Protein Identification Protein Identification using 2D LC/MS/MS using 2D LC/MS/MS

based on pH Gradient based on pH Gradient and Reverse Phase and Reverse Phase

SeparationSeparationColumn Technology Inc.,Column Technology Inc.,

pH Based 2D-LC-MS/MS: pH 3.0-8.0, Buffer (5-10mM) pH Based 2D-LC-MS/MS: pH 3.0-8.0, Buffer (5-10mM) v.s.v.s. Salt Based 2D-LC-MS/MS: salt 0-2000 mM, pH ~2.5Salt Based 2D-LC-MS/MS: salt 0-2000 mM, pH ~2.5

Part I. Comparison of salt and pH step Part I. Comparison of salt and pH step gradientgradient

Thermo ProteomeX System

pH or salt off-linepH or salt off-line► Off-line ion exchange peptide fractionation by Off-line ion exchange peptide fractionation by

either salt or pH step gradient follow by reverse either salt or pH step gradient follow by reverse phase separation and tandem mass spectroscopy.phase separation and tandem mass spectroscopy.

► 50% of Aceteonitrile was added in the pH gradient 50% of Aceteonitrile was added in the pH gradient buffer to denature peptides.buffer to denature peptides.

► Due to solubility issue, only 30% of Acetonitrile was Due to solubility issue, only 30% of Acetonitrile was used in the salt gradientused in the salt gradient

pH or salt on-linepH or salt on-line► One ten port valve, one ion exchange and two reverse C18 One ten port valve, one ion exchange and two reverse C18

columns were used in the 2D separation.columns were used in the 2D separation.► First dimension is pH or salt step gradient.First dimension is pH or salt step gradient.► Second dimension is reverse phase separation.Second dimension is reverse phase separation.► Peptides are first loaded onto the ion exchange column.Peptides are first loaded onto the ion exchange column.► Peptide fractions elute to reverse phase column by pH or salt Peptide fractions elute to reverse phase column by pH or salt

step gradient.step gradient.► Follow by reverse phase separation and tandem mass Follow by reverse phase separation and tandem mass

spectroscopy.spectroscopy.

pH_Off-linepH_Off-line pH_On-linepH_On-line Salt_Off-lineSalt_Off-line Salt_On-lineSalt_On-lineSpectraSpectra 694891694891 718518718518 627352627352 682664682664PeptidePeptide 5091650916 6631766317 4156341563 4855148551

Unique_PeptideUnique_Peptide 1251812518 1205712057 81548154 99629962

Protein_GroupProtein_Group 50935093 47654765 36293629 43914391

pH step 2D-LC-MS/MS pH step 2D-LC-MS/MS v.s.v.s. Salt step 2D-LC-MS/MSSalt step 2D-LC-MS/MSMouse Liver Proteome ~ 3% FPR

pH gradients identify more proteins than salt gradientSalt off-line is the worst one due to the sample lose during desaltingpH off-line is benefit by adding 50% of Acetonitrile

Protein identification

OverlapOverlap pH_Off-linepH_Off-line pH_On-linepH_On-line Salt_Off-lineSalt_Off-line Salt_On-lineSalt_On-line

>=4>=4 2.05%2.05% 4.34%4.34% 2.85%2.85% 3.86%3.86%

33 2.17%2.17% 2.49%2.49% 2.24%2.24% 3.42%3.42%

22 9.22%9.22% 7.22%7.22% 7.40%7.40% 8.30%8.30%

11 86.57%86.57% 85.95%85.95% 87.50%87.50% 84.43%84.43%

pH step 2D-LC-MS/MS pH step 2D-LC-MS/MS v.s.v.s. Salt step 2D-LC-MS/MSSalt step 2D-LC-MS/MS

Peptide Overlapping Effect

Part II. Fully-automatic, On-line, pH Continuous Gradient 2D-LC-MS/MS

•One ten port valve, one ion exchange, two trap column and one capillary (0.75 x 150 mm) column were used for the separation.•Peptides were injected into the cation exchange column at pH 2.5.•Ion exchange separation was done by the continuous pH gradient from pH 2.5 to 8.5 in 36 hours.•Every four hours, SCX flow was switched to another trap column.•The previous SCX-targeted trap column is connected to the capillary C18 column for the RP separation follow by tandem mass spectroscopy.•The two trap column take turns for loading and separation

