Institute for Systems Biology

1. Institute for Systems Biology Hplc: Performance of self made columns vs. chip system Jeff mangalin Nano 250 Spring 2013

2. Overview ISB Refresher Goals for the Quarter Instruments/Techniques Interpreting Data Conclusion

3. Institute for Systems Biology • Four Areas of Focus • P4 Medicine • predictive, preventive, personalized and participatory medicine. • Global Health • Sustainable Environment • Education & Outreach • Moritz Lab • Proteomics Core Facility • Protein Identification

4. Goals for the Quarter • Get familiar with the equipment and computer programs. • Quameter • Peakview • LC-MS Unit • Determine Performance of Self Made Columns • Determine the performance of the Chip Based LC System • Self Made Columns vs Chip

5. What is a Peptide? [1] • Short chains of amino acid monomers • Contain 50 Amino Acids or Less • Shortest peptide is a chain of 2. • Enzyme breaks protein down to peptides. • Chains of about 10 amino acids

6. High Performance Liquid Chromatography •  Used to separate a mixture of compounds, for identification purposes. • Column packed with hydrophobic beads • In our case C18 resin of 3 µm diameter • Separates compounds based on hydrophobicity • More hydrophobic molecules move slower because they are attracted to the beads. • Gradually increase hydrophobicity of buffer to sequentially elute peptides based on their hydrophobicity

7. HPLC [2]

8. EksigentnanoLC 400 Systems/cHiPLC System [3] Docking station, pumps, valves 3 microfluidic chips Can also process self made columns

9. How it works (Self Made Columns)

10. How it works (Self Made Columns)

11. How it works (Self Made Columns)

12. How it works (Chip System) • 1. Jumper Chip • plumbing • 2. Trap • Collects peptides • 3. Column • Gradient sequentiallyEludes peptides fromtrap to column • Out to MS

13. How it works (Chip System) • 1. Jumper Chip • plumbing • 2. Trap • Collects peptides • 3. Column • Gradient sequentiallyEludes peptides fromtrap to column • Out to MS

14. AB SCIEX TripleTOF® 5600 • Used for determining elemental composition of a sample • Three Components • Ion Source • Mass Analyzer • Detector

15. Mass Spectrometry [4]

16. Self Made Columns • How to make a column. • Cut tubing to desired length • Make frit • polymer • Bomb • 3 µm beads packed into tubing via pressure. • Takes about 30 minutes to pack

17. Chip Easy to connect Dead volume free connections Claims to have reproducible results

18. The Experiment How will we compare the chip vs self made columns?

19. Experimental Variables • Different Samples • Complex • E. Coli • Simple • BetaGal • Different size ID tubing • 50 µm ID • 75 µm ID • 100 µm ID • 4 Runs each • Not all runs turned out. • Compare the results

20. BetaGal [5] Beta-galactosidase AbSciex uses this as their standard sample Relatively simple sample Easy to look at in analysis

21. E. Coli [6] • Complex Sample • Thousands of Peptides • Bacteria • Found in lower intestines • Very important for molecular/synthetic biology • Easy to grow • Well studied

22. Software How do we interpret the data?

23. Peakview Used for qualitative review of LC/MS and MS/MS systems Enables data investigation by showing mass differences between peaks Generate background subtracted spectra, merging related spectra, or creating contour plots Can show multiple runs at once

24. …and this is what it looks like! But what does it all mean?

25. XIC (Extracted Ionic Chromatagram)

26. Parameters to look at… Retention Time Peak Width Width at 50% Max Height Tailing

27. BetaGal Self Made Column

28. BetaGal Chip

29. Do other peptides behave similarly? Self Made Chip

30. E. Coli Self Made and Chip Runs Self Made Chip System

31. Self Made Column Peakview

32. Chip System Peakview

33. Do we really have to compare ALL those peptides?!!

34. Quameter • Created to generate National Institute of Standards and Technology (NIST) standard metrics for LC-MS/MS experiments • Metrics • Generated from Data representing arbitrary complex samples. • IDFree • Does not give values for individual peaks/peptides • Creates metrics based on distribution of entire XIC.

35. Key Quameter Parameters • XIC-FWHM Q1-3 • Q1- The width where 25% of peak widths are narrower • Q2 – The median peak width • Q3 – The width where 75% of widths are narrower • RT-TIC Q1-4 • Q1 – The time it takes to reach 25% of the TIC • Q2 – Time to 50%...and so on

36. Quameter Graphs

37. Quameter Graphs

38. Results • Similar results from Self-made columns and Chip System • Chip system • Narrower width at 50% half max • More Tailing • Does the tailing matter? • About 30 cm extra tubing on chip connection • Chip runs are more consistent • Not as much shift in retention time

39. Conclusion • Is Chip system better than self made columns? • Need more info • More runs • How long will chips last vs columns? • Cost efficient? • Consistently produces similar results to self made column. • Slightly narrower FWHM • Easy set up • Conclusion: Viable alternative to self made columns

40. Summary ISB Refresher Goals for the Quarter Instruments/Techniques Interpreting Data Results Conclusion

41. References 1.“Biology-Forums.com." Peptide Bonding. N.p., n.d. Web. 09 May 2013. 2. "Protein Facility." The of the Iowa State University Office of Biotechnology:. 09 May 2013 <http://www.protein.iastate.edu/>  3. http://www.eksigent.com/images/Fixed%20size/Prod-180x224-40x40/eksigent-cHiPLC-System.jpg 4. "Mass Spectrometry." Mass Spectrometry. 09 May 2013 http://www.lamondlab.com/MSResource/LCMS/MassSpectrometry/massSpectrometry.php. 5. Beta-galactosidase." Wikipedia. 05 Sept. 2013. Wikimedia Foundation. 09 May 2013 http://en.wikipedia.org/wiki/Beta-galactosidase. 6. "E. coli." Wikipedia. 05 Sept. 2013. Wikimedia Foundation. 09 May 2013 http://en.wikipedia.org/wiki/E._coli.

42. Questions?