Amino acids, Peptide bond and Amino acid analysis

1
Fundamentals of Protein/Peptide Separation
Technique High-Performance Liquid Chromatography
Masaru Miyagi, Ph.D. Case Center for
Proteomics January 23, 2008
2
General Flow Scheme for Proteomic Analysis
Top-down method
2D-PAGE
Protein mixture
Proteins
Separation
Digestion
Digestion
Bottom-up/Shotgun methods
Electrospray/MALDI
Peptide mixture
HPLC
Peptides
MS analysis
Separation
Protein identification
MS data
Database search software
Post-translational modification
3
Problem in Shotgun Proteomics

• Too many peptides
• 2,000 proteins x 40 80,000 peptides
• 10,000 proteins x 40 400,000 peptides
• A huge range of peptide abundances
• gt 109 order of difference in concentration

Goal Analyze all of the peptides generated
4
How Mass Spectrometer Analyzes Peptides Eluted
from a RP-column (LC-MS/MS Analysis)

• Step 1 acquire MS spectrum (1 s)
• Step 2 subject 3-10 most intense peaks to MS/MS
  analysis (1 s per peak)
• Repeat Step 1 and 2

It roughly takes one second to analyze one
peptide peak by MS/MS. (therefore, the capacity
is 60 peptides/min)
Mass spectrometer adds another dimension for the
separation of peptides!!
5
How to Overcome the Problem

• Reduce the protein complexity
• e.g., isolation of cellular organelles
• Reduce the peptide complexity prior to LC-MSMS
• e.g., strong cation exchange (SCX)-chromatography
• Increase the resolution of RP-HPLC separation
• e.g., small particle HPLC
• Use rapid scanning instruments
• e.g., linear ion trap

6
Outline of Todays Talk

• Fundamentals of high-performance liquid
  chromatography
• Reverse-phase chromatography
• Strong cation exchange chromatography
• Multidimensional HPLC

7
What is Chromatography?

• Chromatography is an analytical method that is
  used for the separation, identification, and
  determination of the chemical components in
  complex mixtures.
• All of chromatography methods have in common the
  use of a stationary phase and a mobile phase.
• Stationary phase a phase that is fixed in place
  either in a column or on a planar surface.
• Mobile phase a phase that moves over or through
  the stationary phase, carrying the analyte with
  it.
• The components of a mixture are separated based
  upon the rates at which they are carried through
  a stationary phase by a mobile phase.

8
Column Chromatography
Principles of Instrumental Analysis, Fifth
edition, Thomson Learning
9
Classification of Chromatographic Methods

• Column chromatography
• Gas chromatography
• Liquid chromatography
• Planar chromatography
• Paper chromatography
• Thin-layer chromatography

10
High-Performance Liquid Chromatography (HPLC)

• HPLC is a type of chromatography that employs a
  liquid mobile phase and a very finely divided
  stationary phase.
• The diameter of the particles of the solid
  stationary phase is 3 10 mm.
• In order to obtain satisfactory flow rates, the
  liquid must be pressurized to several hundred
  pounds per square inch or more.

11
Advantages of HPLC

• Speed
• High resolution
• Sensitivity
• Reproducibility
• Accuracy
• automation

UV/VIS MS
Reverse-phase HPLC
Miyagi et al,, Eur. J. Biochem, 2000
12
Low Pressure Mixing System
Shimazu Co., Users manual
13
High Pressure Mixing System
Shimazu Co., Users manual
14
Column Size vs Sensitivity?
Detectors used for HPLC are concentration
dependent!
If we can exactly scale down the performance
with 4.6 mm column to the smaller columns!!
15
Reverse-Phase Chromatography

• Stationary phase nonpolar (hydrocarbon, e.g.
  n-octyl or n-octyldecyl)
• Mobile phase relatively polar (water, methanol,
  or acetonitrile)
• Separation based on hydrophobicities of the
  analytes
• Peak capacity 500 peptides

Silica based resin
R C4, C8, C18 etc.
Skoog, D. A., et al., (1998) Principles of
Instrumental Analysis, Fifth Edition, Thomson
Learning
16
The Theory of Reverse-Phase Chromatography

• Key Hydrophobic Effect
• Hydrophobic effect is an entropy driven effect.

Streitwieser, A. et al. (1992) Introduction to
Organic Chemistry, Forth Edition, Macmillan
Publishing Company
17
Reverse-Phase Chromatography

• Stationary Phases
• Octadecyl (C18) (CH2)17CH3
• Octyl (C8) (CH2)7CH3
• Butoxy (C4) (CH2)3CH3
• Phenyl (CH2)2C6H5

18
Reverse-Phase Chromatography

• Sample
• High polarity
• Elution
• decrease polarity (increase hydrophobicity) of
  the mobile phase

19
Ion-exchange Chromatography

• Stationary phase ion-exchange resin (e.g. RSO3-,
  RN(CH3))
• Mobile phase polar (water)
• Separation based on charges of the analytes

Polystylene ion-exchange resine
Skoog, D. A., et al., (1998) Principles of
Instrumental Analysis, Fifth Edition, Thomson
Learning
20
The Theory of Ion Exchange Chromatography
NaCl
at pH 3
peptide
peptide

SO3
SO3
SO3
Cl
Na
21
Ion-exchange Chromatography

• Cation exchnge stationary phases
• Sulfonate conjugate SO3-
• Carboxylate conjugate COO-
• Anion exchnge stationary phases
• Quaternary ammonium conjugate NR3
• Polyalkyl amine conjugate NHR2

22
Ion-exchange Chromatography

• Sample
• low ionic strength
• Consider pH of the sample solution
• Elution
• increase salt concentration or change pH of the
  mobile phase

23
Multidimentional HPLC Ion Exchange RP-HPLC
system

• Peptides are separated in 2D-capillary HPLC
  according to their net charge and hydrophobicity.
• The peptides are loaded to the 1st dimension
  cation exchange column then the peptides are
  eluted by step gradient of increasing of salt
  concentration.
• Each step releases a group of peptides, which
  then pass on the reverse-phase column.
• Each peptide group then is separated by gradient
  of increasing of organic solvent concentration.

Washburn, M. P., et al. (2001) Nat. Biotechnol.
19, 242
24
Injection Mode
A)
2
micro pump module
2
micro pump module
3
3
MS
MS
1
1
1
1
1
1
2
2
2
2
10
10
10
10
Isocratic pump
3
3
3
3
9
9
9
9
Micro
Autosampler
Micro
Autosampler
4
4
4
4
8
8
8
8
waste
waste
7
7
5
5
7
5
5
7
6
6
6
6
waste
waste
Step Elution Mode from SCX column
B)
Gradient elution mode from RP-column
C)
Experimental set-up for multidimensional LC.
Column 1 SCX column (1 mm i.d. x 1 cm) Column
2 Reverse-phase trapping column (300 mm i.d. x 1
mm) Column 3 Reverse-phase analytical column
(50 mm i.d. x 5 cm).
25
Ultra performance liquid chromatography (UPLC)
Conventional HPLC 3.5 mm, C18
UPLC 1.7 mm, C18
Wilson et al., J Proteome Res. 2005, 4, 591-598
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RP-HPLC Peak Capacities
Peak capacity maximum number of peaks
(components) that can be theoretically separated
on a given LC column within a gradient time
Gilar et al., J Chromatogr A. 2004 Dec
241061(2)183-92
27
Current peptide separation techniques are not
sufficient!!

• We need better peptide separation techniques for
  proteomics