The Proteomic Analytical Challenge Future Potential of Ion ... · VP Pharmaceutical and Life Science Business Operations ©2010 Waters Corporation | COMPANY CONFIDENTIAL 2. The Proteomic

©2010 Waters Corporation | COMPANY CONFIDENTIAL

The Proteomic Analytical ChallengeFuture Potential of

Ion Mobility Enhanced High Definition LC-MSE

Tim Riley, Ph.D.

VP Pharmaceutical and Life Science Business Operations

©2010 Waters Corporation | COMPANY CONFIDENTIAL 2

The Proteomic Data ChallengeMost proteomic researchers

underestimate the complexity and dynamic range of their samples and

overestimate the power of their mass spectrometers


SYNAPT G2 HDMS


Alternate Scanning LC-MS (LC-MSE) …time resolved accurate mass measurements

MSE is a UNBIASED process

1 sec

LE

EE

LE

EE

EEEE

D.

m

Gas cell collision energy alternates between low (5 eV) and elevated energy (linear ramp from 15 eV -

42 eV)

LC-MSE alternate scanning provides quantitative accurate mass

measurement for all detectable peptide precursor and product ions

throughout UPLC separation


E coliUPLC Separation


E coli Precursor MH+ Density

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

400 nanogram digest injection; 27K RP, Ion Mobility On, 2-5-2 search criteria


E coli Product Ion Density

~600K Total Product Ion Detections


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 relative to the most abundant ion detections.

MS Detection Range > 104

Log Ion Intensity

Ion C

ount

~2/3


E coli Lysate DigestPeptide 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

E coli Lysate Digest m/z Distribution of All Ions

~60%


ChimericyChimericy defines a situation in a data dependent MS/MS experiment when a precursor ion similar in m/z to the precursor analyte of interest, i.e. within the precursor mass selection window of the mass spectrometer, elutes within +/-one-half of a chromatographic peak width of that analyte. This situation produces a contaminated product ion spectra.


Magnified view of Precursor Ion DensityIo

n C

ount

Binned m/z

~900 ions per each unit mass bin or ~2700 ions per 3 Da mass bin are distributed across the entire chromatographic range


DDA MS/MS Precursor Selection Ion Density Chimeric Challenge

Prec

urs

or

Ion C

ount

Binned Retention Time, 30 sec bins

Many precursor ions can be detected in any +/- 1.5 Da DDA MS/MS mass selection window between m/z 400 to 2000 across the entire chromatographic space

561-564 m/z

~50 precursor ions in m/z 561-564 in 30 sec DDA MS/MS precursor ion

selection window


Data Complexity Chimeric Challenge Mass Resolution and Accuracy is Critical

Precursor m/z Product m/z

Mas

s Res

olu

tion


How Does LC-MSE Address Data Complexity?


Alternate Scanning LC-MS (LC-MSE) …time resolved accurate mass measurements

MSE is a UNBIASED process

1 sec

LE

EE

LE

EE

EEEE

D.

m

Gas cell collision energy alternates between low (5 eV) and elevated energy (linear ramp from 15 eV -

42 eV)

LC-MSE alternate scanning provides quantitative accurate mass

measurement for all detectable peptide precursor and product ions

throughout UPLC separation


Precursor and Product Ion Chromatographic Time Alignment

prod

uct M

H+

p

recu

rsor

MH

+

Retention Time


prod

uct M

H+

p

recu

rsor

MH

+

9 Second Retention Time Region

Precursor/Product Apex Time Align to +/-1/10 of 15 sec Chromatographic Peak Width

+/- 1/10 Peak Width (3 sec)Time Alignment Window


Process MSE Raw Data• Precursor and product ion charge state

and monoisotopic accurate mass• Chromatographic retention time apex• RT align precursor and product ions

• Combine intensity for all isotopes and charge states for each peptide

• Mobility drift time apex (optional)• Drift time align precursor and product

ions (optional)

Rank and Allocate Peptides• Extract tentative peptides

• Rank peptide quality based on 14 physicochemical properties

• Tentatively assign peptides to proteins

Rank Proteins• Find best identified protein

• Deplete all precursor and product ions associated with best protein

• Re-rank and re-score remaining protein ID’s

• Select next best identified protein• Repeat process until protein False

Positive Rate limit is reached

Multi-PassModified Protein Query

• Search for modified forms of previously identified proteins

− Missed Cleavages− Post translational modifications

− Chemical modifications− Etc.

IdentityE MSE Search Engine Workflow


IDENTITYE …More Rigour !


