FluorescenceFluorescenceFluorescenceFluorescenceSensitivitySensitivitySensitivitySensitivity

Enhancements forEnhancements forEnhancements forEnhancements forProteins and PeptidesProteins and PeptidesProteins and PeptidesProteins and Peptides

Using AQCUsing AQCUsing AQCUsing AQCDerivatization Derivatization Derivatization Derivatization withwithwithwith

Capillary LiquidCapillary LiquidCapillary LiquidCapillary LiquidChromatographyChromatographyChromatographyChromatography

Jeffrey L. Holyoke and Steven A.Cohen

PITTCON 2000March 14, 2000March 14, 2000March 14, 2000March 14, 2000

030501

Why Switch to Capillary LC?

• Analysis Sensitivity– Microanalytical– Microprep

• Sample Limited

• Excellent MS Compatibility, Especially ESI

• Small Fraction Volume

030502

Overview

• Applications with Native FluorescentMolecules

• Derivatization of Proteins and Peptideswith 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)

030503

Photomultiplier tube

Fiber optic coupling device

Dichroic mirror

Fluorescence signalFiber optic

ObjectiveCollimating device Laser

beam Ball lens

Column/Capillary

Cell

Set of filters

Laser

LIF Detector Optical Path

030504

Conditions for Small MoleculeStudy

• Waters CapLC System, Liconix Helium CadmiumLaser, Picometrics Zetalif Fluorescence Detector

– 15 cm x 0.32 mm Symmetry C18 (100 Å, 5 �m)– 10 �l/min, A: 0.01% TFA, B: 100% MeOH– 30 to 70% B in 20 min, 50�C Column Temperature– 1 �l Injection– PDA from 220 to 400 nm– Excitation at 325 nm, Emission Detection at 395 nm– Total Run Time: 30 min

Samples– Nucleotide adducts and precursor analog: deoxyguanosyl-amino-biphenyl

(dG-8-ABP), tetrol and benzo[a]pyrene diol deoxyguanosine (BPdG),samples courtesy of Dr. Radoslav Goldman, NIH)

030505

UV Spectra of Small Molecules

Waters CapLC PDA

250 300 350nm0

100

%

250 300 350nm0

100

%

250 300 350nm0

100

%

BPdG

dG-8-ABP

Tetrol

Scanning UV Detector

300250 350 400

250 300 350 400

4003503002500

4

8

12

0

2

4

8

0

5

10

15

20

nm

nm

nm

mAu

mAu

mAuBPdG

dG-8_ABP

Tetrol

N

N

N

NH

O

NH2

O

O H

NH

C

CH

O H

C

CH

C

CH

CHCH

OH

OH

OH

OH

N

N

N

NH

O

NH

O

OHOH

C

CH

C

CH

CHCH

OH

OH

OH

BPdG

dG-8-ABP

Tetrol

030506

UV Detection for Small MoleculeMixture

167 fmol SampleFull Spectrum and Extracted Wavelength

Chromatograms

9 10 11 12 13Minutes

0

9

mAU

344 nm

0

6

mAU

300 nm

TIC

100

1000

mAU

dG-8-ABP

Tetrol BPdG

030507

26 28 30 32 34 36 Time38

100

%

1: Scan ES+ TIC

1.02e7

300 320 340 360 380 400 420 440 460 480 500 520 540 560 580m/z0

100

%

1: Scan ES+ 2.45e5

319

317435

320 436

0

100

%

1: Scan ES+ 1.21e5

454

303304

570455456

571

BPdG

dG-8-ABPTetrol

5 pmols on Column

MS Scan of Compounds 1, 2 and 3

030508

Comparison of UV and LIFDetection for Small Molecule

Mixture

Minutes

mV

0

4

8

12

9 10 11 12 13-4

Tetrol

BPdG

Laser Induced Fluorescence

17

100

%

Diode Array TIC

1.00e6

BPdG

dG-8-ABPTetrolUV Absorbance

BPdG167 fmol

030509

LIF Response to Tetrolm

V

0

5

mV

0

5

100 fmol

50 fmol

-3.5

-3.0

-2.5Blank

-3.5

-3.0

-2.53 fmol

Minutes

-3.5

-3.0

-2.5 10 fmol

9 10 11 12 13

mV

mV

mV

030510

Response Linearity for Tetrol

y = 1128.7x + 1748.1R2 = 0.9991

0E+0

2E+5

4E+5

6E+5

0 100 200 300 400 500

Concentration of Solution (fmol/ul)

Ave

rage

Pea

k A

rea

030511

Experimental ObjectivesExperimental ObjectivesExperimental ObjectivesExperimental Objectives• Show improvements in sensitivity possible with laser-

induced fluorescence (LIF) detection

• Compare detection modes (UV, LIF and MS) for derivatizedand underivatized peptide mixtures

• Explore potential advantages of peptide derivatization with6-aminoquinolyl-N-hydroxysuccinimiyl carbamate (AQC)– Sensitivity– Detection of weakly retained underivatized components– Orthogonal chromatographic mode for peptide mixtures

• Use LC/MS of derivatized peptides to study derivatizationchemistry 030512

Experimental Protocols• Chromatographic System

– Waters CapLC™ System– Waters ZMD Mass

Spectrometer– Picometrics Zetalif LIF Detector

emission at > 395nm– Liconix HeCd Laser operating

at 325 nm– Waters Symmetry® C18

columns (0.32 x 150 mm)

• Peptide Derivatization

– Digest Preparation: BovineCytochrome c was digested with 1percent Trypsin + CaCL2 in NaHCO3(pH 8.5).

– Sample was incubated at 37 C for 24hours.

