Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
Practical Method Development and Optimization for UHPLC and HPLC Separations of PeptidesPractical Method Development and Optimization for UHPLC and HPLC Separations of Peptidesp p p pfor Peptide Mapping Analyses Using Different Mobile Phase Additivesfor Peptide Mapping Analyses Using Different Mobile Phase Additives
Thomas J. Waeghe1, Benjamin Libert2, Stephanie A. Schuster2, and Barry E. Boyes2Thomas J. Waeghe , Benjamin Libert , Stephanie A. Schuster , and Barry E. Boyes1MAC MOD Analytical Inc 103 Commons Court Chadds Ford PA 19317; 2Advanced Materials Technology Inc Wilmington DE 19810 Presented at EAS 20161MAC-MOD Analytical, Inc., 103 Commons Court, Chadds Ford, PA 19317; 2Advanced Materials Technology Inc., Wilmington, DE 19810 Presented at EAS 2016
Abstract Parameters That Affect Peptide LC MS Separations of Enolase Tryptic Digest LC MS Extracted Ion Chromatograms Total-ion Chromatogram Showing Enolase Tryptic DigestAbstract pReversed-Phase Gradient Separations
LC-MS Separations of Enolase Tryptic Digest Using Different Mobile Phase Additives
LC-MS Extracted-Ion Chromatograms for Gradient Input Runs at Two Temperatures PT
GAK
LI
VDAI
K
Total ion Chromatogram Showing Enolase Tryptic Digest Fragments Obtained Using Near-Optimum Conditionsp
Parameter Determined by Impacts performanceUsing Different Mobile Phase Additives for Gradient Input Runs at Two Temperatures
9.0e6
EM
R
LR
SHFF
K
QEF
MIA
PAE
EALD
L
TICTune File ValuesSource Type: HESI
Peptide mapping is a critical competency and technique for both research anddevelopment and for quality assurance and product release for many new
Parameter Determined by Impacts performance• Most labs choose acetonitrile for viscosity • Peak width
Same LC conditions as shown on previous panel for LC-UV 10 mM Trifluoroacetic Acid20 mM Formic Acid
2.5
0.90
0.95
1.00 (x1,000,000)
1:626.30(+)1:597.30(+)1:572.30(+)1:560.30(+)1:550.80(+)1:546.30(+)1:404.20(+)1:387.20(+)1:378.70(+)1:373.20(+)
15.12
1
14.42
6
15.50
2
30 min 0.90
0.95
1.00 (x1,000,000)
1:626.30(+)1:597.30(+)1:572.30(+)1:560.30(+)1:550.80(+)1:546.30(+)1:404.20(+)1:387.20(+)1:378.70(+)1:373.20(+)
26.62
5
60 min8.0e6
HG
DKL
V
HEA
LE
TAN
K
MK
DLV
VGL
DW
EAW
S
AGG
ALA
LQVA
PN
IQTA
Note: MS spectra were acquired with data-dependent acquisition
Source Type: HESICapillary Temp (C): 225.00Source Heater Temp (C): 150.00Sheath Gas Flow (): 35.00Aux Gas Flow (): 10 00development and for quality assurance and product release for many new
biopharmaceuticals including peptide drugs, monoclonal antibody drugs andOrganic modifier
• Most labs choose acetonitrile for viscosity, mass transfer, efficiency reasons, UV transparency, ionization efficiency in MS
• Selectivity• Retention• Peak capacity
chromatograms
MS Scan: 0 2 s (300–2000 m/z)
2.0
0.65
0.70
0.75
0.80
0.85
1:1247.30(+)1:995.50(+)1:921.50(+)1:911.50(+)1:896.50(+)1:889.40(+)1:878.50(+)1:814.40(+)1:807.90(+)1:789.90(+)1:733.40(+)1:708.70(+)1:707.15(+)1:702.20(+)1:659.40(+)1:644.90(+)6 630( )
