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UniGE, Life Sciences Mass Spectrometry 1

The importance of sample preparation for the

analysis of proteins and peptides by liquid

chromatography with mass spectrometric detection

Gérard Hopfgartner, Antoine Lesur and Emmanuel Varesio

Life Sciences Mass Spectrometry, School of Pharmaceutical Science

EPGL, University of Lausanne, University of Geneva,

20 Bd d‘Yvoy, CH-1211 Geneva 4, Switzerland

EBF Focus Meeting: Large meets Small

Brussels, June 20-21, 2011


Challenges in the analysis

of peptides and proteins

• Selectivity : thousands of proteins and peptides

are present in plasma

• Sensitivity : large dynamic range of

concentrations

• Matrix effects : co-eluting analytes can affect the

ionization process and therefore the limit of

quantification

• Sample throughput : Proteomics deals with

moderate samples sets while bioanalysis deals

with large number of samples


Sample Preparation Strategies for

Peptide/Protein Analysis in Plasma

Peptides

300-1000 Da

Peptides

1000-5000 Da

Proteins

> 30 kDa

Proteins

5-30 kDa

Protein Precipitation

Liquid-Liquid Extraction

Solid-Phase Extraction

Affinity Enrichment

Abundant Protein

Depletion

Protein Precipitation

Solid-Phase Extraction

Affinity Enrichment

Partial Protein

Precipitation

Abundant Protein

Depletion

Protein Digestion

Affinity Enrichment

Abundant Protein

Depletion

Affinity Enrichment

(protein)

Protein Digestion

LLE

SLE

Affinity Enrichment

(peptide)


Quantitative Peptide/Protein Analysis Workflow

LIMS 96 DWP

Study

Sample

List Reduction/Alkylation

Digestion

QqQ

QqQLIT

Isolation

Purification

Peptides

Isolation

Purification

++ + + ++++++ ++ ++


Sample Preparation Steps for Protein

Analysis by LC-SRM/MS

1) Removal of abundant proteins

2) Reduction/alkylation

3) Overnight trypsin digestion

4) Removal of un-digested proteins

Mostly

24 hours

Do we need to optimize the process?


Tryptic Digestion of Whole Plasma

Plasma

Volume

Albumins Globulins

50 µl 2.1 mg 1.23 mg

1000 µl 42 mg 24.5 mg

To digest 50 µl of plasma about 70 µg of trypsin (1/50) are needed per sample,

for 96 samples -> 6.7 mg of trypsin

Promega Trypsin Gold, Mass Spectrometry Grade 100µg US$100.13

6700 US$ for 96 samples


Can We Accelerate Trypsin Digestion ?

Microwave digestion Thermomixer digestion

30 minutes at 37, 60°C ?

Why 18 hours at 37°C ?


Heavy Chain sequence

1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYY 6061 ADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSSAS 120121 TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL 180181 YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS 240241 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 300301 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT 360361 KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 420421 GNVFSCSVMHEALHNHYTQKSLSLSPGK 448

Number of amino acids: 448 MW = 49’253.6 Da pI = 8.49

Light Chain sequence

1 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPS 6061 RFSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKVDIKRTVAAPSVFIFPP 120121 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 180181 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 214

Number of amino acids: 214 MW = 23’357.9 Da pI = 6.49

Recombinant monoclonal antibody (mAb), MW = 145’000 Da

Quantitation of mAb in Human Plasma

A. Lesur et al. (2010) J. Chrom. A, 57, 1217


Internal Standard and Protein Analysis

Protein

No IS

Digestion

Peptides

Signature

Peptide

+

Labeled signature

peptide

SRM LC-MS/MS

quantitation

Protein

+

Labeled Protein (IS)

