2013-04-08 1 New Developments in Capillary Electrophoresis with focus on Bioanalysis Lecture 7 Christian Nilsson Today´s Lecture • Focus on metabolomic studies • CE-MS an alternative to more traditonal techniques (LC-MS, GC-MS, NMR) • cIEF as an alternative to slab gel IEF in pharmaceutical industry Metabolomics • Metabolome: Entire set of low-molecular weight compounds within a biological sample • Metabolomics: Analysis of the metabolome • In plants the amount of metabolites is expected to reach 1 million Metabolomics • Identify and quantify groups of metabolites belonging to different metabolomic pathways Metabolomics • Non-targeted approach – Measure as many metabolites as possible within a single analysis • Targeted approach – Focus on analysis and quantification of a few known metabolites • Both approaches can be combined – First broad approach to discover potential biomarker – Second approach to quantify the compounds identified in the first step Metabolomics • Large difference in: – Type of molecules – Concentrations • Several internal standards often necessary – Each representing a class of compounds 2013-04-08 2 Metabolomics • Metabolomic fingerprint – Changes due to disease – Changes due to therapeutic treatment • Important approach for screening of diagnostic markers for diseases Metabolomics • GC-MS – Not suitable for non-volatile, termolabile or polar compounds – Laborious derivatisation of metabolites often necessary Metabolomics • NMR – Rapid, non-destructive, minimal sample pretreatment – Limited sensitivity – Relatively large sample amount required (micrograms) Metabolomics • LC-MS – Now derivatization required – Can be used for identification and quantification of metabolites – Use of UPLC or monolitic LC to improve efficiency – Less suitable for ionic and polar compounds • Hydrophilic Interaction LC (HILIC) is an alternative CE-MS in metabolomics • Especially suitable for analysis of charged and polar metabolites • Complementary technique to RP-LC • HILIC-MS an alternative Reviews: Electrophoresis 2009, 30, 276-291 Electrophoresis 2011, 32, 52-65 Electrophoresis 2013, 34, 86-98 CE-MS for metabolomics • No extensive sample pretreatment • Low consumption of mobile phase and sample • Fused silica capillaries instead of expensive LC columns • Easier to multiplex CE compared to LC • Low concentration sensitivity – Can be improved by preconcentration 2013-04-08 3 CE-MS Metabolomics • Want to achieve maximum coverage • Have to consider pretreatment of sample • Compounds are lost during extraction of metabolome Sample pretreatment • Separation of low-molecular weight compounds from larger molecules (proteins, lipids and larger peptides) – Ultracentrifugation – Precipitation • Larger molecules can otherwise adsorb to the capillary wall and reduce reproducibility CE-MS in metabolomics • MS compatible buffers can be used as electrolyte for CE separation • Have to think about both separation and detection Sample pretreatment • Minimal to prevent loss of metabolites • Extraction of metabolites from bacterias using organic solvents (hot or cold methanol, ethanol, chloroform-methanol) • Urine can be injected directly Non-targeted approach Sample pretreatment • Adapt procedures to target metabolites • Remove larger compounds • Solid Phase Extraction – Affinity SPE can be used Targeted approach Sample pretreatment • Preconcentration of urinary nucleosides from thyroid cancer patients • Affinity SPE column based on phenylboronic acid – Boronic acid used for recognition of sugars • MEKC analysis – Separation buffer: 25 mM borate, 42.5 mM phosphate, pH 6.7, 200 mM SDS Targeted approach Analytica Chimica Acta 2003, 486, 171-182 2013-04-08 4 Sample pretreatment