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

Overview  What is chromatography?  Principles  Basic layout  Stationary phases HPLC as a Frontend to Mass Spectrometry in  Chromatography in Proteomics Proteomics  Samples  Strategy Biomedical Research Techniques  NanoLC November 8 th 2017, Erasmus Medical Centre  Applications  Recent developments  Summary HPLC? What is Chromatography  Chromatography is the separation of analytes by creating a partitioning High Performance Liquid Chromatography system for which the analytes have a different affinity.  Chromato graphy means color writing  Founding fathers  Partitioning is between the Christian Friedrich Schönbein (1799 - 1868) Mikhail Semyonovich Tsvet (1872-1919)  Mobile phase  transports the analyte Movement of substances through Separation of plant pigments in calcium  Stationary phase  slows down/ retains the analyte filter paper carbonate columns  Common forms are  Gas chromatography  Liquid chromatography  Thin layer chromatography 1

Stationary vs. Mobile Phase Chromatogram  In a suitable separation system the different analytes will have a  The detected signals are presented in a chromatogram different retention by the column  This shows the analytes separated in time Flow of solvent Time  Basic Layout for HPLC Basic Layout for HPLC PWHH: PWHH: PWHH: 7.23 s 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 2

Various Flavours in Stationary Phase Reversed Phase Name Principle Strongest retention  Polar mobile phase (aqueous) Reversed phase Hydrophobicity Hydrophobic molecules  Apolar stationary phase (C 18 , C 8 , C 4 )  Hydrophobic interactions cause retention Normal phase Hydrophilicity Hydrophilic molecules  During gradient analysis the mobile phase is made less Ion exchange Charge Highest charge polar,”loosening” the hydrophobic interactions Size exclusion Size Smallest molecule  In proteomics typically Ion Pair Reversed Phase is used due to the Affinity Key-lock Best affinity charges present on biomolecules  Choice depends on the sample being analysed + 3 HN + – –  Some samples require combinations of stationary phases Bonded Phases Ion Exchange  C-2 Ethyl Silyl -Si-CH2-CH3  Low ionic strength mobile phase  Charged stationary phase  C-8 Octyl Silyl -Si-(CH 2 ) 7 -CH 3  Charge-Charge interactions cause retention • C-18 Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3  Elution is based on increase in mobile phase ionic strength • CN Cyanopropyl Silyl -Si-(CH 2 ) 3 -CN  Both cation and anion exchange columns are available  Choice depends largely on pI of the sample + 3 HN – COO – + – + + – + – + – – + – + – + + – – + – + pH < pI < pH 3

Terminology Samples in Proteomics Abundance difference  Retention A measure for partitioning on the stationary phase is 10 orders  Proteomics samples typically have:  Eluent Solvents used  Complex matrix (10.000’s different High abundant  Mobile phase Location in which an analyte moves proteins)  Huge concentration variation within a  Stationary phase Location where the analyte does not move sample (abundance difference) Only 6 orders of enlargement  Limited sample amount (few µl’s of sample)  Gradient Eluent composition change over time  Resolution Measure of separation between two peaks  This requires: Low  Peak capacity – Efficient separation The number of peaks that can be separated in a gradient abundant – Sensitive measurement techniques  Loadability Amount of material that can be separated efficiently – Detection that provides structural (“What is it?”) information General Strategy Proteomics Effect of Digestion on Sample Complexity Step 1: Isolation of proteins  Tryptic digestion will cleave a protein behind lysine or arginine residue. Step 2: Digestion  1 protein is cleaved into 20-50 peptides. Step 3: Separation Step 4: MS detection Step 5: MS/MS detection  Proteomics samples typically have 1000-10000 proteins Step 6: Protein identification  After digestion the complexity is increased 20 fold. + +/- - 100 1.50 1.50 40 40 mAU 125 75 30 30 100 1.00 1.00 5 7 50 mAU mAU 20 20 mAU mAU mAU 0 5 0.50 0.50 25 10 10 2 5 0 0 0 0.0 0.0 0 0 -20 0 2 0 35 4 0 0 6 70 0 8 100 105 120 140 140 min 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 min min Time (min) Time (min) Time (min) Digested protein Digested protein complex Digested tissue sample From: Ruedi Aebersold & Matthias Mann NATURE 422 (2003) p.198 4

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. Downscaling Factor LC Sensitivity Comparison The concentrating effect of the smaller ID columns compared to standard HPLC can be calculated 4.6 mm 2.1 mm 2   d conventional   Concentration factor   d nano 300 µm Capillary LC (300 µm ID) Nano LC (75 µm ID) 75 µm  4.6   2   2 4.6    = 3800 x = 235 x     0.075 0.3 2 pmol digested myoglobin (injected on each column) 5

1D - Long Gradient RP LC  µColumn switching or Pre-concentration setup  Popular for its simplicity and relatively low time consumption (compared to 2D).  Properties: How to put the theory into practice?  1 sample analyzed in 1 RP run  1 run = 1-4 hours  Limited separation power for very complex samples Preconcentration 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)  The sample is loaded on a trap column where it is concentrated and washed. Nano 75 µm ID x 15 cm Acclaim PepMap C 18 ,  Switching the valve transfers the sample to the analytical column and allows detection 1 pmol BSA digest 4-55 %B in 120 min by UV/MS 6

2D Salt Plug Application 2D Salt Plug Chromatograms Neutral 1+ 2+ 3+ 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 25 750 mM 1000 mM  Consecutive injections of increasing salt concentrations transfer part of 2000 mM 0 the trapped sample from the SCX to the RP trap column 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  The pre-concentration part of the setup will wash the sample and Retention time (min) analyze it by UV/MS Comparison of 2D Methods with E.Coli Tryptic Digest Column Length Variation Intens. 50.0 8 x10 Base Peak Chromatograms SCX x RP 12.0 10.0 mAU mAU 15 cm PMD 120 min 2.0 45.0 5.0 1.5 5.0 40.0 1.0 0.0 308 35.0 0.0 0 25 50 75 100 130 65.0 67.5 70.0 72.5 75.0 12.0 9.0 0.5 mAU mAU 30.0 25 cm PMD 120 min 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 5.0 5.0 Intens. 50.0 8 x10 Base Peak Chromatograms RP x RP 1.5 0.0 338 1.0 45.0 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 1.0 40.0 5.0 5.0 35.0 0.5 0.0 0.0 30.0 443 min min 0.0 0 25 50 75 100 130 65.0 67.5 70.0 72.5 75.0 20 25 30 35 40 45 Time [min] 25.0 9.0 12.0 14.0 16.0 18.0 21.0 7

New developments Particle size comparison 10.0 1 - 20110812 #25 c1: CytoC -- 120min (025) UV_VIS_1 mAU WVL:214 nm  New instrumentation have 2 µm C18 particles column HPLC - 350 bar UHPLC - 800 bar 5.0 high pressure capabilities Cytochrome C digest 50 cm x 75 µm ID 50 cm x 75 µm ID 1 C18 3 µm C18 2 µm 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 75 um x 50 cm, C18, 2 um mAU WVL:214 nm 3 µm column 12.5 75 um x 50 cm,C18,3 um 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 Different Structures Stationary Phases  Porous Increase in MS/MS spectra with longer gradients and smaller particles  Perfusion Fused core Monolithic 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 intensity 90 min 180 min 300 min Gradient time 8

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! 9