Developments in Data Independent MS Acquisition … · 2013. 9. 27. · ©2011 Waters Corporation 30 Synapt G2-S Schematic ©2011 Waters Corporation 31 Ionized Precursors Precursors

©2011 Waters Corporation 1

Developments in Data Independent MS Acquisition

Comprehensive and Reproducible Identification and Label-Free Quantification of Proteins

Tim Riley, PhDVice President, Waters Corporation


The next step in the evolution of High Definition MS


SYNAPT G2-S Geometry


Ion Block Design

Sampling cone aperture— Increased to 0.8mm diameter

o Approx 5x increase in gas/ion flow

New ion block design— No supplemental pumping

— All ions (and gas) enter StepWave guide

o Approx 200x increase in gas flow

o Up to 200x increase in ion flux.


Ring ElectrodesOpposite phase of RF applied to adjacent plates

Ele

ctri

c Fi

eld

Diffuse Ion Cloud

Ions In

Ions Out

Compact Ion Cloud

15mm

5mm

~11mm

~120mm

Novel StepWave ion guide designConjoined Ion Guide


Ions + GasFrom API

Source

Rough Pump

Gas

Ions

DifferentialAperture

Conjoined Ion Guides

Ion Trajectories

Additional design benefitRobustness


Ele

ctri

c Fie

ld

Diffuse Ion

Cloud

Maximising signal

Maximising robustness

Designed to deal with problems associated with a larger sampling orifice


m/z900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700

%

0

100

m/z900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700

%

0

100

1303.2

1234.7

1173.0

1117.2

1066.51233.8 1302.3

1379.8

1310.3

1954.3

1804.01466.0

1387.2

1675.31563.6

2131.8

ESI Positive Ion SensitivitySynapt G2-S

24,000 mw Protein

Synapt G2-S

Synapt G2

Synapt G2

m/z1286 1288 1290 1292 1294 1296 1298 1300 1302 1304 1306 1308 1310 1312 1314 1316 1318 1320 1322 1324

%

0

100 1303.2

1302.31310.2

Synapt G2

Synapt G2-S>10X increase

in signal


Identification …limits of detection

SYNAPT G2-S vs SYNAPT G2

SYNAPT G2-S

SYNAPT G2


Identification…detection in complex mixtures

Intensity 6.04 x 106Intensity 535

In-Spectral Dynamic Range > 4 orders of magnitude

Glu-Fibrinopeptide spiked into Bovine Insulin (1/10,000)


Mix 1 1fmole 25K resolution

LC-MSE

Low Energy Precursor Spectra

Low Energy Precursor Spectra

MSE Synapt G2-S vs Synapt G2


Data Dependent LC-MS/MS

vs

Data Independent LC-MS/MS


Conventional Data Dependent LC-MS/MSPrecursor Survey Scan


Conventional Data Dependent LC-MS/MSProduct Ion Scan


Conventional Data Dependent LC-MS/MS- Weaknesses -

MS2 is a BIASED process

MS2 is a DISCONTINUOUS process

DDA is a SERIAL Process

DDA is a BIASED Process

DDA is a DISCONTINUOUS Process

DDA is subject to INTERFERENCES


Data Independent LC-MS/MS (LC-MSE)


What is the level of Complexity?E coli UPLC Separation


E coli Precursor MH+ Density

~450K Ion Detections = ~36K EMRT’s = ~10K ID’d peptides = 697 ID’d Proteins


Intensity distribution of all ion detections in an E coli lysate digest

~2/3 of all precursor ion detections are less than 2 orders of magnitude in intensity of the most abundant ion detections.

MS Detection Range > 104

Log Ion Intensity

Ion C

ount

~67%


Peptide Retention Time Distribution

Approximately 50% of all tryptic peptides elute in 20% the chromatographic gradient elution time

Peptide

Count

Retention Time, mins


Approximately 60% of all ion detections are between m/z 400-800

Ion C

ount

Binned m/z

~60%

m/z Distribution of All Ions in an E coli Lysate Digest


Data Complexity Mass Resolution and Accuracy is Critical

Precursor m/z Product m/z

Mas

s Res

olu

tion


Ion mobility enhanced UPLC-MSE


Synapt G2-S Schematic


Ionized Precursors

Precursors Transferred to TOF MS

UPLC/HDMSE…deconvoluting chimericy

Co-Eluting Peptides


Ionized Precursors

Precursors & Products Time Aligned

UPLC/HDMSE…deconvoluting chimericy

Co-Eluting Peptides


Ion mobility

UPLC-HDMSEMobility Drift Time + Retention Time Alignment


IMS Off

IMS On

Impact of Ion Mobility Enhanced UPLC-MSE Precursor/Product Drift Time Alignment

With Drift Time Alignment35 product ions tentatively associated with one precursor

