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©2011 Waters Corporation 1

Increasing the Peak Capacity of LC/MS/MS Systems for the Analysis

of Complex Biological Samples

Mark A. McDowall

Pharmaceutical Discovery & Life Sciences

Waters Corporation, Manchester, UK


Discovery Metabolomics Workflow

Differential Analysis

Comprehensive Data Acquisition

ID Metabolites of Interest

Report Generation

Experimental Design


Discovery Proteomics Workflow

Differential Analysis

Comprehensive Data Acquisition

ID Proteins of Interest

Report Generation

Experimental Design

Hi3 Protein Quantification


Binocular Vision of Biological Processes

PROTEOMICS METABOLOMICS


Limitations in Bottom-Up LC/MS/MS

Dynamic range of sample > dynamic range of MS

Many peptides in a small analytical space

Under-sampling of precursor ions in LC/MS/MS

Up to 50% of LC/MS/MS Spectra are chimeric1

Sensitivity

LC / MS / MS I ^ 1. Houel S. et al, J. Proteome Res., 2010, 9 (8), 4152-4160.


Non-Dispersive (Filter) Separations

— SPE

Ion Mobility — FAIMS / DIMS

Mass Spectrometry — Quadrupole

— ITD (Precursor Selection)

— Magnetic Sector

Dispersive Separations

— HPLC / UPLC

Ion Mobility — TW-IMS

Mass Spectrometry — TOF MS

— FT-ICR

— OBT

HPLC (Seconds) > TW-IMS (mSeconds) > TOF-MS (µSeconds)

Dispersive / Parallel Analytical Systems …the timeing is right !


Data-Dependent MS/MS …Targets the Most Abundant Peptides

Michalski et. al., J. of Prot. Res., 2011, 10, 1785-93. HeLa, 1D SDS, LTQ OrbiTrap Velos, >1+


Data-Dependent MS/MS …co-fragmented peptides frequently observed

Michalski et. al., J. of Prot. Res., 2011, 10, 1785-93. HeLa, 1D SDS, LTQ OrbiTrap Velos, >1+

Most of the time, two peptides or more are fragmented at once

Not a problem for abundant peptides

In the median case, only 14% of the peptide current in the isolation window is due to the precursor ion.


Peak Capacity

Separation Space Peak Width

Pc ~


Peak Capacity …multiple dimensions (LC, MS, IMS, etc)

IDEAL SITUATION Fully Orthogonal Separations

Regular Distribution

Pc(system)= Pc1 x Pc2

REAL SITUATION Partially Orthogonal Separations

Uneven Distribution

Pc(system)= Pc1 x Pc2 x F

F = fraction of bins occupied

Giddings JC. J Chromatogr A (1995);703:3 Gilar M. J Sep Sci (2005);28:1694


Increasing Peak Capacity …Travelling Wave Ion Mobility

2

Rmax = maximum resolution for travelling wave ion mobility (Ca 40FWHM) K = ion mobility coefficient Derived from: Shvartsberg AA. Anal Chem (2008);80:9689 Personal communication: Jason Wildgoose

~ 22

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Arrival Time (ms)

Relati

ve Inte

nsity (

%)

(GRGDS)2+ 211.7 Å²

(SDGRG)2+ 222.7 Å²

IMS Resolution (Ω/ΔΩ) = 44 FWHM

SYNAPT G2 2.5mBar N2


2

Increasing Peak Capacity …Travelling Wave Ion Mobility

N(system) = NLC x NIM x NMS 10,000’s (HRMS) > 1000’s (2D-LC) > 100’s (1D-LC) > 10’s (IMS)


2

IMS Increases Peak Capacity …by creating a datacube

Nm/z

N(system) = NLC x x NMS 10,000’s (HRMS) > 1000’s (2D-LC) > 100’s (1D-LC)