Protein

+

Labeled peptide (IS) Threonine labeled with 13C/15N


Evaluation of Accelerated Digestion


Peptides Kinetic Formation

Thermomixer @ 37°C

Thermomixer @ 60°C

Microwave @ 40%

Microwave @ 100%


Signature Peptide for mAb’s Quantitation in

Human Plasma

min

Thermomixer @ 37°C

Thermomixer @ 60°C

Microwave @ 40%

Microwave @ 100%


Accelerated Digestion in Human Plasma


Standard solutions

NameExpected concentration

(µg/ml)Accuracy

(%)

CAL 1 20CAL 2 100CAL 3 400CAL 4 800

CAL 5 1000

10289.8110104

93.5

Calibration Curve in Human Plasma

2 . 0 e 2 4 . 0 e 2 6 . 0 e 2 8 . 0 e 2 1 . 0 e 3

Analyte Concentration [µg/ml]

1 . 0

2 . 0

3 . 0

4 . 0

5 . 0

6 . 0

7 . 0

8 . 0

9 . 0

Analy

te A

rea / IS

Are

a


Capture of targeted

proteins

(in plasma samples)

Immobilization of

antibodies on

magnetic beads

Tryptic digestion

directly on beads

Microwave

assisted digestion

LC-SRM/MS analysis Removal of the

magnetic beads

Immunocapture Techniques coupled to MS


Goat IgG Captured by Magnetic Beads Coated with Protein G

- 10 min digestion assisted by microwave

- 44% coverage for the heavy chain


How Selective is Liquid Chromatography with

Selected Reaction Monitoring Quantitation?

1 MRIAVICFCL LGITCAIPVK QADSGSSEEK QLYNKYPDAV ATWLNPDPSQ 50

51 KQNLLAPQNA VSSEETNDFK QETLPSKSNE SHDHMDDMDD EDDDDHVDSQ 100

101 DSIDSNDSDD VDDTDDSHQS DESHHSDESD ELVTDFPTDL PATEVFTPVV 150

151 PTVDTYDGRG DSVVYGLRSK SKKFRRPDIQ YPDATDEDIT SHMESEELNG 200

201 AYKAIPVAQD LNAPSDWDSR GKDSYETSQL DDQSAETHSH KQSRLYKRKA 250

251 NDESNEHSDV IDSQELSKVS REFHSHEFHS HEDMLVVDPK SKEEDKHLKF 300

301 RISHELDSAS SEVN 314

MW (average) = 33’713.53 Da pI = 4.35 (mature protein)

4 isoforms are described in UniProtKB (www.uniprot.org/uniprot/P10451#section_alternative)

Heavily phosphorylated and glycosylated protein

Protein: Osteopontin (P10451-1 | OSTP_HUMAN)

http://www.uniprot.org/uniprot/P10451


EFHS*HEFHS*HEDMLVVDPK

14 transitions at the same retention

9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8

Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

9.6e4

Inte

nsity,

cp

s

9.64

Precursor m/z 480.8 z=5+

SRM Selectivity in Human Plasma

(Osteopontin phosphopeptide)

