Column Technology

Simplified Multi-Dimensional Separations to Maximize Protein Detection in Poteomic Analyses

Jie Dai Hu Zhou and Rong Zeng
Research Center for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai, 200031, China


  1. pH elution can be an alternative to conventional salt elution to wash peptides from SCX column to reverse phase column on 2D- LC-MS/MS system.
  2. The pH buffer has suitable pH range and is compatible to mass spectrometry.
  3. An integrated column containing SCX and reverse phase column is used for the study.
  4. The pH buffers were injected from auto-sampler which allows 2D-LC/MS/MS analysis on any 1D-LC/MS system.
  5. The peptides can be fractionated by using pH step gradient according to their pI distribution.


Currently most of shotgun identification of protein mixture rely on multi-dimensional liquid chromatography coupled to mass spectrometry. A strong cation exchange based on salt gradient is applied as the first dimension to fractionate peptides followed by a reverse phase separation. This technique requires two HPLC pumps and extensive wash after ion exchange step to remove salt. In this study, we use a pH step gradient follow by reverse phase separation and mass spectrometry for the analysis of complicate protein/peptide mixture. The buffers contain no salt and is compatible to mass spectrometry. The method can be performed on any 1D HPLC/MS system.


Proteins from human liver tissue were digested by trypsin and used for this study.
2D-LC-MS/MS conditions:

  1. Bi-phase Colimns : 0.32 mm x 50 mm SCX + 0.15 mm x 100 mm RP packed in a single columns, were from Column Technology, Inc, Fremont, CA.
  2. SCX pH Buffer: The MS compatible buffer kit was from Column Technology Inc., (Fremont, CA, with different pH steps: 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7 and 8.
  3. RP Buffer: 0.1% formic acid (v/v) aqueous (A) and 0.1% formic acid (v/v) acetonitrile (B).
  4. pH elution of peptides from SCX: The peptide fractions were eluted into the reverse phase column by injecting different pH buffers using a 100 ul loop. After the loadiung, the biphasic column is equilibrated with 0.1% formic acid at ~3┬Ál/min flow rate. The reverse phase separation was performed by a gradient from 5 to 65% solvent B in 115 minutes at 200 ul flow rate with 100:1 split ratio.
  5. Mass spectrometry: LTQ used
  6. Database searching: The acquired MS/MS spectra were automatically searched against IPI database using the TurboSEQUEST program in the BioWorks™ 3.1 software suite (Thermo). An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state). Peptides with a +1 charge state were accepted if they had a cross correlation (Xcorr) of at least 1.9. Peptides with a +2 charge state were accepted if they had an Xcorr >=2.2. Peptides with a +3 charge state were accepted if they had an Xcorr >=3.75.

A Bi-phasic column

Fig. 1. A Bi-phasic column included two sections. The front section is a strong cation exchange column (320um i.d.; 50mm length), and the second section is a reverse phase column (150 um i.d., 100mm length). Designed and manufactured by Column Technology Inc., Fremont, CA.

Table 1. Protein numbers of liver tissues in every fraction Separated by 2D-LC on an Bi-Phase Column on LTQ.

Protein numbers of liver tissues

pH Elution is superior to salt elution for SCX

Beas-peak chromatograms

Fig. 2. Beas-peak chromatograms of nine pH step fractions on LTQ

The distribution of unique peptides

Figure 4. The distribution of unique peptides with different theoretical pI in nine fractions of different pH steps (left) and the overlap of peptides across pH steps (right), which reveal:

Peptides were focused according to their pIs

Peptide overlap across pH steps is low

Comparison of pI profile

Figure 5. Comparison of pI profile of unique peptides eluted by IMDL and database digested theoretically by trypsin.The database has more basic peptides since there are a number of peptides with missed cleavage sites thus contains more K and R.


  1. pH elution can be an alternative to conventional salt elution.
  2. pH elution was applied by on a Bi-phasic column which combines strong cation exchange and reverse phase sections to a single HPLC column.
  3. The peptides can be fractionated by using pH step gradient according to their pI distribution, followed by reverse phase elution and mass spectrometry identification.
  4. The method demonstrated very low peptide overlapping between different pH fractions, has high resolution of peptides with close pIs.
  5. This method can be used to separate complicate protein/peptide mixture by two dimensional HPLC-MS/MS and only use a single HPLC system.