Extended length capillary columns Extended length capillary columns for simple peptide mapping and for simple peptide mapping and complex proteomic separationscomplex proteomic separations

Andrew W. Guzzetta and Allis S. Chien

Vincent Coates FoundationMass Spectrometry Laboratory

Department of Chemistry Stanford University Stanford California

Long Column DataLong Column Data

Long Columns are BetterLong Columns are Better

The Current Big The Current Big KahunasKahunas of the of the Long ColumnLong Column

Richard Smith & Richard Smith & YufengYufeng ShenShen

Old Cap Masters

D. IshiC. HorvathF. YangB. KargerJ. HenionR. SimpsonJ-P. Chervet

Should We Use Long or Should We Use Long or Short Columns?Short Columns?

Core Facility at StanfordCore Facility at Stanford

Gel spot analysisGel spot analysisDetailed peptide mappingDetailed peptide mappingProteomicsProteomics

Simple Peptide MappingSimple Peptide Mapping

Column: 400 or 100 mm in Length, 150 µm IDStationary Phase: Vydac C18, 300A, 5µmTemperature: AmbientFlow Rate: 1 µ l /minMobile Phase: A:H20, B:ACN, 0.1%FATrapping Column:Poros 10, 10um, 10 X 0.15 mm, 10ul/min, 0%BMass Spectrometer: XP plus , IonTrapHPLC: Shimadzu 10ADvp

Myoglobin Tryptic Digest 400 mm vs. 100mm

20 30 40 50 60 70 80

Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

40 cm

10 cm

Better Separation

Separation of Near Isobars and Separation of Near Isobars and Added SelectivityAdded Selectivity

41.0 41.5 42.0 42.5 43.0 43.5 44.0 44.5 45.0 45.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

636.6 650.1

637.6

471.3

1039.1564.1

8.81E8

LFTGHPETLEK resolvedco-eluting

25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0

Time (min)

0

5

10

15

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25

30

35

40

45

50

55

60

65

70

75

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85

90

95

100

Rel

ativ

e Ab

unda

nce

650.1

636.6

637.2

4.50E8

471.2

429.4

100 mm 400 mm

Isotopic data on the Isotopic data on the separated near isobarsseparated near isobars

636.35

636.88

637.33

+MS, 94.3-95.4min (#5297-#5367)

636.57

637.41

637.78

638.24

638.82639.31

+MS, 96.1-96.6min (#5406-#5438)0.0

0.5

1.0

1.5

5x10Intens.

0

1

2

3

4

5x10

635 636 637 638 639 m/z

Long vs. Short Columns Long vs. Short Columns and and

Proteomics DevelopmentProteomics Developmentoror

Optimizing for Complex Peptide MixturesOptimizing for Complex Peptide Mixtures

Serum Proteomics MethodSerum Proteomics MethodColumnColumn: 60 or 10 cm in Length, 75 : 60 or 10 cm in Length, 75 µµm IDm IDStationary PhaseStationary Phase: : VydacVydac C18, 300A, 5C18, 300A, 5µµmmTemperature:Temperature: AmbientAmbientFlow Rate:Flow Rate: 200 200 nlnl/min/minMobile Phase:Mobile Phase: AA:H20, :H20, BB:ACN, 0.1%FA:ACN, 0.1%FAGradient: Gradient: TT %B%B00 5544 55184184 3030200200 8080210210 8080211211 55270270 55

Trapping ColumnTrapping Column::ZorbaxZorbax 300 SB, 5um, 5 X 0.3 mm, 10ul/min, 0%B300 SB, 5um, 5 X 0.3 mm, 10ul/min, 0%BFlow Rate:Flow Rate: 10ul/min10ul/min

Mass Spectrometer:Mass Spectrometer: AgilentAgilent XCT, XCT, IonTrapIonTrapHPLC:HPLC: AgilentAgilent Capillary HPLCCapillary HPLCDatabase Searching:Database Searching: SpectrumMillSpectrumMill

Human Human SerumSerumalbuminalbumin ProteomicsProteomics

0 25 50 75 100 125 150 175 200 225 250 Time [min]0.00

0.25

0.50

0.75

1.00

1.25

7x10Intens.

Chromatographic StabilityChromatographic StabilitySERUMC.D: BPC 100-1800 ±All MS

0

17x10

Intens.

50 100 150 200 250 Time [min]

SERUMD.D: BPC 100-1800 ±All MS

0

1

7x10

50 100 150 200 250 Time [min]

SERUME.D: BPC 100-1800 ±All MS

0.0

0.5

7x10

50 100 150 200 250 Time [min]

SERUMF.D: BPC 100-1800 ±All MS

0.0

0.5

7x10

50 100 150 200 250 Time [min]

SERUMG.D: BPC 100-1800 ±All MS

0.0

0.5

7x10

50 100 150 200 250 Time [min]

SERUMH.D: BPC 100-1800 ±All MS

0.0

0.5

1.07x10

50 100 150 200 250 Time [min]

SERUMI.D: BPC 100-1800 ±All MS

0

1

7x10

50 100 150 200 250 Time [min]

SERUMJ.D: BPC 100-1800 ±All MS

0.0

0.5

7x10

50 100 150 200 250 Time [min]

Eight Serum Digest Replicates

Chromatographic StabilityChromatographic Stability

Same sample separated by 40hrs of continuous running

SERUMC.D: BPC 100-1800 ±All MS

0.00

0.25

0.50

0.75

1.00

1.25

7x10Intens.

