Integrated Multidimensional LC based protein identification method

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Integrated Multidimensional LC based protein
identification method

• Column Technology Inc.,
• WWW.Columntechnology.com

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Bi-Phasic column

• Integrated Multi-dimensional LC (IMDL) Based on
  pH elution

SCX column
C18 column
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IMDL method

• Bi-phasic column contains both ion exchange and
  reverse phase material for the 2D LC separation.
• Inject buffer plugs by autosampler
• Only need one HPLC system for the 2D LC
  separation

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Integrated multi-dimensional LC (IMDL) based on
pH elution
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Integrated multi-dimensional LC (IMDL) based on
pH elution
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Integrated multi-dimensional LC (IMDL) based on
pH elution
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IMDL

• Features
• Two dimensional HPLC separation
• PH step gradient (separation based on
  protein/peptide PIs)
• Mass compatible buffers
• Only need one HPLC system
• No salt
• Large scale protein identification
• Work on any commercial LC/MS instrument

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Separation of mouse liver proteome

• Protein lyses from mouse liver tissue
• 300 ug of total proteins were Tryptic digested to
  peptides
• Load peptides on a biphasic column
• 2D LC separation of peptide mixture
• Peptides and proteins identified by SequestTM
  software

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Separation of mouse liver proteome by IMDL method
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Proteins identified from mouse liver proteome

• Peptide hits 80677
• Unique peptides 14682
• Proteins 4809

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Separation of serum proteins

• Affinity depletion of high abundant proteins.
• The low abundant proteins remains in eluent.
• High abundant proteins washed out from affinity
  column.
• Both high and low abundant proteins undergo
  tryptic digestion
• The tryptic digested peptides were separated by
  IMDL method and identified by mass spectrometry.

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300ug high-abundant fraction of plasma by IMDL
method
2204 proteins (793 with salt 2D)
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300 ug low-abundant fraction
of plasma by IMDL method
3489 proteins (1053 with salt 2D)
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Comparison of Unique peptides, Peptide hits and
Protein group numbers identified by IMDL on an
Integrated-Column and traditional 2D-HPLC coupled
with MS (Sequest parameters Xcorr 1.9 with
charge state 1, Xcorr 2.2 with charge state
2, and Xcorr 3.75 with charge state 3 ?Cn
0.1).
Low-abundant and high abundant fractions