Application of Data Independent
Acquisition Techniques Optimized for
Improved Precursor Selectivity
Jarrett D. Egertson, Ph.D.
MacCoss Lab
Department of Genome Sciences
University of Washington
6/8/2013
Acquisition Methods
Targeted
Data Independent
Acquisition (DIA)
Discovery
Selected Reaction
Monitoring (SRM)
Data Dependent
Acquisition (DDA)
Peptide Quantitation
Peptide Identification
LC–MS/MS: Data Dependent
1
2
Acquisition
3
4
5
m/z
MS Scan
MS Scan
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
500
m/z
900
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
500
Scan 1
m/z
900
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
500
Scan 1
Scan 2
m/z
900
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
500
Scan 1
Scan 2
Scan 3
Scan 4
Scan 5
Scan 6
Scan 7
…
Scan 20
Scan 21
m/z
900
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z
500
900
MS Scan
Time
~2 seconds
~30 seconds
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
Time
500
m/z
900
Data Independent Acquisition (DIA)
20 20 m/z-wide windows = 400 m/z
m/z
Time
500
LGLVGGSTIDIK++ (586.85)
900
Data Independent Acquisition (DIA)
LGLVGGSTIDIK++ (586.85)
LVGGSTIDIK+
VGGSTIDIK+
(1002.58)
GGSTIDIK+
(790.43)
GSTIDIK+
STIDIK+
TIDIK+
IDIK+
(676.39)
(589.36)
(488.31)
(375.22)
(889.50)
Data Independent Acquisition (DIA)
LGLVGGSTIDIK++ (586.85)
LVGGSTIDIK+
VGGSTIDIK+
(1002.58)
GGSTIDIK+
(790.43)
GSTIDIK+
STIDIK+
TIDIK+
IDIK+
(676.39)
(589.36)
(488.31)
(375.22)
(889.50)
Data Independent Acquisition (DIA)
Intensity x 10-6
LGLVGGSTIDIK++ (586.85)
3.5
LVGGSTIDIK+
VGGSTIDIK+
(1002.58)
3.0
GGSTIDIK+
(790.43)
GSTIDIK+
STIDIK+
TIDIK+
IDIK+
(676.39)
(589.36)
(488.31)
(375.22)
2.5
2.0
1.5
1.0
0.5
0.0
48
49
50
Retention Time
51
52
(889.50)
MS/MS
1.02 femtomoles of Bovine Serum Albumin
(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
MS
MS/MS
1.02 femtomoles of Bovine Serum Albumin
(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
MS
MS/MS
1.02 femtomoles of Bovine Serum Albumin
(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate
Theoretical Benefits of DIA
• Comprehensive
Sampling
500 – 900 m/z
– Reproducibility
• Improved
Quantitation
MS
MS/MS
Isolation Window Width
DDA
DIA
Vs.
2 m/z
Vs.
10 m/z
20 m/z
Lower precursor selectivity
• More peptides co-fragmented
• More complex MS/MS spectra
• More interference
Precursor Selectivity
2 m/z
ANFQGAITNR
Precursor Selectivity
10 m/z
ANFQGAITNR
Precursor Selectivity
20 m/z
ANFQGAITNR
Intensity
4e7
Precursor Selectivity
25
10 m/z
ANFQGAITNR
Retention Time (min)
26
Intensity
4e7
Precursor Selectivity
10 m/z
ANFQGAITNR

X
Intensity
4e7
25
 X
 X
20 m/z
Retention Time (min)
26
Precursor Selectivity

890
SLQDIIAILGMDELSEEDKLTVSR+++
(897.8 m/z)
X
900
SLQDIIAILGMDELSEEDKLTVSR+++
(892.47 m/z)

X
Improving Precursor Selectivity
X
Improving Precursor Selectivity
X
X
Improving Precursor Selectivity
Improving Precursor Selectivity
X
Improving Precursor Selectivity
X
X
 ANFQGAITNR X
Intensity
4e7
Overlapped Isolation WindowsX
Intensity
No Overlap
20 m/z
X
4e7
25


Overlapped
Overlapped
20 m/z
Demultiplexed: ~10 m/z
X
Retention Time (min)
26
Improved Quantitation
10 m/z
Demultiplexed
Lower Limit of
Quantitation (fmol)
20
20 m/z
21 Peptides Spiked Into
Yeast Lysate Quantified
15
10
5
0
MS1
Dario Amodei
All
Top 3
Top 5
Transitions Integrated
Top 7
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
Generating a DIA Method Using
Skyline: Generate a Target List
20 20 m/z-wide windows = 400 m/z
500
m/z
900
Generating a DIA Method Using
Skyline: Generate a Target List
Generating a DIA Method Using
Skyline: Generate a Target List
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
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
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
Generating a DIA Method Using
Skyline: Generate a Target List
Generating a DIA Method Using
Skyline: Generate a Target List
Importing Data: Filtering Settings
Acknowledgements
University of
Stanford University
Washington
Dario Amodei
Mike MacCoss
Parag Mallick
Brendan MacLean
Purdue
Dario’s Poster: Tuesday
June University
11th
Don Marsh
(#512) 10:30 AM – 2:30 PM
Olga Vitek
Gennifer Merrihew
Jarrett’s Talk: Monday,
June 10thScientific
Thermo
8:30-8:50AM Exhibit Hall
A Kellmann
Richard Johnson
Markus
Sonia Ting
Andreas Kuehn
& the rest of the
Reiko Kiyonami
lab
Yue Xuan