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. Major Topics. Brief Historical Review of Mass Spectrometry Single Large Biomolecular Ion Detection
Chung-Hsuan (Winston) Chen
Genomics Research Center; Academia Sinica
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
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.
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.
Mass-to-charge ratio Analyzer
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%
Charge Detector for Large Biomolecule Detection
Signal Ratio ≒ 0.7
Advantage: M/Z independent; Quantitative; Pressure resistence and Inexpensive
Disadvantage: Detection limit: ~100 ions
Comparison of Multiplier & Charge Detector
Electron multiplier detector
(MALDI Ion Trap)
－Secondary Ion Measurements
Faraday charge detector
Faraday charge detector
Secondary positive ions
Trapping negative ions
Secondary Ion Ejection Coefficients for different Compounds at various Energies
Approach: Secondary ion production
Single IgG at various Energies+ (M/Z: 350,000) Detection
Average of 11 shots
Accumulation of 15 shots
Single IgM (980 KDa) Ion Detection at various Energies
Accelerator Mass Spectrometer for efficient Collision-induced-dissociation for large biomolecules
Z-gap MCP detector
Secondary electrons and ions
Photo of Biomolecular accelerator
Review of mass range in mass spectrometry Collision-induced-dissociation for large biomolecules
Our MALDI ion trap mass spectrometer
Cell mass spectrometer
Commercial mass spectrometer
(10 ~100K Da)
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.
A high speed MS from atom to cell
Typical Mass Spectrum from CLIAD Spectrometer
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.
Mass distributions of lymphocyte (CD3+ cells), CEM and mixtures of lymphocyte and CEM
Figure 4. mixtures of lymphocyte and CEM 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)
Cellular uptake of nanoparticles mixtures of lymphocyte and CEM
Raw264.7 cell uptake of several NIST polystyrene with Cell-MS
HeLa cell uptake of 30nm gold nanoparticles with Cell-MS and ICP-MS
1 μ m polystyrene
A Portable Multiple Function MS mixtures of lymphocyte and CEM
Size: 26 cm x 24 cm x 20 cm; Weight: 16Kg
Function: MALDI; ESI; LIAD
Mass Range: atom to Cell
Size comparison of PMFMS to a commercial MALDI-TOF (Jung-Lee Lin, Ming-Lee Chu)
Ultraflex II (TOF/TOF)
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.
(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
Tissue Lin, Ming-Lee Chu)
Total protein extraction
Removal of major proteins
Flow chart for proteomic analysis
Other Fragmentation Methods:
IRMPD; ECD; ETD; VUV and etc
2 X 105 cell lysate
In-gel digestion (reduction, alkylation)
LC / LTQ-FT MS
IPI Human 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
MS for Gastric Cancer Marker Search
Stomach Cancer Profile: Pepsinogen (↓); α 1-antitripsin (↑); Albumin (↑); Leucine zipper protein (↓)
Sensitivity and specificity based on the number of peptides meet the prediction of up-and down-regulation.
Sensitivity : < 10 attomole; Disease Threshold: 190 attomole
Dermcidin peptide E-R11 showed differential expression
Tandem mass spectrum of E-R11
Map of the peptide subunits of DCD.
The unprocessed DCD has 110 amino acids and is composed of four polypeptides.
The number represents the amino acid position relative to the start residue of DCD.
Peptide E-R11 aligns with number 43 to 53 in the sequence.
* We have demonstrated a MS method to assay the peptide constituents in exhaled air samples with the sensitivity of peptide detection reaching to attomole level.
Glycoprotein enrich Analysis
(PNGase F release)
Glycosylation sites Identified
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-Hepatocellular carcinoma cells
-A large population of Huh7 cells express progenitor characteristics.
CD133+ cells were detected in 64.9% of Huh7 cells
Table 1. The up-regulated protein candidates in CD133+-Huh7 cells
Validation of DCD expression (I)
Single glycoprotein Patients
(PNGase F release)
Quantitative calibration by using synthetic E-R11
Using the linear equation y=1.04x+2.64 to calculate the quantity of 1.04x105 and 1.82x105, we obtained 196 attomoles and 337 attomoles, respectively.