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Herman C. Lam, Ph.D. Calibration & Validation Group 4th Multidimensional Chromatography Workshop 4th Multidimensional Chromatography Workshop Toronto (January, 2013) Toronto (January, 2013)

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Page 1: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Herman C. Lam, Ph.D.Calibration & Validation Group

4th Multidimensional Chromatography Workshop4th Multidimensional Chromatography WorkshopToronto  (January, 2013)Toronto  (January, 2013)

Page 2: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

MDLC for Shotgun ProteomicsIntroduction

General conceptsAdvantagesChallenges in implementation

ApplicationsShotgun proteomics

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Page 3: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Multidimensional chromatography

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Anal. Chem.

2008, 80, 7187‐7193.

LC

Fraction

s

TLC

Page 4: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

tf

tf –

t1t1

Peak capacityPeak capacity P is the number of peaks that would fit into a separation space 

Separation performance and utilization of separation space

Estimation: P = (tf – t1)/ Wtf

= time for the final peak, t1

= time for the void peakW –

average peak width4

Isocratic Gradient

tR

W4

N = 16 ( tR / W4   )2

Page 5: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Peak capacityFor a multidimensional chromatographic system

P = P1 x P2 X P3 ………….P1 = peak capacity of the first dimensionP2 = peak capacity of the second dimensionP3 = peak capacity of the third dimension

Assumptions: The separation dimensions are orthogonal and no peak broadening

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1st

D, P1

2nd

D, P2

3rd

D, P3

Page 6: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Orthogonality

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Correlation between the mode of separationHigh orthogonality: low correlation between the modes of separation (eg. SCX and reversed phased)

Geometric orthogonality concept

M. Gilar, Anal. Chem., 2005, 77 (19), pp 6426–6434

(A) Non orthogonal system, 10% area coverage represents 0% orthogonality.(B)

Hypothetical orthogonal system, full area coverage. (C) Random, ideally orthogonal, system, area coverage is 63% representing 

the 100% orthogonality.

1D

2D

1D 1D

2D 2D

Page 7: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

LC Column Orthogonality and  Solvent Compatibility

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Orthogonality Solvent

compatibility

Page 8: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Challenges for implementation of  MDLC

Off‐lineVery tediousSample lose

On‐lineDifferent separation mechanisms at work

Optimization of D1 and D2Solvent compatibility

Multiple pumps and complex switch valve systemsLong total run time: For 2D separation (Td1 + nTd2)

T  ‐ Run time       n= number of fractions from D1)

Volume of data and data analysis

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Page 9: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Proteomics Proteomics  deals  with  the  large‐scale  determination  of  gene  and cellular functions directly at the protein level.

Proteomics  includes not only  the  identification and quantification of proteins,  but  also  the  determination  of  their  localization, modifications, interactions, activities, and their functions 

.

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Bottom up

Top down

Page 10: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

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MS/MS

Enzymatic Digestion

LC 

Separation

Complex PeptideMixture

DatabaseSearch

Peptide sequences:

SPAFDSIMAETLKAFDSLPDDIHEGGILAQSPFLIIKIIGHFYDDWCPL

Trypsin: Cleaves  at lysine and arginine, unless 

either is followed by proline in C‐terminal 

direction

Proteins

Cells

Page 11: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Challenges of MS proteomicsThe complexity of biological samples (great dynamic range, huge numbers of proteins)

107‐8 in human cells, and at least 1012 in human plasmaDynamic range of a LC‐MS is about 104‐6

Ion suppression when peptides co‐elution

The limited scanning speed of MS

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Simple mixture Complex 

mixture

Signal is suppressed1.Not being picked for MS/MS2.Poor MS/MS spectrum

abhde

fgc

Page 12: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Advantages for MDLCIncrease in peak capacity to reduce sample complexity

Enable the analysis of very complex biological samplesEliminate interferences Can be automated for 0n‐line applications

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Page 13: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