Targeted approach Analytica Chimica Acta 2003, 486, 171-182 Sample pretreatment Targeted approach Analytica Chimica Acta 2003, 486, 171-182 CE-MS in Metabolomics • Non-targeted metabolomics – Analysis both at high and low pH to improve coverage – Identification based on molecular weight Comparsion of CE-MS and UPLC-MS • CE-MS – Use of triple layer coating of polybrene-dextran sulfate-polybrene – Profiling of human urine – Analysis of urine from 30 females and 30 males • Compared to analysis with reversed phase UPLC-MS • Differences in profile between genders Mol. Biosyst. 2011, 7, 194-199 Comparsion of CE-MS and UPLC-MS • CE-MS – Different compounds was used for gender classification by CE-MS and UPLC-MS – CE: Highly polar compounds with no retention in reversed phase UPLC – CE: A m/z value in the range of 50-150 compared to >150 in UPLC. Mol. Biosyst. 2011, 7, 194-199 Comparsion of CE-MS and UPLC-MS Mol. Biosyst. 2011, 7, 194-199 Only glutamic acid retained in Reversed phase UPLC 2013-04-08 5 CE-MS applications CE-MS in Metabolomics • Optimisation of Electrolyte and Sheath liquid – Anionic metabolytes – Frequently relatively low sensitivity in negative ionization mode – Improved sensitivity – Use of triethylamine in electrolyte and sheath liquid Electrophoresis 2011, 32, 3016-3024 CE-MS in Metabolomics Electrophoresis 2011, 32, 3016-3024 The amount of compounds that was detected was more than doubled by the use of TEA Probably due to less ion suppression with TEA in the buffer compared with ammonium acetate Simultaneous detection of amino acids and carboxylic acid by CE-MS • Improving the coverage in a single run • Amino acids • Carboxylic acids (for example: glycerate, lactate, fumate, succinate, malate, citrate) • Acidic electrolyte (1M Formic acid) • Uncoated capillary • Normal polarity Anal. Chem. 2010, 82, 9967-9976 Simultaneous detection of amino acids and carboxylic acid by CE-MS • A high sheath gas flow pressure was used (20 psi) • The gas flow caused a liquid suction throw the capillary reducing the migration time of the carboxylic acids • The polarity is changed from positive to ngative during the CE run to detect both amino acids and carboxylic acids Anal. Chem. 2010, 82, 9967-9976 Simultaneous detection of amino acids and carboxylic acid by CE-MS • The high sheath gas pressure might cause band broadening • However, it was possible to separate most of the compound evaluated in the described study Anal. Chem. 2010, 82, 9967-9976 2013-04-08 6 Simultaneous detection of amino acids and carboxylic acid by CE-MS Anal. Chem. 2010, 82, 9967-9976 Analysis of pineapple leaf as an example The ionization mode is changed after 29 min Single Cell Metabolomics • Want to get a better understanding of cell functions • Investigate cell-to-cell differences • Low molecular weight compounds that are produced in one cell can be found in many other cells, which complicate predictions • This is also true for macromolecules (such as proteins and DNA) but to less extent Single neuron detection • Single cell is an emerging field in MS metabolomics • See [Current Opinion in Biotechnology 2013, 24, 95-104] for a review of single cell metabolomics in general Anal. Chem. 2011, 83, 6810-6817 Single neuron detection • CE-MS for single neuron analysis • Home-made sheath liquid interface with a flow rate of 750 nl/min • 6 nl from each neuron extract was injected into the capillary using a 500 nl stainless steel sample vial • More than 300 compounds were detected Anal. Chem. 2011, 83, 6810-6817 Single neuron detection • 6 different types of neuron were compared • Could compare the metabolyte levels of the different neurons • Could see chemical similarities among some neurons and other