Vented Column Technology Applied Vented Column Technology Applied to Proteomic to Proteomic MudPITMudPIT Analysis onAnalysis on

Long Capillary ColumnsLong Capillary Columns

Andrew W. Guzzetta and Allis S. Chien

Vincent Coates FoundationMass Spectrometry Laboratory

Department of Chemistry Stanford University Stanford California

Last year…. the long column and proteomics

What if?What if?

Space

What if we could do What if we could do MudPITMudPITon a 60 cm column?on a 60 cm column?

Available Column Technologies

Tri-Phasic MudPIT

SCX Reverse PhaseRP

Vented Technology

Reverse PhaseRP

1. Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. W. Hayes McDonald, Ryoma Ohi, David T. Miyamoto, Timothy J. Mitchison, and John R. Yates, III International Journal of Mass Spectrometry, Volume 219, Issue 1 , 1 August 2002, Pages 245-251

2. Automation of Nanoscale Microcapillary Liquid Chromatography-Tandem Mass Spectrometry a Vented Column. Lawrence J. Licklider, Carson C. Thoreen, Junmin Peng and Steven P. Gygi* Anal. Chem. 2002, 74,3076-3083

MudPITMudPIT Meets Vented Column Meets Vented Column Technology Meets The Long Column.Technology Meets The Long Column.

SCX Reverse PhaseRP RP60 cm

Vent/Plug

Building the Double Vented Tetra PhasicMudPIT Device

Building The Building The TriphasicTriphasic MudPITterMudPITter

1 cm = 7 cm

Method

1. Place screen frit in the tee2. Swage with short piece of PEEK3. Pack Poros 104. Wash5. Pack SCX (Whatman Partisil, 5um)6. Wash7. Pack final section of Poros 108. Unswage9. Cut first segment of Poros 10 to length10. Reswage with new PEEK11. Cut off final length of fused silica capillary

Building The Building The TriphasicTriphasicColumnColumn

Continuous Vent Continuous Vent

The Discontinuous VentThe Discontinuous VentTriTri--PhasicPhasic On The ValveOn The Valve

MudPIT Gradients

Optimized for speed to test different models, not optimized for proteomic performance

LoadWashACN bump

Salt &Separate

Gradient 18

Initial conditions30ul/min 0%B

T %B1 510 570 4080 8080.1 5120 Stop

Purpose: Salt bump. Separate peptides on the analytical column.

Gradient 17

Initial conditions30ul/min 0%B

T %B1 54 510 8012 8012.1 520 Stop

Purpose load sampleonto first RP and washand elute onto SCX

10 cm Continuous Vent Human Serum MudPIT

0 mM

12.5 mM

25 mM

50 mM

250 mM

1000 mM

5000 mM

RT: 0.00 - 116.03

0 10 20 30 40 50 60 70 80 90 100 110Time (min)

0

50

1000

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance 0

50

1000

50

1000

50

100662.0 937.7

900.51068.9

689.2 812.6

1207.81292.7572.0

973.8 673.6481.0519.5 1106.9

1207.8631.5409.9 638.2 992.9

682.7

998.2648.2 993.0637.8625.4831.5

547.5

682.8 831.6764.1656.3 998.1

820.6 831.7

764.21137.8

1246.4

872.6461.9932.9 461.8903.01231.7 1366.1 461.9820.7

60 cm Continuous Vent Human Serum MudPIT

RT: 0.00 - 116.03

0 10 20 30 40 50 60 70 80 90 100 110Time (min)

0

50

1000

50

1000

50

1000

50

100

Rel

ativ

e A

bund

ance 0

50

1000

50

1000

50

100900.3872.8501.1481.1 812.7

1106.9709.1689.1

519.5 1263.4

841.5 1207.9710.5

572.1 556.0 598.6 673.6

409.8 992.91207.9517.4 998.4637.8

841.1573.0

648.3 855.1 831.5868.0543.5565.1

638.2517.3 826.6853.2 841.6

547.6 758.8 831.6683.0660.5

910.5 867.9

831.5

820.8927.6 1137.7 838.0932.9 1352.9851.4

1246.5403.9

872.2950.5

927.6432.2 715.1401.5 406.7

0 mM

12.5 mM

25 mM

50 mM

250 mM

1000 mM

5000 mM

Making the Cut

Comparing 10 and 60 cm Columns at the 25 mMSalt Step Using the Continuous Vent Method

10 CMRT: 22.58 - 95.12

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Tim e (m in)

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce1207.8

631.5

626.7 714.5

714.3409.9

594.1517.4 638.2

992.9

998.2

973.7710.5

522.6522.6 1292.2572.9 841.1520.9 1222.9 1350.0 1118.3 1289.4 1348.4

409.8 992.91207.9517.4

992.8998.4638.4631.5648.1

594.1 637.8

543.6

855.0682.8

841.1

826.5573.01093.7520.9 819.71292.3 1393.6

926.3559.1709.31361.7 533.4

NL:4.03E9Base Peak F: + c ESI Full m s [ 400.00-1400.00] MS 25m m 10cm c

NL:4.11E9Base Peak F: + c ESI Full m s [ 400.00-1400.00] MS 25m m 60cm c

60 CM

XIC Plots For 10 and 60 Columns At The 25 mMSalt Step With The Continuous Vent Method

1.00E10

25 30 35 40 45 50 55 60 65 70Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

517.4409.9

626.7631.5

631.2

1207.8

638.3

850.0

998.1

10 cm

1.00E10

35 40 45 50 55 60 65 70 75 80Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

517.3409.8

626.9

631.5

1207.9 638.4

849.8

998.4

m

m

m

m

m

m

m

m

60 cm

Evaluating The CV and DCV Experiments ByCounting Significant Peptides With Mascot

Formatting Options

CV vs. DCVCV vs. DCVShort vs. LongShort vs. Long

10 cm 10 cm DCVDCV

60 cm 60 cm DCVDCV % Gain% Gain

PeptidePeptideCountCount 537537 635635 18.318.3

10 cm 10 cm CVCV

60 cm 60 cm CVCV

PeptidePeptideCountCount 593593 739739 24.624.6

% Gain% Gain 10.410.4 16.416.4

CV vs. DCVCV vs. DCVShort vs. LongShort vs. Long

Peptide Peptide Count Count CategoryCategory(protein with n peptides)(protein with n peptides) 10 cm 10 cm DCVDCV 60 cm 60 cm DCVDCV % Gain% Gain

6+6+ 447447 498498 11.411.411--55 9090 137137 52.252.2

10 cm 10 cm CVCV 60 cm 60 cm CVCV6+6+ 486486 589589 21.221.211--55 107107 150150 40.240.2

ConclusionConclusion

Long columns are better at showing us Long columns are better at showing us a greater number of low level peptides.a greater number of low level peptides.The CV method is superior to the DCV The CV method is superior to the DCV method.method.Manufacture of the CV device needs to Manufacture of the CV device needs to be standardized and simplified.be standardized and simplified.

AcknowledgementsAcknowledgements

The Vincent & Stella Coates FoundationThe Vincent & Stella Coates FoundationMike Mike MacCossMacCoss, University of Washington, University of WashingtonAndy Andy GieschenGieschen, , AgilentAgilentThermoFinniganThermoFinnigan