Without Drift Time Alignment254 product ions tentatively associated with one precursor


Increases analytical peak capacity/ separation power by approximately 7-10 fold

Reduces chimeric interferences & distraction

— Precursor and product ions are mobility drift time aligned as well as chromatographic retention time aligned .

o Increased specificity, less distracting product ions per peptide

— Improved precursor charge state recognition

o Isotopes for each charge state of each peptide precursor have the same mobility drift time

— More precursor ions detected because of reduced interference from chemical noise

— Improved measurement & confidence in peptide monoisotopic accurate mass

Adds an additional dimension of separation without increasing analysis time

How Does Ion Mobility Improve Protein Identification Rate and Quality


Example UPLC-HDMSE data

The Impact of Ion Mobility

Synapt G2 HDMS


Application 1:High Throughput analysis of Shewanella

Shewanella oneidensis750 ng load, 27 minute cycle time


Increased number of proteins/ peptides identified

MSE684 Proteins

4337 peptides

HDMSE1019 Proteins5432 peptides

622 Proteins91% of MSE in HDMSE

Resolution >20,000 (FWHM)5 ppm precursor window12 ppm product window

Proteins had to be in 2/3 replicates2% FPR at protein level

6 peptides/protein


Absolute Quantitation of Proteins from Shewanella Oneidensis

Observed in MSE and HDMSEObserved in HDMSE only

Silva, et.al., Mol. Cell Proteomics, 2006, 144-156


Application 2: Increased Proteome Coverage of Caenorhabditis elegans

1D Chromatography1 µg C. elegans

< 2 hours

2D-3 Fraction1.5 µg C. elegans

< 4 hours

2D-5 Fraction2.5 µg C. elegans

< 6 hours

13.1% ACN

50% ACN

17.7% ACN

16.7% ACN

50% ACN

20.4% ACN

10.8% ACN

14% ACN


0

200

400

600

800

1000

1200

1400

1600

1800

2000

1D 2D-3 Fraction 2D-5 Fraction

Prot

eins

LC Method

MSE

HDMSE

Increasing Number of Identified Proteins with Increase in Peak Capacity

MSE

HDMSE

Resolution 20,0005 ppm precursor window12 ppm product windowProteins had to be in 2/3 replicates


0

2000

4000

6000

8000

10000

12000

14000

1D 2D-3 Fraction 2D-5 Fraction

Pept

ides

LC Method

MSE

HDMSE

Increasing Number of Identified Peptides with Increase in Peak Capacity

MSE

HDMSE


Acknowledgements

Waters Corporation— Martha Stapels

— Steve Ciavarini

— Craig Dorschel

— Barry Dyson

— Keith Fadgen

— Scott Geromanos

— Lee Gethings

— Dan Golick

— Marc Gorenstein

— Chris Hughes

— Jim Langridge

— Keith Richardson

— Hans Vissers

PNNL— Robby Robinson

— Ljiljana Paša-Tolić

— Richard D. Smith

幻灯片编号 1幻灯片编号 2SYNAPT G2-S GeometryIon Block Design幻灯片编号 5幻灯片编号 6Designed to deal with problems �associated with a larger sampling orifice幻灯片编号 8Identification �…limits of detectionIdentification�…detection in complex mixturesMSE Synapt G2-S vs Synapt G2幻灯片编号 12幻灯片编号 13幻灯片编号 14幻灯片编号 15Conventional Data Dependent LC-MS/MS� - Weaknesses -Data Independent LC-MS/MS (LC-MSE) What is the level of Complexity?�E coli UPLC Separation E coli Precursor MH+ Density�Intensity distribution of all ion detections in an E coli lysate digestPeptide Retention Time Distributionm/z Distribution of All Ions in an E coli Lysate DigestData Complexity �Mass Resolution and Accuracy is Critical 幻灯片编号 29Synapt G2-S SchematicUPLC/HDMSE �…deconvoluting chimericyUPLC/HDMSE �…deconvoluting chimericyUPLC-HDMSE �Mobility Drift Time + Retention Time Alignment Impact of Ion Mobility Enhanced UPLC-MSE Precursor/Product Drift Time AlignmentHow Does Ion Mobility Improve Protein Identification Rate and Quality幻灯片编号 36Application 1:�High Throughput analysis of Shewanella Increased number of proteins/ peptides identifiedAbsolute Quantitation of Proteins �from Shewanella OneidensisApplication 2: Increased Proteome Coverage of Caenorhabditis elegansIncreasing Number of Identified Proteins with Increase in Peak CapacityIncreasing Number of Identified Peptides with Increase in Peak CapacityAcknowledgements

Documents

Developments in Data Independent MS Acquisition … · 2013. 9. 27. · ©2011 Waters Corporation 30 Synapt G2-S Schematic ©2011 Waters Corporation 31 Ionized Precursors Precursors