– Derivatization: The digest was diluted10x with water before derivatization(0.1 mg/ml).

– The diluted sample (20 ul)was mixedwith 60 ul of borate buffer (0.2 M, pH8.8) and 20 ul of AQC.

– The final concentration in the derivativewas 20 ug/ml (1.6 pmol/ul)

030513

Derivatization of Peptides with AQC

Derivatized Amino Acid

H20t1/ 2 ~ 15s

t1/2

au

0.00

0.04

0.08

au

0.00

0.04

0.08

Minutes

14.00 18.00 22.00 26.00 30.00 34.00 38.00 42.00 46.00 50.00

Underivatized, @ 205 nm

Derivatized, @ 260 nm

Analysis of Peptide Mix at 750 fmol

030515

Conditions for Peptide Separations

Chromatographic Conditions

Solvent A: 50 mM NH4Ac, pH 6.90.Solvent B: 60/40 acetonitrile/water .Flow rate: 5 ul/min.Gradient: 15 - 50%B in 50 min

Column: Symmetry C18 (100A, 5 um),0.32x 150 mm

UV Detection: PDA, 248 nm channel

Fluorescence Detection: Excitation 325nm Emission > 370nm

MS

Ionization Mode: ES+Data Type: Compressed centroidMass Range: 500 to 2500

Tuning Parameters: ES+

Source Page (ESI) Capillary: 3.2 kVCone: 50 VExtractor: 4 V

030516

UV Detection for the DerivatizedPeptide Mixture

0

0.3

Au

10 20 30 40 50 60Minutes

0

0.3

Au

Derivatization Blank

3.2 pmol Derivatized Bovine Cytochrome c Hydrolyzate

030517

LIF Detection for DerivatizedPeptides

174

300

mV

10 20 30 40 50 60Minutes

174

300

mV

Derivatization Blank

3.2 pmol Derivatized Bovine Cytochrome c Hydrolyzate

030518

LC/MS Analysis of Derivatized Digest1.6 pmols Bovine Cytochrome c

25.00 30.00 35.00 40.00 45.00 50.00 55.00Time18

100

%

1

100

%

41.20

36.43

34.8033.8232.13

27.08

39.9059.23

58.67

46.3244.00 55.2848.05

49.6251.75 53.48

57.4859.67

T10603

(904, 657)

T7894

(713,642,528)ggk601 Hk624

655

T9670, 585

T131176, 1091T6

899, 814, 729 T2510, 1019T51048, 964, 805

T11653, 1305

T9670, 585

UV at 248FS: 2.10e6

ES+ Scan 600 - 2500FS: 2.41e5

ggk601

Katne903,544

(830) T3659, 1318

T4975

T81120, 975, 561

030519

500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z0

100

%

0

100

%

0

100

%

510M/Z2+ +2 AMQ 1019

M/Z+ + 2 AMQ

910

573

528

814M/Z2+ +1 AMQ

899M/Z2+ + 2 AMQ

603M/Z3+ + 1 AMQ 904

M/Z2+ + 1 AMQ657M/Z3+ + 2 AMQ

521

T2RT = 55.83 minutes

T6RT = 44.48 minutes

T10RT = 41.70 minutes

Mass Spectra for Derivatized Peptides

030520

Details for MS Analysis of DerivatizedPeptides

Worksheet for Tagged PeptidesExpected Retention Tim e

Pe ak AA S e que nce #AA Othe r #Tag MW MW/2 MW/3 with NH4Ac Ob served MW

2 YIPGTK 6 2 1018.7 509.4 339.6 55.83 510, 10191 848.6 424.3 282.9

6 TGQAPGFSYTDANK 14 2 1797.0 898.5 599.0 44.48 729, 814, 8991 1626.9 813.4 542.3

10 CAQCHTVEK 9 heme 614.5 2 1803.0 901.5 601.0 41.7 603, 657, 9041 1973.0 986.5 657.7

030521

Minutes

6.0 10.0 14.0 18.0 22.0 26.0 30.0 34.0

mV

0

4

8

12

16

20

24

AMQ

Asp

Ser

Glu Gly His NH

3

Arg T

hrAl

a

Pro Aa

ba

Cys

2Ty

r

Val

Met

Lys

Ile Leu

Phe

mV

15

25

35

45

55

Minutes20.0 24.0 28.0 32.0 36.0 40.0 44.0 48.0

AMQ

Asp

Ser

Glu G

lyH

is

NH

3Ar

gTh

rAl

a

Pro

Aaba

Cys

2 Tyr

Val

Met

Lys Ile

Leu

Phe

Future Analysis: AQC DerivatizedAmino Acids with LIF - 2 pmols on

Column474 Fluorescence Detector5 uL cell volumeEx: 250Em: 395

LIF Fluorescence DetectorTransverse IlluminationEx: 325Em: >370

030522

Summary and Conclusions• Capillary LC analysis can provide low or even sub-femtomole

detection limits for a variety of analytes• LIF detection is highly suitable for capillary LC and is one of

the most sensitive detection methods for molecules withfluorescence at available laser wavelengths

• Derivatization of peptide mixtures with AQC offers a highlysensitive alternative to analysis of underivatized samples

• MS results demonstrate successful production of fully taggedpeptides with little evidence of partial derivatization

• Derivatization of tryptic digests provides retention forhydrophilic small peptides and simplifies LC and LC/MSanalysis of these components

• Derivatization can provide improved sequence coverage ofthe protein by analysis of the hydrophilic peptides

030523

Acknowledgments:Cozette Cuppett, - Waters

Harold O. Johnson III, - 3M

Radoslav Goldman, NIH now at Georgetown

HongJi Liu, - Northeastern University

030524