Download - Fluorescence sensitivity enhancements for proteins and peptides … · 2007. 11. 21. · Met Lys Ile Leu Phe mV 15 25 35 45 55 Minutes 20.0 24.0 28.0 32.0 36.0 40.0 44.0 48.0 AMQ

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  • 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