1 Stationary vs. Mobile Phase Chromatogram In a suitable - - PDF document

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HPLC as a Frontend to Mass Spectrometry in Proteomics

Biomedical Research Techniques November 8th 2017, Erasmus Medical Centre

Overview

• What is chromatography?
• Principles
• Basic layout
• Stationary phases
• Chromatography in Proteomics
• Samples
• Strategy
• NanoLC
• Applications
• Recent developments
• Summary

HPLC? High Performance Liquid Chromatography

• Chromato graphy means color writing
• Founding fathers

Christian Friedrich Schönbein (1799 - 1868) Mikhail Semyonovich Tsvet (1872-1919) Movement of substances through Separation of plant pigments in calcium filter paper carbonate columns

What is Chromatography

• Chromatography is the separation of analytes by creating a partitioning

system for which the analytes have a different affinity.

• Partitioning is between the
• Mobile phase

 transports the analyte

• Stationary phase  slows down/ retains the analyte
• Common forms are
• Gas chromatography
• Liquid chromatography
• Thin layer chromatography

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Stationary vs. Mobile Phase

• In a suitable separation system the different analytes will have a

different retention by the column

Flow of solvent

Chromatogram

• The detected signals are presented in a chromatogram
• This shows the analytes separated in time

Time 

Basic Layout for HPLC

Column with stationary phase Heart of the system

B A

Pumping system for the mobile phase Sample introduction Detection

UV-VIS, Fluorescence, MS

PWHH: 7.19 s PWHH: 7.23 s PWHH: 5.82 s PWHH: 6.24 s

Basic Layout for HPLC

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Various Flavours in Stationary Phase

Name Principle Strongest retention Reversed phase Hydrophobicity Hydrophobic molecules Normal phase Hydrophilicity Hydrophilic molecules Ion exchange Charge Highest charge Size exclusion Size Smallest molecule Affinity Key-lock Best affinity

• Choice depends on the sample being analysed
• Some samples require combinations of stationary phases

Reversed Phase

• Polar mobile phase (aqueous)
• Apolar stationary phase (C18, C8, C4)
• Hydrophobic interactions cause retention
• During gradient analysis the mobile phase is made less

polar,”loosening” the hydrophobic interactions

• In proteomics typically Ion Pair Reversed Phase is used due to the

charges present on biomolecules

+

+ 3HN

– –

Bonded Phases

• C-2

Ethyl Silyl

• Si-CH2-CH3
• C-8

Octyl Silyl

• Si-(CH2)7-CH3
• C-18

Octadecyl Silyl

• Si-(CH2)17-CH3
• CN

Cyanopropyl Silyl

• Si-(CH2)3-CN

Ion Exchange

• Low ionic strength mobile phase
• Charged stationary phase
• Charge-Charge interactions cause retention
• Elution is based on increase in mobile phase ionic strength
• Both cation and anion exchange columns are available
• Choice depends largely on pI of the sample

+ –

pH < pI < pH

+

+ 3HN

– – – – – – – – – + + + + + + + + +

–

COO–

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Terminology

• Retention

A measure for partitioning on the stationary phase

• Eluent

Solvents used

• Mobile phase

Location in which an analyte moves

• Stationary phase

Location where the analyte does not move

• Gradient

Eluent composition change over time

• Resolution

Measure of separation between two peaks

• Peak capacity

The number of peaks that can be separated in a gradient

• Loadability

Amount of material that can be separated efficiently

Samples in Proteomics

• Proteomics samples typically have:
• Complex matrix (10.000’s different

proteins)

• Huge concentration variation within a

sample (abundance difference)

• Limited sample amount (few µl’s of sample)

Only 6 orders

• f enlargement

High abundant

Abundance difference is 10 orders

Low abundant

 This requires: – Efficient separation – Sensitive measurement techniques – Detection that provides structural (“What is it?”) information

General Strategy Proteomics

Step 1: Isolation of proteins Step 2: Digestion Step 3: Separation Step 4: MS detection Step 5: MS/MS detection Step 6: Protein identification

From: Ruedi Aebersold & Matthias Mann NATURE 422 (2003) p.198

Effect of Digestion on Sample Complexity

• Tryptic digestion will cleave a protein behind lysine or arginine residue.
• 1 protein is cleaved into 20-50 peptides.
• Proteomics samples typically have 1000-10000 proteins
• After digestion the complexity is increased 20 fold.

+

Digested protein

5.00 6.00 7.00 8.00 9.00 0.0 0.50 1.00 1.50 mAU min 5.00 6.00 7.00 8.00 9.00 0.0 0.50 1.00 1.50 mAU min 10 20 30 40 10 20 30 40 10 20 30 40 Time (min) 10 20 30 40 mAU

Digested protein complex

Time (min) mAU

+/-

Digested tissue sample

2 4 6 8 100 120 140

• 20

2 5 5 7 5 100 125 mAU min

• 35

70 105 140 25 50 75 100 Time (min) mAU

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Concentration vs. Sample Amount

• The concentrating effect of Nano LC is required when the sample amount is

limited.