10.38
2
11.92
0
19.51
9
30 min60 C
0.65
0.70
0.75
0.80
0.85
1:1247.30(+)1:995.50(+)1:921.50(+)1:911.50(+)1:896.50(+)1:889.40(+)1:878.50(+)1:814.40(+)1:807.90(+)1:789.90(+)1:733.40(+)1:708.70(+)1:707.15(+)1:702.20(+)1:659.40(+)1:644.90(+)6 630( )
24
35.20
5
25.50
6
27.32
4
60 min60 C
7.0e6
K DK
TTEK
GEN
FHH
GAS
TGV
K 5]
SHR
ISLD
GT
DLY
HS
LM
ED
TFIA
D
PFA
EDD
SHFF
K
LNG
GSH
AV
GD
EG
GVAux Gas Flow (): 10.00
POSITIVE POLARITYSource Voltage (kV): 2.20Source Current (uA): 100.00
antibody-drug conjugates (ADCs). New 2-µm superficially porous columns,capable of pressures up to 1000 bar can deliver high resolution /peak capacity
• Peak capacity
Mobile phase • UV transparency• Peak shape• Peak width
MS Scan: 0.2 s (300–2000 m/z)
Multiple Ion Chromatogram scale (x 106)1.5
0.40
0.45
0.50
0.55
0.601:1629.30(+)
( )
10.78
0
0.600
12.27
1
11.06
6
12.81
8
0.40
0.45
0.50
0.55
0.601:1629.30(+)
( )
17.45
318
.22
38.09
4
.420
4
22.72
3
2
5 0e6
6.0e6
AIE
K
TAIE
K
VY
HN
LKKN
PN
S
DK
TV
EV
ELT
NAV
FAG
SIV
PSG
PA
FVK
DLT
VTN
PKm
ethy
l [C
5G
WG
VMVS
AV
DD
FL
GP
QLA
D
SG
ETE
VS
IED
P
WEA
WS
PVPF
LNV
LG
ASA
GN
V
S-Lens RF Level (%): 69.00FTMS Full Micro Scans: 1FTMS Full Max Ion Time (ms): 50.00
capable of pressures up to 1000 bar, can deliver high resolution /peak capacityseparations, and can be used at temperatures up to 60 C. Moreover, a variety
padditive type and concentration
UV transparency• LC-MS sensitivity (S/N)• Column stability at pH and temperature
Peak width• Loading (dynamic range)• Selectivity
LC MS i l
g ( )All chromatogram are on the same scale. 1.0
0.15
0.20
0.25
0.30
0.35
4.721 9.354
13.49
7
8.634
13.74
7
0
9.066
882 3
8.900
10.74
7
438
015
0
390 23
.699
13.76
6
14.43
0
6 5.880
7.930
0.15
0.20
0.25
0.30
0.35
25.86
823.39
024
.098
2 372
2.647
1.
13.94
8
75
34.88
0
4
21.60
4
24.11
3 25.51
118.11
0
55
3.493 4.0e6
5.0e6
SK
IATA
RIA
TA
VK IG
SEV
KYD
LDF
LR
KNPN
SD
GN
PT
ALLL
K
EEL
GD
NV
ND
VIA
P
GIQ
IVAD
Dar
bam
idom
QD
SFAA
GR
A
WLT
G
YP
IV
FAE
DD
W
TSP
YVLP
min
) R
YG
LLR
LSK
Data-Dependent Acquisition SettingsScan Event Details:1: FTMS + positive high res=30000
of mobile phase additives include trifluoroacetic acid (TFA), ammoniumformate/formic acid buffers and a new additive, difluoroacetic acid (DFA), can
• LC-MS signal
Gradient steepness• User experience
S l l it• Selectivity
P k it0.5
1.00 (x1,000,000)
1:37870(+)1:373.20(+)
662 5 1.00 (x1,000,000)
1:37870(+)1:373.20(+)
5
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min0.00
0.05
0.10
0.15
2.513
14.
15.115. 18
.
2.250 7.8
12.97
36.4 24.0
8.307
23.3
19.49
2
7.373
21.87
3
9.076 15
9.416
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 min0.00
0.05
0.10
0.15
17.19
3
2
17.19
3
25.89
5
9.893
11.79
32.262
46.94
0
1.077
14.312
15.03
315
.9079.4
75
29.65
3 44.16
42
16.20
0
28.05
3.0e6
AR
DS
R
DV
K H
LAD
LS
GVL
HA
DG
KTF
AEA
LYD
LDFK
AA
DA
LSES
IK
IEE
NV
TAG
SEFF
K C AA
QG
VSLA
AS
VS
IED
PF T
(19.