had more distinct features • The described platform is adapted to other nanoliter samples Anal. Chem. 2011, 83, 6810-6817 Single neuron detection Anal. Chem. 2011, 83, 6810-6817 White bars = 1 mm. Extraction of the intracellular analytes from a single neuron 2013-04-08 7 Single neuron detection Anal. Chem. 2011, 83, 6810-6817 Single neuron detection Anal. Chem. 2011, 83, 6810-6817 Comparsion among four different types of neurons MS couplings CE-MS with platinum ESI spray needle • Improvement of sheath flow CE-MS for anionic metabolites – Using platinum ESI spray needle instead of stainless steal needle – Negative ionization mode – CE in reversed polarity due to positively charged capillary coating Anal. Chem. 2009, 81, 6165-6174 CE-MS with platinum ESI spray needle • Stainless steel needle showed oxidation and corrosion due to electrolysis • Iron oxides precipitate and plugged the capillary outlet – Shorter capillary life time Anal. Chem. 2009, 81, 6165-6174 CE-MS with platinum ESI spray needle • Many anionic metabolites formed complexes with iron oxides and nickel ions from the stainless steel tip. • Metal-metabolite complexes caused ionization suppression and reduced sensitivity • Platinum is not oxidized by electrolysis Anal. Chem. 2009, 81, 6165-6174 2013-04-08 8 CE-MS with platinum ESI spray needle Anal. Chem. 2009, 81, 6165-6174 CE-MS with platinum ESI spray needle Nickel(II)- and Iron(II)-anion complexes formed using a stainless steel needle [Fe(II)-citrate]- [Ni(II)-citrate][Ni(II)-GTP]2- [Fe(II)-CoA]2- [Ni(II)-GTP]CE-MS with platinum ESI spray needle Anal. Chem. 2009, 81, 6165-6174 CE-MS with platinum ESI spray needle Anal. Chem. 2009, 81, 6165-6174 Example of analysis of metabolytes from: -Glycolysis -Pentose phosphate pathway -Tricarboxylic acid cycle CE-MS with platinum ESI spray needle Anal. Chem. 2009, 81, 6165-6174 Metabolites in mouse liver Quantification of compounds from the central metabolic pathways 32 compounds detected Extraction -Liver tissue was put in methanol -Homogenized , 2 min -300μl was mixed with 500μl water and 200μl cloroform -Ultracentrifugation, 15000 rpm, 15 min -Aqueous layer was filtered to remove proteins CE-MS with platinum ESI spray needle Metabolites in mouse liver Quantification of compounds from the central metabolic pathways 32 compounds detected Extraction -Liver tissue was put in methanol -Homogenized , 2 min -300μl was mixed with 500μl water and 200μl cloroform -Ultracentrifugation, 15000 rpm, 15 min -Aqueous layer was filtered to remove proteins 2013-04-08 9 CE-MS with porous sheathless MS connection • Improvement of sensitivity with sheathless detection Analytical Chemistry 2012, 84, 885-892 CE-MS with porous sheathless MS connection • Sheathless MS interface • Polyimide coating was removed at the outlet side of the capillary • HF was used to etch the capillary wall to thickness of about 5 μm • The etched part that now is conductive is insert into a ESI needle • The ESI needle is filled with electrolyte Analytical Chemistry 2012, 84, 885-892 CE-MS with porous sheathless MS connection Analytical Chemistry 2012, 84, 885-892 CE-MS with porous sheathless MS connection • The described connection is useful for narrow capillaries and low flow nano-ESI-MS • The analytes are not diluted as with a sheath flow connection • Improved coverage of the urinary metabolome Analytical Chemistry 2012, 84, 885-892 CE-MS with porous sheathless MS connection Analytical Chemistry 2012, 84, 885-892 ~900 compounds is detected instead of ~300 compounds with a sheath flow interface Flow-through microvial interface Electrophoresis 2010, 31, 1130-1137 • The capillary is inserted in a stainless steel hollow electrospray emitter • The small