m/z ion charge

501.2 b4 1

588.3 y14 3

377.8 b8 3

922.3 b14 2

461.7 b14 3

511.2 b16 4 401.2 y7 2

757.7 y18 3 251.1 b4 2

633.9 y15 3 277.1 b2 2

414.2 b3 3

577.5 y13 3 497.5 y16 4

b

4

1 1

Product


Product Ion MS/MS Spectrum of m/z 480.8

400 500 600 700 800 900 1000 1100 1200

m/z, amu

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

Inte

nsity

, cp

s

587.5

754.6 686.6

520.4 500.5

683.5 480.7

920.8 574.1 726.5 427.3 853.6 471.2 655.7 835.7 1050.0

m/z ion charge

501.2 b4 1

588.3 y14 3

377.8 b8 3

922.3 b14 2

461.7 b14 3

511.2 b16 4 401.2 y7 2

757.7 y18 3 251.1 b4 2

633.9 y15 3 277.1 b2 2

414.2 b3 3

577.5 y13 3 497.5 y16 4

b

4

1 1

585 586 587 588 589 590 m/z, amu

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

Inte

nsity

, cps

587.5

588.5

496 497 498 499 500 501 502 503 504 505 506 507 m/z, amu

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

Inte

nsity, c

ps

500.5

501.2

756.0 756.5 757.0 757.5 758.0 758.5 759.0 759.5 760.0 m/z, amu

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.3e5

Inte

nsity, c

ps

757.7

758.3 758.8

910 912 914 916 918 920 922 924 926 928 930 932 934 936 938 940 m/z, amu

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

Inte

nsity, c

ps

920.8

922.7

408 409 410 411 412 413 414 415 416 417 418 419 m/z, amu

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

Inte

nsity, c

ps

413.0

414.0 412.6

Product


Found “Albumin” peptide

RHPEYAVSVLLR m/z 480.6 z=3+

Target peptide

EFHSHEFHSHEDMLVVDPK phosphorylated m/z 480.8 z=5+

Database Search (UniProtKB_SP)


What Can We Do To Improve Selectivity

To Avoid False Positive Results?

Sample preparation

Chromatography (UHPLC, multidimensional, …)

1) Multiple MS (SRM3)

2) High Resolution: but How Much ?

3) Ion Mobility Spectrometry (DMS)

………………


Ghrelin in Rat Plasma

GSS1FLSPEHQKAQQRKESKKPPAKLQPR

S1) with octanoyl 3314.6 u

S) without 3188.6 u

7+

6+

5+

200 400 600 800 1000 m/z

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

Inte

nsity,

cps

532.3

638.7 456.5

7+

6+

5+

Desacyl Ghrelin

Full Scan MS Spectrum


y4+

200 400 600 800 1000 m/z

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.8e6

Inte

nsity,

cp

s

675.2

703.6

653.5

740.3

606.4

453.7

y4+

Product Ion Spectrum from 5+ Precursor


0.0 1.0 2.0 3.0 4.0 5.0 min

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

1.20e5

Inte

nsity,

cps

2.38

2.66

m/z 638.7 (5+) -> m/z 675.2 (4+)

m/z 663.7 (5+) -> m/z 675.2 (4+)

Ghrelin

Desacyl Ghrelin

LC-SRM/MS Chromatogram (standard)


0.0 1.0 2.0 3.0 4.0 5.0 min

2000

4000

6000

8000

1.00e4

1.19e4

Inte

nsity,

cp

s

3.52

2.90

3.89

2.38

m/z 638.7 (5+) -> m/z 675.2 (4+)

m/z 663.7 (5+) -> m/z 675.2 (4+)

LC-SRM/MS Analysis Rat Plasma after

Protein Precipitation with HClO4


200 400 600 800 1000 m/z, Da

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

Inte

nsity, c

ps

675.7

513.2 906.5

809.4 454.2

249.1 641.4

m/z 663.7 (5+) -> m/z 675.2 (4+) ->

1. MS3 Spectrum of Desacyl Ghrelin (standard)


Max. 1.4e7 cps.

1.0 2.0 3.0 4.0 5.0 0.0

5.0e6

1.0e7

1.4e7

cps

2.66

Max. 4.4e7 cps.

1.0 2.0 3.0 4.0 5.0 6.0

Time, min

0.0

2.0e7

4.0e7

cps

2.38

LC-MS3 traces (standard)

Ghrelin

Desacyl Ghrelin


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 0.00

1.00e4 2.38

0.0

1.0e7 2.39

1.0 2.0 3.0 4.0 5.0 6.0 Time, min

0.0

5.0e5 2.40

2.83

LC-MS analysis Rat Plasma after Protein

Precipitation with HClO4

SRM3

XIC 454

XIC 513

TIC MS3

SRM

m/z 638.7 (5+) -> m/z 675.2 (4+) -> m/z 454

m/z 638.7 (5+) -> m/z 675.2 (4+) -> m/z 513

Desacyl Ghrelin


2. High Resolution and Accurate Mass Measurements

TripleTOF 5600

AB Sciex

TOF MS and MS/MS ≤ 100 msec

Resolution ≥ 30’000

Mass Accuracy ≤ 2 ppm

Rat Ghrelin (6+)

FWHM = 0.015


LC-TOF/MS

2.6

2.8

Human Plasma

Tryptic Digest

Ghrelin

Desacyl Ghrelin


Human Plasma Digest Spiked with Rat Ghrelin

TIC

2.5 ng on-column

?