7
LNQ
K KH
LAD
Lp g(200.0–2000.0)Collision Voltage = 0.0V
2: ITMS + p norm Dep MS/MSformate/formic acid buffers and a new additive, difluoroacetic acid (DFA), canbe applied for effective development and optimization of rugged and robustseparations of comple en me digests
Gradient steepness • Sample complexity• Experimentation and optimization software
• Peak capacity• Peak height
• Selectivity 0 0 5 0 10 0 15 0 20 0 25 0 30 0 35 0 min0 0 5 0 10 0 15 0 20 0 25 0 30 0 35 0 min0.0Comparison of multiple-ion f C S
0.75
0.80
0.85
0.90
0.95
1:80790(+)1:789.90(+)1:733.40(+)1:708.70(+)1:707.15(+)1:702.20(+)1:659.40(+)1:644.90(+)1:626.30(+)1:597.30(+)1:572.30(+)1:560.30(+)1:550.80(+)1:546.30(+)1:404.20(+)1:387.20(+)1:378.70(+)
15.66
913
20.69
1
16.61
5
30 min30 C 0.75
0.80
0.85
0.90
0.95
1:80790(+)1:789.90(+)1:733.40(+)1:708.70(+)1:707.15(+)1:702.20(+)1:659.40(+)1:644.90(+)1:626.30(+)1:597.30(+)1:572.30(+)1:560.30(+)1:550.80(+)1:546.30(+)1:404.20(+)1:387.20(+)1:378.70(+)
27.94
527
.984 37
.956
60 min30 C
2.0e6
TGAP
A
SVY
D
AN
ID T Y
NQ
IGTL
IGLD
CAS
LGAN
AILG
RY
PIV
AAD
ALLL
K
NVP
LYK2: ITMS + p norm Dep MS/MS
(110.0–2000.0) Most intense ion from (1)
separations of complex enzyme digests.Column temperature
• Analyte requirements (recovery, stability)• Column stability requirements• Flow rate (thermal heating)
• Selectivity• Recovery• Analysis time
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min
10 mM Difluoroacetic Acid10 mM Ammonium Formate
10 mM Formic Acid
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 minchromatograms from LC-MS analyses of enolase tryptic digests using formic acid ammonium 050
0.55
0.60
0.65
0.70
1:1629.30(+)1:1247.30(+)1:995.50(+)1:921.50(+)1:911.50(+)1:896.50(+)1:889.40(+)1:878.50(+)1:814.40(+)1:807.90(+)
11.33
2
11.9
13.48
7
15.04
9
133
30 C0.55
0.60
0.65
0.70
1:1629.30(+)1:1247.30(+)1:995.50(+)1:921.50(+)1:911.50(+)1:896.50(+)1:889.40(+)1:878.50(+)1:814.40(+)1:807.90(+)
19.51
2
20.67
2
26.95
5
9.726
30 C1.0e6
VN
L
KA
N
Activation Type: CIDMin. Signal Required: 500.0Isolation Width: 4.00Normalized Coll Energy: 35 0
We will demonstrate the usefulness of these columns and additives in thep • Flow rate (thermal heating)
• Instrument capability and pressure limit • Peak width• Peak capacity
Note: 2.2–52.2% in 40 minusing formic acid, ammonium formate/formic acid, trifluoroacetic acid, and difluoroacetic acid as 0.30
0.35
0.40
0.45
0.50
14.05
2
15.34
9
10.10
7
7
12.06
8
14.
0.30
0.35
0.40
0.45
0.50
19.55
3
27.49
6
23
48.00
7
24.22
6
20.29
1 25.50
7 29
0.0e0Normalized Coll. Energy: 35.0 Default Charge State: 2Activation Q: 0.250Activation Time: 30.000CV 0 0Vdevelopment of peptide mapping separations, and will offer a stepwise method
development and optimization strategy for such separations. Flow rate• User choice• Column and Instrument pressure limits
C
• Selectivity• Peak capacity
mobile phase additives.
