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Protein Identification using 2D LC/MS/MS based on pH Gradient and Reverse Phase Separation

Protein Identification using 2D LC/MS/MS based on pH Gradient and Reverse Phase Separation. Column Technology Inc.,. Part I. Comparison of salt and pH step gradient. pH Based 2D-LC-MS/MS: pH 3.0-8.0, Buffer (5-10mM) v.s. Salt Based 2D-LC-MS/MS: salt 0-2000 mM, pH ~2.5.

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Protein Identification using 2D LC/MS/MS based on pH Gradient and Reverse Phase Separation

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  1. Protein Identification using 2D LC/MS/MS based on pH Gradient and Reverse Phase Separation Column Technology Inc.,

  2. Part I. Comparison of salt and pH step gradient pH Based 2D-LC-MS/MS: pH 3.0-8.0, Buffer (5-10mM) v.s. Salt Based 2D-LC-MS/MS: salt 0-2000 mM, pH ~2.5 Thermo ProteomeX System

  3. pH or salt off-line • Off-line ion exchange peptide fractionation by either salt or pH step gradient follow by reverse phase separation and tandem mass spectroscopy. • 50% of Aceteonitrile was added in the pH gradient buffer to denature peptides. • Due to solubility issue, only 30% of Acetonitrile was used in the salt gradient

  4. pH or salt on-line • One ten port valve, one ion exchange and two reverse C18 columns were used in the 2D separation. • First dimension is pH or salt step gradient. • Second dimension is reverse phase separation. • Peptides are first loaded onto the ion exchange column. • Peptide fractions elute to reverse phase column by pH or salt step gradient. • Follow by reverse phase separation and tandem mass spectroscopy.

  5. Protein identification pH step 2D-LC-MS/MS v.s. Salt step 2D-LC-MS/MS Mouse Liver Proteome ~ 3% FPR pH gradients identify more proteins than salt gradient Salt off-line is the worst one due to the sample lose during desalting pH off-line is benefit by adding 50% of Acetonitrile

  6. Peptide Overlapping Effect pH step 2D-LC-MS/MS v.s. Salt step 2D-LC-MS/MS

  7. 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

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

  9. Gradient Illustration

  10. RT: 0.00 - 240.06 93.89 100 138.35 11.93 155.82 131.47 24.80 108.83 35.48 50 79.72 78.19 158.07 39.62 177.24 188.82 220.58 0 188.84 100 198.46 221.56 5.50 169.75 50 30.30 32.26 13.01 219.60 163.67 44.54 111.14 131.67 57.02 75.39 105.36 0 145.16 100 206.04 202.17 120.31 137.80 212.74 79.18 50 148.39 160.70 97.18 110.84 62.36 231.47 18.49 25.68 57.17 0 89.04 100 144.06 111.88 97.60 183.65 174.05 85.68 117.34 221.40 50 161.73 70.69 41.80 60.95 199.47 31.56 223.48 26.27 0 105.74 100 154.65 120.70 165.14 134.80 167.14 50 94.05 182.51 82.07 222.37 74.49 211.91 53.69 48.65 1.32 20.20 Relative Abundance 0 83.96 129.30 100 72.37 206.58 110.79 147.47 87.74 170.65 188.49 50 68.61 211.79 59.02 224.07 49.82 18.05 6.46 0 205.77 100 63.87 109.50 98.84 77.55 113.79 169.29 30.51 163.94 50 59.92 176.42 51.30 183.21 212.23 12.99 229.14 0 82.13 100 116.21 73.07 71.03 88.66 112.90 145.66 56.14 211.29 220.88 127.60 50 189.43 164.47 28.16 26.36 238.67 0 157.07 100 163.98 152.15 169.64 50 6.65 186.70 27.80 146.72 196.55 35.22 50.54 78.34 88.01 98.62 106.80 224.70 0 0 20 40 60 80 100 120 140 160 180 200 220 240 Time (min) Base Peak Chromatogram for the Continuous Gradient 2D/LC/MS/MS

  11. More peptides, Less overlap Mouse Liver Proteome

  12. Can pH 2.5-to-8.5 Elute Basic Peptides Efficiently? Theoretical pI Distribution of Identified Peptides More basic peptides eluted by buffer to pH 8.5 than to 2000mM salt

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

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