XIC m/z 553.1507 +/- 0.35

2.8


553.0 553.5 554.00

400

800

1200

1600

2000

553.1507


2.8

XIC m/z 553.1507 +/- 0.005


3. Differential Ion Mobility Spectrometry –

Triple Quadrupole Linear Ion Trap

(DMS-QqQLIT)

+ ++++ + ++

+++ ++ +

GSS1FLSPEHQKAQQRKESKKPPAKLQPR

Rat Ghrelin and Desacyl Ghrelin

1st Dimension

(HPLC)

2nd Dimension

(DMS)

3rd Dimension

(MS or MS/MS)


Differential Ion Mobility Spectrometry (DMS)

b) When Vc is a function of time,

DMS provides spectral

information DMMP+ Lut

-30 -20 -10 0 10

Compensation voltage (V)

a) When Vc is constant and is

tuned for certain ions species,

DMS is operated as ions filter

Esf(t) Vc

K(E)=Ko(1+α(E))

Ions To

MS

B. Schneider et al. Anal. Chem. (2010), 82, 1867


LC-DMS-Single Ion Monitoring/MS

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0e0

2e5

4e5

6e5

8e5

I n t e

n s i t y

2.7

2.6 DMS OFF

XIC m/z 532.4 (6+)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Time, min

0e0

1e5

2e5

I n t e

n s i t y

2.6

DMS ON

XIC m/z 532.4 (6+)

+ + + +


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Time, min

0

10000

20000

I n t e

n s i t y

2.6

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Time, min

0e0

1e5

2e5

I n t e

n s i t y

2.6

DMS ON

XIC m/z 532.4 (6+)

MS/MS

XIC m/z 532.4 (6+) -> m/z 563.6

LC-SRM/MS vs. LC-DMS/SIM

+ + + +

+ ++++ + ++

+++ ++ +


Differential Ion Mobility Spectrometry (DMS)

With Organic Modifiers (Additional Selectivity)


LC-DMS SIM/SIM Analysis of Rat Ghrelin

Spiked in Human Plasma

Modifier: methanol 1.5 % in N2, SV 2800 Volts


Conclusions

• For proteins and peptides LC-SRM/MS analysis, the sample

preparation strategies strongly depend on the size of the

protein/peptide.

• Protein digestion can be accelerated by increasing the

temperature. The advantage of microwave versus Thermomixer is

still not clear.

• Contrary to small molecules, the Selected Reaction Monitoring

mode is not as selective for multiply charged peptides.

• Selectivity can be improved from the MS side by applying high

resolution approaches or using differential ion mobility

spectrometry (DMS). Waiting for the combination of both……!

• Organic modifiers in DMS positively affects the resolution power

and the selectivity but can also affects the ionization process.


University of Geneva

Tobias Bruderer

Chantal Grivet

Markus Himmelsbach

Tiffany Porta

Tatjana Sajic

Jonathan Sidibé

David Tonoli

Michel Wagner

Novartis

Dieter Zimmer

Stefan Bek

Dionex

Martin Markus

Frank Steiner

AB Sciex

Ron Bonner

Nic Bloomfield

Lyle Burton

Tom Covey

Igor Chernuchevic

Eva Duchoslav

Tanya Gamble

Dominic Gostick

Gordana Ivosev

J.C. Yves Le Blanc

Sasha Loboda

Brad B. Schneider

Stephen Tate

Acknowledgments

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