DFA offered 2 3 fold better signal 0.05
0.10
0.15
0.20
0.25
15.36
9
6.381
17.40
7
1
3.374
1.554
11.10
4
3.260
8.707
10.60
1
7.289
26.00
1
23.04
623
.419
15.36
715
.037
4.267
10.60
4
0.05
0.10
0.15
0.20
0.25
27.43
0
52.71
3
36.68
7
9.243
24.71
9
33.11
3
7.120
10.65
3
2
44.34
0
7.380
3.638
17.51
0
15.44
316
.42
11.19
6 45.17
1
43.24
1 43.96
5
2.813
11.00
7
24.82
0
30.28
1
51.58
0
27.47
627
.932
36.82
0
26.95
027
.000
41.90
7
2.687
12.58
6
21.54
7
30.56
9
18.34
6
2 4 6 8 10 12 14 16 18
-1.0e6min
CV = 0.0V
Number of Scan Events: 6
86% Coverage: Enolase 1 OS=Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GN=ENO1 PE=1 SV=3
development and optimization strategy for such separations. • Column particle morphology and diameter Peak capacity
• Packing particle size
DFA offered 2–3 fold better signal intensity compared to TFA for LC-MS, with comparable narrow peaks
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min0.00
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 min0.00
Sample: Enolase Tryptic Digest in 0.5% HCOOH, 2 L of 1 µg/µL Column: HALO 2 Peptide ES-C18, 2.1 x 100 mm, 2 m Flow: 0 3 mL/min
Instrument: Shimadzu Nexera UHPLCMS: Thermo Orbitrap Velos Pro ETD
PressurePacking particle size
• Column length• Flow rate
Column temperature
• Retention• Selectivity
MS, with comparable narrow peaks and retention. For LC analysis conditions, see panel 8
Flow: 0.3 mL/minMobile Phase A: 0.1% (v/v) DFA in waterMobile Phase B: 0.1% (v/v) DFA in CH3CNGradient: 4–40% CH3CN in 18.34 min.
1
• Column temperature
4 10 13
G ad e t 0% C 3C 8 3Column temp.: 35 CUV Detection: 220 nmData Rate: 10 Hz
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min Response Time: 0.05 s
Method Development Approaches Recommended Method Development Strategies forMethod Development Approachesfor Peptide Mapping Separations
Recommended Method Development Strategies for RPLC-UV and RPLC-MS of Protein Enzymatic Digestsfor Peptide Mapping Separations
Systematic Approach (DryLab) Wang, Carr et al. Approach*Systematic Approach using Computer Simulation (DryLab 4)
• ColumnHALO 2 Peptide ES C18
Systematic Approach (DryLab)1. Select desired column geometry.2 Choose initial flow rate based on column ID
Wang, Carr et al. Approach1. Choose desired column geometry.2 S t fl t f lComputer Simulation (DryLab 4)
• Two-dimensional Design– HALO 2 Peptide ES-C18
2.1 x 100 mm, 2 m 2. Choose initial flow rate based on column ID.3. Select two different gradient times varying
by 2- or 3-fold
2. Set flow rate for column max. pressure or 75% of max. pressure.Two dimensional Design
– tG x T • Mobile PhaseA S l t 0 1% DFA i t
by 2- or 3-fold.4. Select two column temperatures varying by
20–40 C
3. Set final so that last peak of interest elutes at very end of gradient.
– 2–3 gradient steepnesses– 2–3 column temperatures
– A Solvent: 0.1% DFA in water– B Solvent: 0.1% DFA in CH3CN
20–40 C.5. Set gradient range of 0–5 to 60% CH3CN or
0–5 to 50% CH3CN4. Choose temperature based on column
stability and “fluidity” 60C?2 3 column temperatures 3
– initial: 2% B 50% B30 min 60 min45 min
0 5 to 50% CH3CN.6. Carry out gradient runs at T1 and T2 with
two different gradient times.5. For example, use 2–50% CH3CN for initial
run.– final : 50% B
• Column Temperatures60 C
30 min. 60 min.45 min. two different gradient times.– For example, 30 and 60 min. at 30 and 60 C.
7. Carry out an additional gradient run at 6. Set gradient time for maximum allowable
time.Column Temperatures– 30 and 60 C
y gintermediate temperature and gradient time.– For example, 45 min. at 45 C.
8 E t RT d i t D L b ft
time.7. Calculate %B at elution of last peak of
interest (using RT %B/min t0 and tD)• Gradient Times
30 d 60 i45 C
8. Enter RTs and areas into DryLab software.– Note: Peak matching can be tricky and difficult
9 Compare the predicted intermediate
interest (using RT, %B/min, t0, and tD).8. Rerun sample with the 2–X %B limit for
gradient time corresponding to that final– 30 and 60 min.– 45-min x 50 C run used for
45 C 9. Compare the predicted intermediate gradient run vs. actual intermediate run to assess peak matches.
gradient time corresponding to that final %B.
