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