50 100 150 200 250 Time [min]

SERUMJ.D: BPC 100-1800 ±All MS

0.0

0.2

0.4

0.6

0.8

7x10

50 100 150 200 250 Time [min]

Chromatographic StabilityChromatographic Stability

SERUMC.D: BPC 100-1800 ±All MS, Smoothed (7.3,1, SG)

0.0

0.2

0.4

0.6

0.8

1.0

7x10Intens.

110 120 130 140 150 160 Time [min]

SERUMJ.D: BPC 100-1800 ±All MS, Smoothed (6.6,1, SG)

0

2

4

6

8

6x10

110 120 130 140 150 160 Time [min]

Database Searching Program, Spectrum Mill

Highest-scoring peptide

Lowest-scoring peptide

Serum ProteinsSerum Proteins

Serum Albumin 35Serum Albumin 35--45 mg ml45 mg mlCeruloplasminCeruloplasmin 0.3 mg/ml0.3 mg/ml

Column Load Range Finding Column Load Range Finding ExperimentExperiment

600 mm

100 mm

Range Finding Range Finding

NODIL.D: BPC 100-1800 ±All MS

0

2

8x10Intens.

50 100 150 200 250 Time [min]

DILA.D: BPC 100-1800 ±All MS

0

1

8x10

50 100 150 200 250 Time [min]

DIL1.D: BPC 100-1800 ±All MS

0.0

0.5

1.0

8x10

50 100 150 200 250 Time [min]

DIL2.D: BPC 100-1800 ±All MS

0

2

4

6

7x10

50 100 150 200 250 Time [min]

DIL3.D: BPC 100-1800 ±All MS

0

1

2

37x10

50 100 150 200 250 Time [min]

DIL4.D: BPC 100-1800 ±All MS

0

1

7x10

50 100 150 200 250 Time [min]

1

Dilu

tion

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3

4

5

6

Vydac, 300A, 5um C18 0.075ID X 600 mm length

100 X 0.075 mm column

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32

1

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8x10Intens.

600 X 0.075 mm column

10

9

8

7

6

5

4

32

1

0 25 50 75 100 125 150 175 200 225 Time [min]

0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

Column comparison in the context of

Range FindingRange FindingSerum Digest Dilution

12.5 ug

1 ug

0.5 ug

0.25 ug

Sample 1

0.125 ug

0.0625 ugSample 6

1:12.5

stress the columnstress the columnstress the trapstress the trap1:200

Identified Peptides at Each Identified Peptides at Each Dilution in the 100 and 600mm Dilution in the 100 and 600mm Range Finding ExperimentsRange Finding Experiments

Identified Peptides at Each Dilution

More Dilute

Pept

ides

600 mm Peptides

0

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40

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80

100

120

140

160

180

200

1 2 3 4 5 6

Dilution

100 mm Peptides

Pick The Right Column LengthPick The Right Column Length

Cumulative Peptide Count For all Dilutions in the Range

Finding Experiments700

600

416

600

500

400

300

200

100

0

10 cm 60 cm

10 extracted peaks

Why is the long column winning?

0 25 50 75 100 125 150 175 200 225 Time [min]

0.00

0.25

0.50

0.75

1.00

1.25

8x10Intens.

600 mm 5E7 counts

0 25 50 75 100 125 150 175 200 225 Time [min]

0.0

0.2

0.4

0.6

0.8

8x10Intens.

5E7100 mm

Range Finding Range Finding Why is short column failing, is it capacity?

Serum Digest Dilution

12.5 ug

1 ug

0.5 ug

0.25 ug

Sample 1

0.125 ug

0.0625 ugSample 6

1:12.5

1:200

Cumulative Extracted Cumulative Extracted Area CountsArea Counts

600 mm

0

2E+10

4E+10

6E+10

8E+10

1E+11

1.2E+11

1.4E+11

1.6E+11

1 2 3 4 5 6Dilution

100 mm

Pick The Right LoadPick The Right Load

Peptide Count in Range Finding Experiment

53

185

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Dil 6 Dil1

ConclusionConclusion

Long columns are better for peptide Long columns are better for peptide mapping and more complex mixturesmapping and more complex mixturesMethod development is essentialMethod development is essentialData analysis is the bottle neck in the Data analysis is the bottle neck in the study of complex mixturesstudy of complex mixturesDo better Do better

AcknowledgementsAcknowledgements

The Vincent & Stella Coates FoundationThe Vincent & Stella Coates FoundationAgilentAgilentThermoFinniganThermoFinnigan