New Developments on Mass Spectrometry and Their Applications 質譜儀的新發展和其應用

1. New Developments on Mass Spectrometry and Their Applications質譜儀的新發展和其應用 Chung-Hsuan (Winston) Chen 陳仲瑄 Genomics Research Center; Academia Sinica 中山大學化學所 1/5/2011

2. Major Topics • Brief Historical Review of Mass Spectrometry • Single Large Biomolecular Ion Detection • Biomolecular Ion Accelerator • Particle Mass Spectrometer • Portable Mass Spectrometer • MS for Proteomic Analysis • Biomarker Discovery • Future Perspective

3. Major Categories for Nobel Prize • Hypothesis & Theory (25%): Relativity; Quantum Theory; Evolution (2) Breakthrough Discovery (35%): Structure of DNA, protein, ribosome; micro-RNA; H-pylori (3) Critical Materials (13%): Polymer, Semiconductor, Superconductor, Liquid Crystal, Optical Fiber,GFP, Antibiotics (4) New Technologies & Instruments (27%): X-Ray, NMR, EKG, MRI, Laser, Sequencer, Microscopy, Mass Spectrometry

4. Nobel Laureates due to Achievements in MS-related Research J. J. Thomason (1906) : Gaseous Electronics F. W. Aston (1922): isotope Measurements E. O. Lawrence (1939): Cyclotron Y. T. Lee (1986): Chemical Dynamics Wolfgang Paul (1989): Ion Trap J. B. Fenn & K. Tanaka (2002): Biomolecules *Alder Nier made the first 3 magnetic sector mass spectrometers for medical applications with the budget of $257. He chipped in $100 of his own money.

5. What a mass spectrometer can do? A mass spectrometer can only be used to measure mass-to-charge (M/Z) ratio and subsequently to obtain the mass of a particle. Nevertheless, mass is usually the most important information. There are several methods can be used to break up the particles into smaller fragments. From the mass of fragments, molecular structures can often be determined. Therefore MS has become the most valuable analytical tool. Its applications include nearly every research field and every industry.

6. Schematic of Mass Spectrometry Ionization → Desorption (solid) ↓ Mass-to-charge ratio Analyzer ↓ Detection

7. Limitation on MS Detection Sensitivity • Although MS has been considered a very sensitive instrument, the overall detection sensitivity is often much less than 0.01. Capability of detecting 1 attomole usually means detecting 1 in ~106 molecules in the sample. • Desorption efficiency:~100% with a careful design • Ionization Efficiency: <<1%; Key factor for low detection sensitivity • Mass Analyzer: ~100% is possible; TOF • Detection: small M/Z: OK; Large M/Z: poor

9. Laser & MS for Biomolecule Detection MALDI (Matrix-assisted Laser Desorption/Ionization) Laser ablation of a solid sample which contains most small molecules plus a little bit of large molecules. Small molecule is served as matrix. Desorption is due to the strong absorption of laser photons by matrix which carries the large analyte molecules into gas phase. Ionization mechanism is still not well known. Pion / Pneutral << 0.1%

10. Large DNA Detection

11. Low Secondary ejection efficiency for ions with low velocity

13. Laser Induced Acoustic Desorption (LIAD) • Broad energy distribution is one key factor for poor mass resolution for MALDI. • With laser acoustic desorption, matrix can possibly be eliminated so that broad energy distribution as well as adducts and fragmentation can mostly be prevented. All major factors which cause poor mass resolution by MALDI can be mostly eliminated by laser induced acoustic desorption. Thus, better mass resolution is expected.

16. 1.95V 1.38V RF Charge Detector for Large Biomolecule Detection He 30mtorr Left Faraday Plate Right Signal Ratio ≒ 0.7 355nm Yag laser Advantage: M/Z independent; Quantitative; Pressure resistence and Inexpensive Disadvantage: Detection limit: ~100 ions

17. Quantification Comparison of Multiplier & Charge Detector Electron multiplier detector (MALDI TOF) Charge detector (MALDI Ion Trap) Cyto+ Cyto+ BSA+ BSA+

18. － － － Secondary Ion Measurements Schematic diagram 10mm ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ Faraday charge detector ＋ ＋ ＋ ＋ ＋ － － － － － － － 10mm ＋ 3mm － － － ＋ 10mm Stainless steel (+10~30KV) Faraday charge detector Ion Trap

19. Secondary positive ion ejecting ratio Secondary positive ions Trapping negative ions

20. Secondary Ion Ejection Coefficients for different Compounds at various Energies

21. Charge Amplification Detector for Large Biomolecular Ions Approach: Secondary ion production RF shielding High voltage

22. Single IgG+ (M/Z: 350,000) Detection Single ion laser power=1.2μJ Resistance=2kΩ laser power=1.2μJ Resistance=2kΩ Average of 11 shots laser power=3.8μJ Resistance=1MΩ Accumulation of 15 shots

23. Single IgM (980 KDa) Ion Detection

24. Biomolecular Ion Accelerator

25. Biomolecular Ion Accelerator for Lactoferrin Detection

26. Acceleration for Fibrinogen (MW: 350 kDa)

27. Accelerator Mass Spectrometer for efficient Collision-induced-dissociation for large biomolecules Z-gap MCP detector Secondary electrons and ions Conversion dynode Acceleration stage Ion trap MALDI source Photo of Biomolecular accelerator

28. Biomolecule Accelerator • Biomolecule as large as IgM (980 KDa) and gold nanoparticles (6 nm) were successfully detected. • We aim to produce biomolecular ion with energy as high as 2 megavolt for singly charged ion and gigavolt for ions produced from ESI

29. Review of mass range in mass spectrometry Mass Range Our MALDI ion trap mass spectrometer (1K~1000K Da) Cell mass spectrometer (500G~10P Da) Commercial mass spectrometer (10 ~100K Da) 10 Da 1K 100K 10M 1G 100G 10T 1000T 10P peptide protein immunoglobin Huge glycoprotein virus cell

30. Single Cell Light Scattering Detector Mass Spectrometer(Huan Chang at IAMS in Sinica) M/Z = 4Ve/(qr02ω2) By frequency scanning, very high M/Z can be achieved. When ω is reduced by 4 orders of magnitude, M/z increase by 8 orders of magnitude.

