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

2. 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 ~2.5 Salt Based 2D-LC-MS/MS: salt 0-2000 mM, pH ~2.5 Part I. Comparison of salt and pH step gradient 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. pH_Off-linepH_On-lineSalt_Off-lineSalt_On-line Spectra694891718518627352682664 Peptide50916663174156348551 Unique_Peptide125181205781549962 Protein_Group5093476536294391 pH step 2D-LC-MS/MS v.s. v.s. Salt step 2D-LC-MS/MS 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 Protein identification

6. Overlap pH_Off-linepH_On-lineSalt_Off-lineSalt_On-line >=4 2.05%4.34%2.85%3.86% 3 2.17%2.49%2.24%3.42% 2 9.22%7.22%7.40%8.30% 1 86.57%85.95%87.50%84.43% pH step 2D-LC-MS/MS v.s. v.s. Salt step 2D-LC-MS/MS Salt step 2D-LC-MS/MS Peptide Overlapping Effect

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. Base Peak Chromatogram for the Continuous Gradient 2D/LC/MS/MS RT: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 50 100 93.89 138.35 11.93155.82 131.47 108.8324.80 35.48 79.72 78.19 158.07 39.62 220.58177.24188.82 188.84 198.46 221.56 5.50 169.75 30.30 32.26 13.01 219.60 163.67 44.54111.14131.67 57.02 105.3675.39 145.16 206.04 202.17 120.31 212.74137.80 79.18 148.39 160.70 97.18 110.84 62.36 231.47 25.6857.1718.49 89.04 144.06 111.88 183.6597.60 174.05 85.68 117.34 221.40 161.73 70.69 41.80 60.95 199.47 31.56 223.48 26.27 105.74 154.65 120.70 165.14 134.80 167.14 94.05 182.51 82.07 222.37 74.49211.91 53.69 48.65 20.201.32 83.96 129.30 72.37206.58 110.79 147.47 87.74 170.65 188.49 211.7968.61 59.02 224.07 49.82 18.05 6.46 205.77 63.87 109.50 98.84 77.55 113.79169.29 30.51163.94 59.92 176.42 212.23183.2151.30 12.99 229.14 82.13 116.21 73.07 71.0388.66 112.90 145.66 56.14 211.29220.88 127.60 189.43 164.47 28.16 26.36 238.67 020406080100120140160180200220240 Time (min) 0 50 100 157.07 163.98 152.15 169.64 6.65 186.70 196.55146.7227.80 224.7035.2250.5498.62106.8088.0178.34

11. pH_Off-linepH_On-lineSalt_Off-line Salt_On- line Gradient_pH Spectra694891718518627352682664680405 Peptide5091666317415634855160834 Unique_Peptide12518120578154996213106 Protein_Group50934765362943915043 OverlappH_Off-linepH_On-lineSalt_Off-lineSalt_On-line Gradient_pH>=42.05%4.34%2.85%3.86%1.72% 32.17%2.49%2.24%3.42%2.11% 29.22%7.22%7.40%8.30%6.27% 186.57%85.95%87.50%84.43%89.91% More peptides, Less overlap Mouse Liver Proteome

12. 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?

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.