Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity...

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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

Transcript of Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity...

Page 1: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 2: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Acquisition Methods

Targeted DiscoveryData Dependent

Acquisition (DDA)

Peptide Identification

Data IndependentAcquisition (DIA)

Selected Reaction Monitoring (SRM)

Peptide Quantitation

Page 3: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

LC–MS/MS: Data Dependent Acquisition

m/z

MS Scan

MS Scan

1 234 5

Page 4: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Data Independent Acquisition (DIA)

20 20 m/z-wide windows = 400 m/z

m/z500 900

Page 5: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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.

Data Independent Acquisition (DIA)

Scan 1

20 20 m/z-wide windows = 400 m/z

m/z500 900

Scan 2

Page 7: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 8: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Data Independent Acquisition (DIA)

20 20 m/z-wide windows = 400 m/z

m/z500 900

Tim

e

~2 secondsMS Scan

~30 seconds

Page 9: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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.

Data Independent Acquisition (DIA)

20 20 m/z-wide windows = 400 m/z

m/z500 900

Tim

e

LGLVGGSTIDIK++ (586.85)

Page 11: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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.

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.

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)

Page 14: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

MS/MS

1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

Page 15: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

MS MS/MS

1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

Page 16: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

MS MS/MS

1.02 femtomoles of Bovine Serum Albumin(LVNELTEFAK++) in 1.2 ug of S. cerevisiae lysate

Page 17: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Theoretical Benefits of DIA

• Comprehensive Sampling– Reproducibility

• Improved Quantitation

500 – 900 m/z

MS MS/MS

Page 18: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 19: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor Selectivity

2 m/zANFQGAITNR

Page 20: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor Selectivity

10 m/zANFQGAITNR

Page 21: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor Selectivity

20 m/zANFQGAITNR

Page 22: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor SelectivityIn

ten

sity

4e7

Retention Time (min)25 26

10 m/zANFQGAITNR

Page 23: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor SelectivityIn

ten

sity

4e7

10 m/z

Retention Time (min)

Inte

nsi

ty

4e7

25 26

20 m/z

ANFQGAITNR

X

X

X

Page 24: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Precursor Selectivity

SLQDIIAILGMDELSEEDKLTVSR+++(892.47 m/z)

SLQDIIAILGMDELSEEDKLTVSR+++(897.8 m/z)

890 900

X

X

Page 25: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Improving Precursor Selectivity

X

Page 26: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Improving Precursor Selectivity

X

X

Page 27: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Improving Precursor Selectivity

Page 28: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Improving Precursor Selectivity X

Page 29: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Improving Precursor Selectivity X

X

Page 30: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 31: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 32: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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

Page 33: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Generating a DIA Method Using Skyline: Generate a Target List

20 20 m/z-wide windows = 400 m/z

m/z500 900

Page 34: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

Generating a DIA Method Using Skyline: Generate a Target List

Page 35: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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.

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 37: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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.

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.

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.

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.

Importing Data: Filtering Settings

Page 42: Application of Data Independent Acquisition Techniques Optimized for Improved Precursor Selectivity Jarrett D. Egertson, Ph.D. MacCoss Lab Department of.

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