HPLC as a Frontend to Mass Spectrometry in Proteomics Biomedical - PowerPoint PPT Presentation

HPLC as a Frontend to Mass Spectrometry in Proteomics Biomedical Research Techniques October 31 th 2018, 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

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 PWHH: PWHH: 7.23 s PWHH: PWHH: 6.24 s 5.82 s 7.19 s A B Pumping system for the mobile phase Detection UV-VIS, Fluorescence, MS Column with stationary phase Heart of the system Sample introduction

Basic Layout for HPLC

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 (C 18 , C 8 , C 4 )  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 + 3 HN + – –

Bonded Phases  C-2 Ethyl Silyl -Si-CH2-CH3  C-8 Octyl Silyl -Si-(CH 2 ) 7 -CH 3 • C-18 Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3 • CN Cyanopropyl Silyl -Si-(CH 2 ) 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 + COO – 3 HN – + – + – + – + + – – + – + – + – + – + – + pH < pI < pH

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 Abundance difference is 10 orders  Proteomics samples typically have:  Complex matrix (10.000’s different High abundant proteins)  Huge concentration variation within a sample (abundance difference) Only 6 orders of enlargement  Limited sample amount (few µl’s of sample)  This requires: Low – Efficient separation abundant – 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. + - +/- 100 40 40 1.50 1.50 mAU 125 75 30 30 100 1.00 1.00 7 5 50 mAU mAU 20 20 mAU mAU mAU 5 0 0.50 0.50 25 10 10 2 5 0 0 0 0.0 0.0 0 0 -20 0 2 0 4 0 6 0 8 0 100 120 140 min 35 70 105 140 5.00 5.00 6.00 6.00 7.00 7.00 8.00 8.00 9.00 9.00 0 0 10 10 20 20 30 30 40 40 Time (min) min min Time (min) Time (min) Digested protein Digested tissue sample Digested protein complex

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 4.6 mm 2.1 mm 300 µm 75 µm 2 pmol digested myoglobin (injected on each column)

Downscaling Factor LC The concentrating effect of the smaller ID columns compared to standard HPLC can be calculated 2   d conventional   Concentration factor   d nano Capillary LC (300 µm ID) Nano LC (75 µm ID) 2 2     4.6  4.6    = 235 x = 3800 x     0.075 0.3

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 mAU WVL:214 nm 6.0 5.0 Signal intensity 4.0 3.0 2.0 1.0 0.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 Retention time (min) Nano 75 µm ID x 15 cm Acclaim PepMap C 18 , 1 pmol BSA digest 4-55 %B in 120 min

2D Salt Plug Application Neutral 1+ 2+ 3+  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

2D Salt Plug Chromatograms 200 mAU WVL:214 nm 175 150 PMD 1 mM 125 2 mM Signal Intensity 5 mM 100 10 mM 20 mM 75 50 mM 100 mM 50 200 mM 500 mM 750 mM 25 1000 mM 2000 mM 0 min -20 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 Retention time (min)

Comparison of 2D Methods with E.Coli Tryptic Digest Intens. 50.0 8 x10 Base Peak Chromatograms SCX x RP 2.0 45.0 1.5 40.0 1.0 35.0 0.5 30.0 0.0 25.0 20 25 30 35 40 45 Time [min] 2.0 5.0 7.5 10.0 12.5 15.0 19.0 Intens. 50.0 8 x10 Base Peak Chromatograms RP x RP 1.5 45.0 1.0 40.0 35.0 0.5 30.0 0.0 20 25 30 35 40 45 Time [min] 25.0 9.0 12.0 14.0 16.0 18.0 21.0

Column Length Variation 12.0 10.0 mAU mAU 15 cm PMD 120 min 5.0 5.0 0.0 308 0.0 0 25 50 75 100 130 65.0 67.5 70.0 72.5 75.0 12.0 9.0 mAU mAU 25 cm PMD 120 min 5.0 5.0 0.0 338 1.0 0 25 50 75 100 130 65.0 67.5 70.0 72.5 75.0 12.0 8.9 mAU mAU 50 cm PMD 120 min 5.0 5.0 0.0 0.0 443 min min 0 25 50 75 100 130 65.0 67.5 70.0 72.5 75.0

New developments  New instrumentation have HPLC - 350 bar UHPLC - 800 bar high pressure capabilities 50 cm x 75 µm ID 50 cm x 75 µm ID C18 3 µm C18 2 µm 75 um x 50 cm,C18,2 um 75 um x 50 cm,C18,3 um

Particle size comparison 10.0 1 - 20110812 #25 c1: CytoC -- 120min (025) UV_VIS_1 mAU WVL:214 nm 2 µm C18 particles column 5.0 Cytochrome C digest 1 1 pmol -2.0 8.0 2 - 20110812 #26 . c2: CytoC -- 120min (026) UV_VIS_1 mAU WVL:214 nm 3 µm C18 particles column 5.0 2 min -2.0 0 13 25 38 50 63 75 88 100 113 130 24.9 1 - 2011-09-09 #5 c1: Fab-90min-3 (005) UV_VIS_1 mAU WVL:214 nm 2 µm column Fab fragment digest 12.5 1 pmol 1 -6.0 25.0 2 - 2011-09-09 #6 . c2: Fab-90min-3 (006) UV_VIS_1 mAU WVL:214 nm 3 µm column 12.5 2 min -5.0 26.2 40.0 50.0 60.0 70.0 80.0 90.0 100.0 108.3

Effect on MS signal Increase in MS/MS spectra with longer gradients and smaller particles Average MS intesity dependance on gradient time 10000000 9000000 8000000 7000000 Average MS intesity 6000000 3µm column 5000000 2µm column 4000000 Effect of gradient length and 3000000 2000000 particle size on signal 1000000 0 90 min 180 min 300 min intensity Gradient time

Different Structures Stationary Phases  Porous  Perfusion Fused core Monolithic