The Future!Ion Mobility Enhanced UPLC-IMS-MSE

“High Definition LC-MSE”


IMS – Transfer Region CID for Mobility Drift Time Alignment of Precursor and Product ions

Drift time

Precursor ions separated by IMS

m/z

Precursor ionsDrift time

m/z

Precursor and products share same drift time

Q1 RF only


How Does Ion Mobility Improve Protein Identification Rate and Quality

Reduces interferences— Improved precursor charge state recognition – molecular isotope

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

— More ions detected

— Improved determination of peptide monoisotopic accurate mass

Reduces false positive distraction — Precursor and associated product ions are aligned on the basis of

mobility drift time as well as chromatographic retention time for more secure identifications

— Reduced opportunity for mis-assigning random product ions to a parent ion and producing a false positive


Precursor Ion Spectra

Expanded m/z Spectral Range

Mobility Drift Time Alignment of Precursor Molecular Ion Isotope Data for Improved Charge

State Assignment and Interference Removal


Precursor Ion Spectra

Expanded m/z Spectral Range

Mobility Drift Time Alignment of Precursor Molecular Ion Isotope Data for Improved Charge

State Assignment and Interference Removal


Precursor and Product Ion Chromatographic and Mobility Drift Time Alignment

prod

uct M

H+

p

recu

rsor

MH

+

Retention Time


prod

uct M

H+

p

recu

rsor

MH

+


Precursor/Product Apex Time Align to +/-1/10 of 15 sec Chromatographic Peak Width




prod

uct M

H+

p

recu

rsor

MH

+


Precursor/Product Ion Chromatographic Time AlignmentPrecursors and Identified Product Ions Color-Coded



prod

uct M

H+

p

recu

rsor

MH

+




prod

uct M

H+

p

recu

rsor

MH

+



3 sec Time Alignment Window



Ion

Mob

ility

Drif

t Tim

ePrecursor ions Square , Products ions Round

Parent and Associated Product Ions Align Vertically as a Function of their Similar Mobility

Mobility Drift Time Alignment of Precursor and Product Ion Data


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


Impact of Mass Resolution and Mobility Separation on Protein Identification Rate


High Definition UPLC-MSE Study

E coli lysate digest sample

Identical chromatography for all analyses

Replicate 400 ng digest injections

Three different mass resolution - mobility conditions:—“Sensitivity Mode” ~18,000 RP

—“Resolution Mode” ~ 27,000 RP

—“Resolution Mode” ~ 27,000 RP plus Mobility Drift Time


Protein Identification RateImpact of Resolution Ion Mobility

396

468

674

94% of the 674 proteins ID’d at 29K RP with mobility on are in common with proteins ID’d for same sample using 1500 ng loading and 5 cut 2D-LC analysis.

0

100

200

300

400

500

600

700

800

18k RP, No Mobility 27k RP, No Mobility 27k RP with Mobility

Pro

tein

s Id

en

tifi

ed

400 ng Ecoli Digest


Peptide Identification RateImpact of Resolution and Ion Mobility

18K RP, no mobility 27K RP, no mobility 27K RP with mobility

396Proteins

468Proteins

674Proteins


Summary

Proteomic “System” sample digests are extremely complex and high in dynamic range. This challenges both the detection limit and the selectivity of the MS analytical system

High resolution, accurate mass measurement of both parent and product ion data are critical requirements for reducing protein ID errors when analyzing complex protein digests

For complex, high dynamic range digest samples, protein identification rate increases with increasing mass resolution; however, mass resolution alone is not adequate to deal with the chimeric nature of the data

Ion mobility drift time alignment of precursor/product ion information in combination with chromatographic time alignment and higher mass resolution greatly improves protein ID rate and ID quality


Acknowledgments

Manchester

Jim Langridge

Therese McKenna

Chris Hughes

Iain Campuzano

Hans Vissers

Phill Young

Barry Dyson

Keith Richardson

Richard Denny

Milford

Scott Geromanos

Martha Stapels

Craig Dorschel

Marc Gorenstein

Dan Golick


END


Peptide Identification RateImpact of Resolution and Ion Mobility

18K RP, no mobility 27K RP, no mobility 27K RP with mobility

396Proteins

468Proteins

674Proteins

In-Source Frag. 1739 12%Missed Cleavage 1070 7%Neutral Loss 287 2%Tryptic (pass1) 7897 54%Variable Mods 3837 26%

Documents

The Proteomic Analytical Challenge Future Potential of Ion ... · VP Pharmaceutical and Life Science Business Operations ©2010 Waters Corporation | COMPANY CONFIDENTIAL 2. The Proteomic