31. Charge Monitoring Laser Induced Acoustic Desorption Mass Spectrometer A high speed MS from atom to cell

32. Typical Mass Spectrum from CLIAD X-coordinate (time) determines mass-to-charge ratio (M/Z); Y axis indicates the number of charges on each particle. No commercial MS has this feature.

33. Mass and charge distributions for various sizes of polystyrene microparticles

34. Mass distributions of lymphocyte (CD3+ cells), CEM and mixtures of lymphocyte and CEM

35. Figure 4. Mass histograms of human red blood cells from (a) a healthy male adult, (b) a patient with iron deficiency anemia, and (c) a patient with thalassemia. Insets: Photos of the corresponding glutaraldehyde-fixed cells. The scale bar is 10 μm. Mass histograms of human red blood cells from (a) a healthy male adult, (b) a patient with iron deficiency anemia, and (c) a patient with thalassemia. Insets: Photos of the corresponding glutaraldehyde-fixed cells. The scale bar is 10 μm. (Huan Chang at IAMS)

36. Cellular uptake of nanoparticles Nanoparticle 60nm polystyrene (Number= 135,000) Raw264.7 cell uptake of several NIST polystyrene with Cell-MS HeLa cell uptake of 30nm gold nanoparticles with Cell-MS and ICP-MS Cell Cell 100nm polystyrene (Number= 28,000) 1 μ m polystyrene (Number= 30) 300nm polystyrene (Number= 1,200) Cell Cell

37. A Portable Multiple Function MS Size: 26 cm x 24 cm x 20 cm; Weight: 16Kg Function: MALDI; ESI; LIAD Mass Range: atom to Cell

38. Size comparison of PMFMS to a commercial MALDI-TOF (Jung-Lee Lin, Ming-Lee Chu) Portable MS BRUKER Ultraflex II (TOF/TOF)

39. Portable Multiple Function MS • Putting MALDI, ESI and LIAD in one MS. No other MS can do due to the incompatibility of MALDI to ion trap. Conventional ion trap can only measure M/Z up to ~4000. For MALDI, M/Z can easily reach to 100,000. Therefore, ion trap cannot be used to replace time-of-flight (TOF). All bio labs need to have a MALDI-TOF and an ESI-ion trap for proteomic analysis. • Mass Range can cover from atom to cell. The mass range can be covered is 10 orders of magnitude higher than commercial mass spectrometer. • It can measure a single virus, cell, nanoparticle, and microparticle. For small molecules, the number of ions can be directly measured. • It can measure charge directly few commercial MS can do.

40. Molecular Imaging by MS

41. Dual Polarity Mass Spectrometer (Y. S. Wang) MALDIESI

42. Proteomics All ~omics aims to analyze all compounds in a biological system which can be cell, tissue, organ or body fluid such as serum, plasma, urine, sweat, exhaled air and etc. Proteomic aims to analyze all proteins. Approach: (1) Bottom-up: from peptide analysis to identify proteins through protein ID. Advantage: Easy & Fast; Disadvantage: Difficult to analyze mutated or PTM- proteins. (2) Top-Down: Detecting the entire proteins and identify the protein by fragments. Advantages: All proteins can be analyzed in principle. Disadvantages: Time-consuming & Some technical barriers need to be overcome

43. Tissue Serum Total protein extraction Supernatant fraction Removal of major proteins No treatment PAGE separation In-Solution digestion In-Gel digestion IEF separation Mass analysis MASCOT search Quantitative analysis Flow chart for proteomic analysis

44. Collision induced Dissociation (CID) Other Fragmentation Methods: IRMPD; ECD; ETD; VUV and etc

46. Major Processes for Comparative Liver Cancer Stem Cell Analysis

47. Analysis the proteome of liver cancer stem cells • 2. Determination of proteome of CD133+/--Huh7 cells by SDS-PAGE and MS analysis 2 X 105 cell lysate 12% SDS-PAGE 16 sections In-gel digestion (reduction, alkylation) LC / LTQ-FT MS IPI Human database

48. Analysis the proteome of liver cancer stem cells • 3. Identification of the proteome by Mascot and International Protein Index (IPI) database Database search criteria: 1. IPI Human database 2. Peptide tolerant:30 ppm 3. Fragment tolerant: 0.8 Da 4. Modification:Carbamidomethyl (C), Deamidated (NQ), Oxidation (M) 5. Missed cleavages:2 • Protein validation criteria: • Significance threshold: P < 0.01 • Individual ions scores > 40 • Require bold red: These hits represent the highest scoring protein that contains one or more top ranking peptide matches.

49. Peptidomic Analysis

50. Biomarker Search MS for Gastric Cancer Marker Search Stomach Cancer Profile: Pepsinogen (↓); α 1-antitripsin (↑); Albumin (↑); Leucine zipper protein (↓)