LC / MS / MS I

^

Data Independent UPLC/MS/MSE …increasing peak capacity & MS duty cycle

E VERYTHING

^


Waters TriWave Technology

2 3 1 1 1 1


Precursors IMS Separated

Ionized Precursors

Precursors Transferred to TOF MS

Increasing Peak Capacity UPLC/IMS/MSE …deconvoluting chimericy

2 3 4a

Co-Eluting Peptides

1


Precursors IMS Separated

Ionized Precursors

Precursors & Products Time Aligned

Increasing Peak Capacity UPLC/IMS/MSE …deconvoluting chimericy

3 4b

Co-Eluting Peptides

1 2


Shewanella oneidensis …IMS Separation of Co-eluting (UPLC) Peptides

2x PRECURSORS WITHIN +/-1 DA (i.e. THEORETICAL MS/MS WINDOW)

AT SAME UPLC RETENTION TIME

Drift Time (bins)

MOBILITY SEPARATION

Peptide A Peptide B

PEPTIDE A

PEPTIDE B

Drift Time

Peptide A

Peptide B


Impact of Ion Mobility Enhanced Precursor/Product Alignment

IMS OFF

IMS ON


Increasing Peak Capacity …2D RPxRP UPLC (High pH/Low pH)

0-56% B in 70 minutes 20 mM NH4OH pH 10

1

100

%

Bovine_Hemoglobin_Digest_Stored_091803_1 1: Scan ES+ TIC

4.51e928.55

18.75

17.36

16.3010.99

8.91

4.704.29

6.29

11.4013.24

11.9314.09

23.86

22.79

22.39

19.6119.93

27.0026.68

26.51

26.06

35.0530.68

31.41

34.27 36.19

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00Time1

100

%

g TIC

4.37e830.68

18.95

15.79

8.53

6.775.705.214.10

14.0310.38

9.64

13.2111.73

16.41

18.58

18.21

25.68

22.39

21.0419.89 24.20

29.41

29.0025.92

41.26

35.85

35.48

37.65

39.5841.92

pH 10

pH 2.6

0.1% Formic acid, pH 2.6 0-42% acetonitrile in 50 min

20mM ammonium formate, pH 10 0-42% acetonitrile in 50 min

Gilar M. et al, J. Sep. Sci. 2005, 28, 1694-1703

neutral acidic acidic

basic basic

+ +

+ +

+

- -

-

-

Δ pI


Increasing Peak Capacity …2D RPxRP UPLC (High pH/Low pH)

Gilar M. et. al, J. Sep. Sci. 2005, 28, 1694-1703

Acidic peptides are more effectively retained in RP at

acidic pH.

Basic peptides are more effectively

retained at basic pH.


Increasing Peak Capacity …Caenorhabditis elegans

1D Chromatography 1 µg C. elegans < 2 hours

2D-3 Fraction 1.5 µg C. elegans < 4 hours

2D-5 Fraction 2.5 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

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


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 20,000 5 ppm precursor window 12 ppm product window Proteins had to be in 2/3 replicates


Increased Proteome Coverage …of Caenorhabditis elegans

1D UPLC/HDMSE

914 Proteins

1D UPLC/MSE

503 Proteins

475 Proteins



2D(3) UPLC/HDMSE

1415 Proteins

2D(3) UPLC/MSE

798 Proteins

732 Proteins



2D(5) UPLC/HDMSE

1902 Proteins

2D(5) UPLC/MSE

1017 Proteins

934 Proteins


Hi3 Absolute Quantification of Proteins …C. elegans

Identified in 1D, 2D-3Fraction, and 2D-5Fraction Identified in 2D-3Fraction and 2D-5Fraction Identified in 2D-5Fraction only

[ ]stnd Internalstnd. Internalintensity peptide

XProtein intensity peptide3

1i

3

1i ⋅∑

∑

=

=


Qualititative & Quantitative Analysis …mouse brain

Sample — Tryptic protein digests of 4 biological replicates of double

knock-out mice and 4 controls

— Estimated protein concentration ∼ 2-6 µg/µl

1D UPLC/IMS/MSE conditions — 120 min gradient from 1 to 40% acetonitrile (0.1% formic acid)

