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
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