Herman C. Lam, Ph.D.Calibration & Validation Group
4th Multidimensional Chromatography Workshop4th Multidimensional Chromatography WorkshopToronto (January, 2013)Toronto (January, 2013)
MDLC for Shotgun ProteomicsIntroduction
General conceptsAdvantagesChallenges in implementation
ApplicationsShotgun proteomics
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Multidimensional chromatography
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Anal. Chem.
2008, 80, 7187‐7193.
LC
Fraction
s
TLC
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
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
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
LC Column Orthogonality and Solvent Compatibility
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Orthogonality Solvent
compatibility
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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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
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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
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
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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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
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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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
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
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
HILIC‐RP combinationRat pheochromocytoma lysate
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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
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
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
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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
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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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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