• In LC a volume of a certain concentration is injected, this is a fixed amount.
• Upon injection this amount is “dissolved” in the LC volume, to create a new

concentration. A low volume LC will generate a higher concentration for the same injected amount.

Sensitivity Comparison

2 pmol digested myoglobin (injected on each column)

4.6 mm 2.1 mm 300 µm 75 µm

Downscaling Factor LC

Concentration factor

dnano dconventional

2

     

2

0.3 4.6       = 235 x

2

0.075 4.6       = 3800 x

The concentrating effect of the smaller ID columns compared to standard HPLC can be calculated

Nano LC (75 µm ID) Capillary LC (300 µm ID)

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How to put the theory into practice?

1D - Long Gradient RP LC

• µColumn switching or Pre-concentration setup
• Popular for its simplicity and relatively low time consumption (compared

to 2D).

• Properties:
• 1 sample analyzed in 1 RP run
• 1 run = 1-4 hours
• Limited separation power for very complex samples

Preconcentration

• The sample is loaded on a trap column where it is concentrated and washed.
• Switching the valve transfers the sample to the analytical column and allows detection

by UV/MS

Separation Example

Nano 75 µm ID x 15 cm Acclaim PepMap C18, 1 pmol BSA digest 4-55 %B in 120 min

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0

mAU min WVL:214 nm

Retention time (min) Signal intensity

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2D Salt Plug Application

• Consecutive injections of increasing salt concentrations transfer part of

the trapped sample from the SCX to the RP trap column

• The pre-concentration part of the setup will wash the sample and

analyze it by UV/MS

Neutral 1+ 2+ 3+

2D Salt Plug Chromatograms

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0

• 20

25 50 75 100 125 150 175 200 mAU min WVL:214 nm

PMD 1 mM 2 mM 5 mM 10 mM 20 mM 50 mM 100 mM 200 mM 500 mM 750 mM 1000 mM 2000 mM

Retention time (min) Signal Intensity

RP x RP SCX x RP

9.0 12.0 14.0 16.0 18.0 21.0 25.0 30.0 35.0 40.0 45.0 50.0 2.0 5.0 7.5 10.0 12.5 15.0 19.0 25.0 30.0 35.0 40.0 45.0 50.0 20 25 30 35 40 45 Time [min] 0.0 0.5 1.0 1.5 2.0 8 x10 Intens.

Base Peak Chromatograms

20 25 30 35 40 45 Time [min] 0.0 0.5 1.0 1.5 8 x10 Intens.

Base Peak Chromatograms

Comparison of 2D Methods with E.Coli Tryptic Digest Column Length Variation

65.0 67.5 70.0 72.5 75.0 0.0 5.0 8.9 mAU min 0.0 5.0 12.0 mAU

25 cm PMD 120 min

25 50 75 100 130 0.0 5.0 12.0

15 cm PMD 120 min

mAU 25 50 75 100 130 0.0 5.0 12.0

50 cm PMD 120 min

mAU min 25 50 75 100 130 0.0 5.0 10.0 mAU 65.0 67.5 70.0 72.5 75.0 1.0 5.0 9.0 mAU 65.0 67.5 70.0 72.5 75.0

308 338 443

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New developments

• New instrumentation have

high pressure capabilities

50 cm x 75 µm ID C18 2 µm 50 cm x 75 µm ID C18 3 µm UHPLC - 800 bar HPLC - 350 bar 75 um x 50 cm, C18, 2 um 75 um x 50 cm,C18,3 um

Particle size comparison

Cytochrome C digest 1 pmol Fab fragment digest 1 pmol

• 2.0

5.0 10.0 1 - 20110812 #25 c1: CytoC -- 120min (025) UV_VIS_1 mAU 1 WVL:214 nm 13 25 38 50 63 75 88 100 113 130

• 2.0

5.0 8.0 2 - 20110812 #26 . c2: CytoC -- 120min (026) UV_VIS_1 mAU min 2 WVL:214 nm

2 µm C18 particles column 3 µm C18 particles column

• 6.0

12.5 24.9 1 - 2011-09-09 #5 c1: Fab-90min-3 (005) UV_VIS_1 mAU 1 WVL:214 nm 26.2 40.0 50.0 60.0 70.0 80.0 90.0 100.0 108.3

• 5.0

12.5 25.0 2 - 2011-09-09 #6 . c2: Fab-90min-3 (006) UV_VIS_1 mAU min 2 WVL:214 nm

2 µm column 3 µm column

Effect on MS signal

Average MS intesity dependance on gradient time 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 9000000 10000000 90 min 180 min 300 min Gradient time Average MS intesity 3µm column 2µm column

Increase in MS/MS spectra with longer gradients and smaller particles Effect of gradient length and particle size on signal intensity

Different Structures Stationary Phases

• Porous
• Perfusion

Fused core Monolithic

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Summary

• HPLC is used to separate analytes of interest from each other and a

matrix.

• This sample complexity decrease is essential for current

Proteomics research

• Reversed phase remains the most applied separation strategy
• SCX is widely used as well, affinity techniques for selective

enrichment are becoming more mainstream.

• Dedicated LC instrumentation allow a combination of various

separation steps as a front end to mass spectrometer.

• Sample determines separation strategy!