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
20
25
30
35
40
45
50
55
60
65
70
75
80
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
2
3
4
5
6
Vydac, 300A, 5um C18 0.075ID X 600 mm length
100 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]
0.0
0.2
0.4
0.6
0.8
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
20
40
60
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
0
20
40
60
80
100
120
140
160
180
200
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