SCX‐RP 2D combinationHigh orthogonality and relatively easy to implementMudPIT (Multi Dimensional Protein Identification Technology)

Low resolution in the SCX separationPeptides carryover to subsequent fractions

ESI/MS‐MS

Cation exchange(elution with 

salt solution)

Reverse‐phase(elution withacetonitrile)

Lau, E.; Lam, M. P. Y.; Siu, S. O.; Lo, C.; Chu, I. K., Mol. BioSyst. 2011, 7, 1399-1408

Page 14: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

High pH RP ‐

Low pH RP SystemReversed phase separation is based on hydrophobicity Orthogonality is related to differences in retention of peptides in RP under different pH 

Different charges of amino acid side chain at different pH  RP‐RP provides comparable performance to SCX‐RP with a higher peak capacity in the first‐dimension 

Salt free eluents ‐minimized risk of ion suppression

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Page 15: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

High pH RP ‐

Low pH RP System

15IK Chu: PROTEOMICS: Volume 11, Issue 11, pages 2308‐2319, 2011 

Step 1:  Partial filling of the loop with fraction  eluted 

from the 1st

Dimension high pH RP columnStep 2:  Transfer to 2nd

Dimension  low pH RP 

column

Step 3:  Elution from  2nd

Dimension low pH RP 

column

Step 4:  Refill the loop with low pH mobile phase

Page 16: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

High pH RP ‐

Low pH RP SystemAnalysis 0f Mouse embryonic fibroblasts and Zebra fish embryo lysates 

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Hydro

phobicity

Mouse embryonic fibroblasts

Zebra fish embryo

Page 17: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

HILIC‐RP combinationChallenges: 

Solvent compatibility and peptide solubility

17IK Chu: J. Sep. Sci., 2012, 35, p 1755

Peptides 

trapping 

by a RP 

columns 

Transfer 

to HILIC 

via a 

mixing 

loop

HILIC 

elution 

and 

trapping 

by a SCX

column

RP 

elution

Page 18: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

HILIC‐RP combinationRat pheochromocytoma lysate

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Page 19: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

3DLC High pH RP‐SCX‐

Low pH RPEvolved from using SCX as trapping column to separation column

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1st Dimension 2nd Dimension 3rd Dimension

Page 20: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Analysis of STO mouse cell digest3D RP‐SCX‐RP (8 high‐pH RP fractions x 3 SCX fractions, 24 fractions in total)In comparison, 2D RP‐SCX (as trap column)‐RP (12 high‐pH fractions)

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No. of unique peptide (15523 vs. 26391)70.0% increase

Protein sequence coverage13.8% (5.2 peptides/protein)18.9% (7.3 peptides/protein)

No. of proteins(2985 vs. 3613)17.4 % increase

2D2D3D3D

Page 21: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Peptide carryoverCharge separation in SCX fractionation

Sufficient for tryptic peptides (+2,+3 and +4)

Correlation of hydrophobicity across fractions

2D RP-SCX-RP 3D RP-SCX-RP

Page 22: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

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1st DimensionCapillary-flow

2nd DimensionCapillary-flow

3rd DimensionNano-flow

Peptide identification Protein identification

Capillary-/nano-flow 3D RP-SCX-RP

~ 50% protein of yeast proteome

MS/MS

Page 23: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

Outlook and SummaryMDLC has  a key role in the analysis of highly complex samples in proteomics applications Suitable of Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) base quantitative proteomics applicationsFuture areas of MDLC research in Dr. I.K. Chu GroupOther column combinationsRuntime reduction4DLC separation Applications on system biology

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Page 24: 4th Multidimensional Chromatography Workshop Toronto ... · Herman C. Lam, Ph.D. Calibration & Validation Group. 4th Multidimensional Chromatography Workshop Toronto (January, 2013)

AcknowledgementsDr. Ivan Chu and members of his research group at the University of Hong Kong

Dr. Ricky NgDr. Ricky KongDr. S. O. SiuDr. Maggie LamYun ZhaoEdward LauHenry LawQuan Quan

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