Part II. Fully-automatic, On-line, pH Continuous Gradient 2D-LC-MS/MS

Gradient IllustrationGradient Illustration

Base Peak Chromatogram for the Continuous Gradient

2D/LC/MS/MSRT: 0.00 - 240.06

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

Relative Abundance

0

50

100

0

50

100

0

50

10093.89

138.3511.93 155.82131.47108.8324.80 35.48 79.7278.19 158.0739.62 220.58177.24 188.82

188.84198.46

221.565.50 169.7530.30 32.2613.01 219.60163.6744.54 111.14 131.6757.02 105.3675.39

145.16

206.04202.17120.31212.74137.8079.18 148.39 160.7097.18 110.8462.36 231.4725.68 57.1718.49

89.04144.06111.88 183.6597.60 174.0585.68

117.34 221.40161.7370.6941.80 60.95 199.4731.56 223.4826.27

105.74154.65120.70 165.14

134.80167.14

94.05 182.5182.07 222.3774.49 211.9153.6948.6520.201.32

83.96129.3072.37 206.58110.79 147.4787.74

170.65 188.49 211.7968.6159.02 224.0749.8218.056.46

205.7763.87 109.5098.8477.55

113.79 169.2930.51 163.9459.92 176.42 212.23183.2151.3012.99

229.14

82.13116.21

73.0771.03 88.66 112.90 145.6656.14 211.29 220.88127.60 189.43164.4728.1626.36 238.67

0 20 40 60 80 100 120 140 160 180 200 220 240

Time (min)

0

50

100157.07

163.98152.15 169.64

6.65 186.70 196.55146.7227.80 224.7035.22 50.54 98.62 106.8088.0178.34

pH_Off-linepH_Off-line pH_On-linepH_On-line Salt_Off-lineSalt_Off-line Salt_On-linSalt_On-linee Gradient_pH

SpectraSpectra 694891694891 718518718518 627352627352 682664682664 680405

PeptidePeptide 5091650916 6631766317 4156341563 4855148551 60834

Unique_PeptideUnique_Peptide 1251812518 1205712057 81548154 99629962 13106

Protein_GroupProtein_Group 50935093 47654765 36293629 43914391 5043

OverlapOverlap pH_Off-linepH_Off-line pH_On-linepH_On-line Salt_Off-lineSalt_Off-line Salt_On-lineSalt_On-line Gradient_pH

>=4>=4 2.05%2.05% 4.34%4.34% 2.85%2.85% 3.86%3.86% 1.72%

33 2.17%2.17% 2.49%2.49% 2.24%2.24% 3.42%3.42% 2.11%

22 9.22%9.22% 7.22%7.22% 7.40%7.40% 8.30%8.30% 6.27%

11 86.57%86.57% 85.95%85.95% 87.50%87.50% 84.43%84.43% 89.91%

More peptides, Less overlap

Mouse Liver Proteome

0.00%0.20%0.40%0.60%0.80%1.00%1.20%1.40%1.60%1.80%2.00%

3.0~3.5 3.5~4.0 4.0~4.5 4.5~5.0 5.0~5.5 5.5~6.0 6.0~7.0 7.0~8.0 >8.0

pI range

Perc

enta

ge

Salt Online Salt Offline pH Online pH Offline Continuous pH

Theoretical pI Distribution of Identified Peptides

More basic peptides eluted by buffer to pH 8.5 than to 2000mM salt

Can pH 2.5-to-8.5 Elute Basic Peptides Efficiently?

Advantages of Continuous pH ElutionAdvantages of Continuous pH Elution

► Mobile phases contain only buffers and are compatible to the mass spMobile phases contain only buffers and are compatible to the mass spectroscopy.ectroscopy.► No need to wash column after ion exchange step.No need to wash column after ion exchange step.► Proteins, peptides elute according to their pI value.Proteins, peptides elute according to their pI value.► Continuous gradient provides better reproducibility and less overlap.Continuous gradient provides better reproducibility and less overlap.► Fully-automatic and easy to use 2D-LC-MS/MSFully-automatic and easy to use 2D-LC-MS/MS► Easy to isolate target protein and peptides.Easy to isolate target protein and peptides.