897.8 m/z)
890 900
X
X
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Improving Precursor Selectivity
X
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Improving Precursor Selectivity
X
X
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Improving Precursor Selectivity
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Improving Precursor Selectivity X
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Improving Precursor Selectivity X
X
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Overlapped Isolation WindowsIn
ten
sity
4e7
20 m/z
XOverlappe
d
20 m/z
Retention Time (min)25 26
Inte
nsi
ty
4e7
ANFQGAITNR
Overlapped
X
Overlapped
Demultiplexed: ~10 m/z
Overlapped
XNo
Overlap
X
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Improved Quantitation
MS1 All Top 3 Top 5 Top 70
5
10
15
20
10 m/z Demultiplexed20 m/z
Low
er
Lim
it o
f Q
uan
tita
tion
(f
mol)
Transitions Integrated
21 Peptides Spiked IntoYeast Lysate Quantified
Dario Amodei
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Conclusions
Overlapping Windows Improves Selectivity and Sensitivity of DIA• Easily applicable to virtually any DIA-capable
instrument• De-multiplexing implemented in Skyline (multi-
vendor support)• These experiments can be done now with Skyline-
daily
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Generating a DIA Method Using Skyline: Generate a Target List
20 20 m/z-wide windows = 400 m/z
m/z500 900
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Generating a DIA Method Using Skyline: Generate a Target List
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Generating a DIA Method Using Skyline: Generate a Target List
![Page 36: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/36.jpg)
Generating a DIA Method Using Skyline: Generate a Target List
1.00045475 m/z
Mass Excess
H 1.00078 0.00078
C 12 0.0
O 15.9949 0.9949
N 14.0031 0.0031
S 31.9721 0.9721
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Generating a DIA Method Using Skyline: Generate a Target List
1.00045475 m/z
Mass Excess
H 1.00078 0.00078
C 12 0.0
O 15.9949 0.9949
N 14.0031 0.0031
S 31.9721 0.9721
![Page 38: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/38.jpg)
Generating a DIA Method Using Skyline: Generate a Target List
1.00045475 m/z
Mass Excess
H 1.00078 0.00078
C 12 0.0
O 15.9949 0.9949
N 14.0031 0.0031
S 31.9721 0.9721
![Page 39: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/39.jpg)
Generating a DIA Method Using Skyline: Generate a Target List
![Page 40: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/40.jpg)
Generating a DIA Method Using Skyline: Generate a Target List
![Page 41: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.](https://reader036.fdocuments.in/reader036/viewer/2022070411/56649f4f5503460f94c70e13/html5/thumbnails/41.jpg)
Importing Data: Filtering Settings
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AcknowledgementsStanford University
Dario AmodeiParag Mallick
Purdue UniversityOlga Vitek
University of Washington
Mike MacCossBrendan MacLean
Don MarshGennifer MerrihewRichard Johnson
Sonia Ting& the rest of
the lab
Thermo Scientific
Markus KellmannAndreas KuehnReiko Kiyonami
Yue Xuan
Dario’s Poster: Tuesday June 11th
(#512) 10:30 AM – 2:30 PMJarrett’s Talk: Monday, June 10th 8:30-8:50AM Exhibit Hall A