peak matchingassess pea atc es
10. Determine whether there are any optimum regions of robust resolution in the 2-D * Xi li W D i ht St ll Ad S h lli d P t C
30 Cg
resolution map.11. Optimize linear gradient in gradient editor
* Xiaoli Wang, Dwight Stoll, Adam Schellinger, and Peter CarrPeak Capacity Optimization of Peptide Separations in Reversed‐Phase Gradient Elution Chromatography: Fixed Column Format
l h
8Optimization was carried out to identify a robust gradient time
and temperature combination for a run time < 40 minutes
p g gor choose a two or three segment gradient to find optimum conditions. 14
Anal. Chem. 2006, 78, 3406‐3416
p
LC UV Ch t f G di t I t R Summary and ConclusionsPeptide Identification Using Proteome Discoverer SoftwareTypical Peptide Mapping Conditions and LC-UV Separations of Enolase Tryptic Digest LC-UV Chromatograms for Gradient Input Runst T T t F D L b I t
yConfidence Single Letter
Amino Acid SequenceRT [min]
Sequest HT Modifications # PSMs
# Missed Cleavages Theo. MH+ [Da] XCorr
Sequest HT∆M [ppm]
Sequest HTDesirable Mobile Phase Additive Properties Using Different Mobile Phase Additives at Two Temperatures For DryLab Input30 i di t • A sample of enolase was digested with trypsin, and was analyzed
using gradient UHPLC using a 100-mm HALO 2 Peptide ES-C18High TGAPAR 1.99 1 0 572.31509 1.85 -1.32High ANIDVK 5.22 1 0 659.37227 1.99 -2.06High HLADLSK 5 59 2 0 783 43593 2 87 -0 89
10 mM TFASample: Enolase Tryptic Digest in 0.5% HCOOH, 1 µg/µL Column: HALO 2 Peptide ES-C18, 2.1 x 100 mm, 2 m Flo 0 3 mL/min
30 min. gradient60 C
60 min. gradient60 C using gradient UHPLC using a 100-mm HALO 2 Peptide ES-C18
column.High HLADLSK 5.59 2 0 783.43593 2.87 -0.89High IATAIEK 6.32 1 0 745.44543 2.26 -1.14High IGSEVYHNLK 7.76 2 0 1159.6106 2.98 0.04High DGKYDLDFKNPNSDK 8 46 3 2 1755 81842 5 11 -0 7
Typical Conditions for RPLC Separations of Peptides
Desirable properties for additives
Flow: 0.3 mL/minInjection vol.: 1µLMobile Phase A: 10 mM DFA in waterM bil Ph B 10 M DFA i CH CN
• Various mobile phase additives were compared in terms of LC-UV d LC MS i l k h k idth d t ti
High DGKYDLDFKNPNSDK 8.46 3 2 1755.81842 5.11 -0.7High LNQLLR 8.75 2 0 756.47265 2.01 1.28High TFAEALR 8.94 1 0 807.43593 2.06 -1.23High YDLDFKNPNSDK 9 13 1 1 1455 67505 3 81 0 89
Separations of Peptides• 150 or 250 mm C18 column
M bil h A t ith 0 02
additives • Very high purity, quality, stability
and reproducibility18 20 22
Time (min)
Mobile Phase B: 10 mM DFA in CH3CNGradient: 2–47% B in 40 min., except as indicated belowUV Detection: 220 nm
and LC-MS signal, peak shape, peak width and retention.• Difluoroacetic acid (DFA) offers peak shape peak width and
High YDLDFKNPNSDK 9.13 1 1 1455.67505 3.81 -0.89High DGKYDLDFK 9.22 2 1 1100.52587 2.52 -1.5High KAADALLLK 9.97 2 1 942.59824 2.61 -0.38High YDLDFK 10 10 1 0 800 3825 1 74 1 28
• Mobile phase A: water with 0.02–0.2% (v/v) additive
and reproducibility• Low UV absorbance with
t bl b li d bl k
( )Data Rate: 10 HzResponse Time: 0.05 s
• Difluoroacetic acid (DFA) offers peak shape, peak width and retention comparable to TFA and ammonium formate/formic acid,
High YDLDFK 10.10 1 0 800.3825 1.74 -1.28High GNPTVEVELTTEK 10.29 1 0 1416.72167 3.57 -0.73High VNQIGTLSESIK 10.68 2 0 1288.71071 3.67 -1.3Hi h SIVPSGASTGVHEALEMR 10 76 1×O id ti [M17] 1 0 1856 91709 4 23 0 29
• Mobile phase B: CH3CN with same % additive or 10–30% lower than in
acceptable baselines and blank runs p ,