volume between the capillary end and the inner wall of the electrode tip act as a flow-through micro vial (outlet vial) • Addition of a chemical modifier solution at a low flow rate is possible • Ensure stable flow to the tip with a minimum of sample dilution 2013-04-08 10 Flow-through microvial interface Electrophoresis 2010, 31, 1130-1137 Flow-through microvial interface Electrophoresis 2010, 31, 1130-1137 • Amino acid analysis at low pH (pH 3.1) • Uncoated capillary • 5-fold improvement of the detection limits compared to a conventional sheath liquid interface Flow-through microvial interface Electrophoresis 2010, 31, 1130-1137 Capillary Isoelectric Focusing of Protein Isoforms Outline • Background – Capillary Isoelectric Focusing – Sample Preparation – Sample Injection – Focusing – Mobilization • Results • Conclusions Capillary Isoelectric Focusing • Separation based on isoelectric point • Capillary Electrophoresis Equipment • Carrier ampholytes to establish pH gradient 2013-04-08 11 cIEF – Sample Preparation Urea-GelProtein sample: 5-10 mg/ml < 50 mM salt Anodicstabilizer Cathodicstabilizer Peptide pI markers Ampholytes Protein sample DILUTION cIEF - Injection Sample UV Detection Capillary Pressure cIEF - Focusing UV Detection Capillary Applied Voltage: 25 kV Anolyte Catholyte -+ cIEF - Focusing Anolyte Catholyte UV Detection Anodic Stabilizer Cathodic Stabilizer pH Gradient 3 4 5 6 7 8 9 10 Capillary Applied Voltage: 25 kV -+ Anolyte Chemical Mobilizer UV Detection Anodic Stabilizer Cathodic Stabilizer pH Gradient 3 4 5 6 7 8 9 10 Capillary cIEF - Mobilization Applied Voltage: 30 kV 280 nm -+ Results: Peptide markers -0,005 0 0,005 0,01 0,015 0,02 0,025 0,03 0 5 10 15 20 25 30 Time (min) Abs(280nm) pI 10.0 9.5 7.0 5.5 4.1 FOCUSING MOBILIZATION 2013-04-08 12 Results: Peptide markers Current -5 0 5 10 15 20 25 0 5 10 15 20 25 30 Time (min) Current(microA) FOCUSING MOBILIZATION Results: 3 batches of proteins Batch 1 Batch 1 - Run 1 -0,005 0 0,005 0,01 0,015 0,02 15 17 19 21 23 Time (min) Abs Batch 1 - Run 2 -0,005 0 0,005 0,01 0,015 0,02 15 17 19 21 23 Time (min) Abs Results: 3 batches of proteins Batch 2 Batch 2 - Run 1 0 0,005 0,01 0,015 0,02 0,025 15 17 19 21 23 25 Time (min) Abs Batch 2 - Run 2 -0,002 0,003 0,008 0,013 0,018 0,023 15 17 19 21 23 25 Time (min) Abs Results: 3 batches of proteins Batch 3 Batch 3 - Run 1 -0,002 0,003 0,008 0,013 17 19 21 23 25 Time (min) Abs Batch 3 - Run 2 -0,002 0,003 0,008 0,013 17 19 21 23 25 Time (min) Abs Results: 3 batches of proteins Batch 1-3 Batch 1 - Run 1 -0,005 0 0,005 0,01 0,015 0,02 15 17 19 21 23 Time (min) Abs Batch 2 - Run 1 0 0,005 0,01 0,015 0,02 0,025 15 17 19 21 23 25 Time (min) Abs Batch 3 - Run 1 -0,002 0,003 0,008 0,013 17 19 21 23 25 Time (min) Abs Results: Gel IEF of proteins 1 2 3 4 5.1 4.75 4.65 4.45 Legend: 1.PI marker. 2.Ref. material 09800031 14 mg. 3.DS 08800050 14 mg. 4.DS 09PD80010 14 mg. 5.1 4.75 4.65 4.45 1 2 3 4 5 Legend: 1.2192-122 (Tox 2) 20 mg. 2.2192-121 (Undiluted Tox 2) 15 mg. 3.1178-160 (Tox 1) 15 mg. 4.1178-139 (STD) 15 mg. 5.PI marker. 2013-04-08 13 Results: Gel IEF of proteins 1 2 3 4 5.1 4.75 4.65 4.45 Legend: 1.PI marker. 2.Ref. material 09800031 14 mg. 3.DS 08800050 14 mg. 4.DS 09PD80010 14 mg. Batch 3 - Run 1 -0,002 0,003 0,008 0,013 17 19 21 23 25 Time (min) Abs Results: rFSH – Narrow pH gradient -0,001 0 0,001 0,002 0,003 0,004 15 17 19 21 23 25 Abs(280nm) Time (min) Batch 1 - pH 2.5-5.0 gradient Results: rFSH – Narrow pH gradient -0,001 0 0,001 0,002 0,003 0,004 15 20 25 Abs(280nm) Time (min) Batch 1 - pH 2.5-5.0 gradient Batch 1 - Run 1 -0,005 0 0,005 0,01 0,015 0,02 15 17 19 21 23 Time (min) Abs Conclusions • High Resolution • Reproducibility • Old CE equipment used