— 1 µl injected; calculated amount 1.4 µg/injection

— UPLC/IMS/MSE (Data Independent Acquisition)

o IMS = 40 FWHM

o ToF MS = 25,000 FWHM


1D UPLC/IMS/MSE

2597 Proteins


Technical Reproducibility

Loading 1.4 µg Gradient time 120 minFDRprotein 0.71%

1961 proteins (> 2/3 technical replicates)

1615

270289

276

11399

134


PCA of UPLC/IMS/MSE Data

-50

-40

-30

-20

-10

0

10

20

30

40

50

-50 -40 -30 -20 -10 0 10 20 30 40 50

t[2]

t[1]

J48_

001

J48_

002

J48_

003

J49_

001

J49_

002

J49_

003

J52_

001

J52_

002 J5

2_00

3

J51_

001

J51_

002

J51_

003

12 independent UPLC-IMS-MSE experiments. Variance of technical replicates is significantly smaller than biological replicates.


Hi3 Quantification …of the 1961 Replicating Proteins

Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Silva et al. MCP 5 (2006) 144

[ ]standard internalstandard internal intensity peptide

x protein intensity peptide3

1i

3

1i ⋅∑

∑

=

=


Increasing Sensitivity


Increasing Sensitivity …by incorporating a conjoined Ion Guide


Increasing Sensitivity …by incorporating a conjoined TW Ion Guide

No Electric Field

N2 Flow

1 mbar N2


With Electric Field (25 V between guides)

N2 Flow

E


1 mbar N2 T-Wave

T-Wave




Synapt G2-S vs. Synapt G2 …the effect of the StepWave Ion Guide


UPLC/HDMSE E.coli …Peptide Identification Rates

# peptides

0

2000

4000

6000

8000

10000

12000

14000

10 ng 50 ng 400 ng

Pept

ides

Column Load

G2-S

G2

E. coli (Cytosolic Fraction ) Tryptic Digest UPLC/HDMSE 75 µm column 90 min gradient


UPLC/HDMSE E.coli …Protein Identification Rates

0

100

200

300

400

500

600

700

800

10 ng 50 ng 400 ng

Prot

eins

Column Load

G2-S

G2

E. coli (Cytosolic Fraction ) Tryptic Digest UPLC/HDMSE 75 µm column 90 min gradient


UPLC/HDMSE E.coli …Peptide ‘Precursor’ Resolution

Peptide Resolution >45K FWHM for ‘Precursor’ Ions Across Wide m/z Range

m/z 401 m/z 552 m/z 800


UPLC/HDMSE E.coli …Mass Error Distribution

RMS Error: 0.9ppm


Summary

Protein digests can be extremely complex.

Number of unique precursors >100 ions/sec.

LC/MS/MS is a (self limiting) serial process.

LC/IMS/MS is a multiplexed alternative.

Chimericy is a major limitation in LC/MS/MS.

LC/IMS/MS significantly reduces chimericy.

StepWave significantly increase sensitivity.


Acknowledgements

Waters Corporation, Manchester UK

Tim Riley Jim Langridge Chris Hughes Lee Gethings Jonathan Williams Barry Dyson Keith Richardson

Waters Corporation, Milford MA

Scott Geromanos Craig Dorschel Martha Stapels Dan Golick Steve Ciavarini Jose De Corral

PLGS (UPLC/IMS/MSE)Interest Group

Konstantinos Thalassinos University College London, UK

Stefan Tenzer Mainz University, Germany

Twan America Plant Research International, NL

Arthur Moseley & Will Thompson Duke University, USA

Andrew Ottens Virginia Commonwealth University, USA

Greg Cavey Southwest Michigan Innovation Center, USA

Yishai Levin Weizmann Institute of Science, Israel

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