but with much better LC-MS ionization efficiency (signal-to-noise).High SIVPSGASTGVHEALEMR 10.76 1×Oxidation [M17] 1 0 1856.91709 4.23 -0.29High IEEELGDNAVFAGENFHHGDK 11.36 2 0 2328.05273 4.73 -2.91High AAQDSFAAGWGVMVSHR 11.71 1×Oxidation [M13] 1 0 1805.83878 4.72 -1.41Hi h SIVPSGASTGVHEALEMR 12 17 2 0 1840 92217 4 22 0 27
A solvent• Temperature: 30 to 60 C
• Ability to provide good retention and symmetrical peak shapes for 10 20 30
10 20 10 20 30 40
• Systematic method development for optimizing peptide separations allows one to find temperature and gradient time
High SIVPSGASTGVHEALEMR 12.17 2 0 1840.92217 4.22 -0.27High IEEELGDNAVFAGENFHHGDKL 12.70 1 1 2441.13679 5.34 -0.91High NVNDVIAPAFVK 13.40 2 0 1286.71031 2.67 -1.51
Temperature: 30 to 60 C• Flow rate set so that linear velocity
is 2 mm/sec
y p pacidic, basic and zwitterionic analytes
Time (min)10 mM NH4COOH +10 mM HCOOH
10 mM DFA10 20
Time (min)10 20 30 40
Time (min)
60 min gradient30 min. gradient Major peaks were identified in LC-UV chromatograms and entered f separations allows one to find temperature and gradient time
combinations for robust, effective analyses.High IGLDCASSEFFK 13.60 1×Carbamidomethyl
[C5] 1 0 1373.64058 3.78 -0.46
High AAQDSFAAGWGVMVSHR 13.63 1 0 1789.84386 5.28 -0.45Hi h TAGIQIVADDLTVTNPK 14 31 2 0 1755 9487 4 97 0 18
is ~2 mm/sec• Initial % CH3CN: 5%
y• Volatile• Acceptably low or no suppression
60 min. gradient30 C
g30 C
into Microsoft Excel. Peaks were matched, and then RTs and areas were entered into DryLab 4 software, along with elution conditions.
, y• HALO 2 Peptide ES-C18 columns facilitate high resolution peptide
High TAGIQIVADDLTVTNPK 14.31 2 0 1755.9487 4.97 0.18High LGANAILGVSLAASR 15.02 1 0 1412.82199 4.57 -1.33High AVDDFLISLDGTANK 15.40 3 0 1578.80098 5.83 -0.48Hi h WLTGPQLADLYHSLMK 16 89 1 O id ti [M15] 1 0 1888 96258 4 8 1 3
• Final % CH3CN: 40–60%• Gradient slope: ranges from 0 1%
• Acceptably low or no suppression of LC-MS signalMi ibl ith t d i
16 18Time (min) 16 18 20
Time (min)For conditions, see panel 8
mapping of complex tryptic digests with short analysis times (20 30 minutes)
High WLTGPQLADLYHSLMK 16.89 1×Oxidation [M15] 1 0 1888.96258 4.8 -1.3High WLTGPQLADLYHSLMK 18.55 2 0 1872.96767 5.32 -0.18High SGETEDTFIADLVVGLR 19.42 1 0 1821.92288 6.42 -0.33
Gradient slope: ranges from 0.1% to 2% CH3CN per min.
• Gradient time: can vary from 30 to
• Miscible with water and organic modifiers (20–30 minutes).
A k l d t S t d i t b NIH G t GM116224 (BEB)
High RYPIVSIEDPFAEDDWEAWSHFFK 20.64 1 1 2984.38898 7.39 -1.18High YPIVSIEDPFAEDDWEAWSHFFK 21.51 2 0 2828.28787 5.46 -0.78
High YGASAGNVGDEGGVAPNIQTAEEALDLIVDAIK 24.66 1 0 3257.61721 4.97 -0.88
• Gradient time: can vary from 30 to 240 min.
Note: 2.2–52.2 % B in 40 min. Acknowledgements: Supported in part by NIH Grant GM116224 (BEB). Opinions expressed are solely those of the authors. HALO and Fused-
g LIVDAIK
Data Set was generated using the 30-min, 60 C Xcalibur software raw file PSM = Peptide Spectrum Match
XCorr = Cross Correlation Factor
0 10 20 30Time (min)
0 10 20 30Time (min) 6
Note: 2.2 52.2 % B in 40 min.
15Opinions expressed are solely those of the authors. HALO and FusedCore are registered trademarks of Advanced Materials Technology, Inc.
Spectrum Match Correlation Factor
123
( ) e ( ) 6DFA, TFA and NH4COOH/HCOOH give narrow peaks, similar retention but different selectivities for peptide mapping. 10 20
Time (min)10 20 30 40
Time (min) 9