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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

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New developments on mass spectrometry and their applications

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
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


Major categories for nobel prize
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


Nobel laureates due to achievements in ms related research
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.


What a mass spectrometer can do
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.


Schematic of Mass Spectrometry

Ionization

Desorption (solid)

Mass-to-charge ratio Analyzer

Detection


Limitation on ms detection sensitivity
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


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%




Laser induced acoustic desorption liad
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.


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


Quantification

Comparison of Multiplier & Charge Detector

Electron multiplier detector

(MALDI TOF)

Charge detector

(MALDI Ion Trap)

Cyto+

Cyto+

BSA+

BSA+


Secondary ion measurements

Secondary Ion Measurements

Schematic diagram

10mm

Faraday charge detector

10mm

3mm

10mm

Stainless steel

(+10~30KV)

Faraday charge detector

Ion Trap


Secondary positive ion ejecting ratio
Secondary positive ion ejecting ratio

Secondary positive ions

Trapping negative ions



Charge amplification detector for large biomolecular ions
Charge Amplification Detector for Large Biomolecular Ions at various Energies

Approach: Secondary ion production

RF shielding

High voltage


Single IgG at various Energies+ (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



Biomolecular ion accelerator
Biomolecular Ion Accelerator at various Energies




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


Biomolecule accelerator
Biomolecule Accelerator Collision-induced-dissociation for large biomolecules

  • 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


Review of mass range in mass spectrometry Collision-induced-dissociation for large biomolecules

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


Single cell light scattering detector mass spectrometer huan chang at iams in sinica
Single Cell Light Scattering Detector Mass Spectrometer Collision-induced-dissociation for large biomolecules(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.


Charge Monitoring Laser Induced Acoustic Desorption Mass Spectrometer

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.




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

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


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)

Portable MS

BRUKER

Ultraflex II (TOF/TOF)


Portable multiple function ms
Portable Multiple Function MS Lin, Ming-Lee Chu)

  • 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.


Molecular imaging by ms
Molecular Imaging by MS Lin, Ming-Lee Chu)



Proteomics
Proteomics Lin, Ming-Lee Chu)

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


Tissue Lin, Ming-Lee Chu)

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


Collision induced dissociation cid
Collision induced Dissociation (CID) Lin, Ming-Lee Chu)

Other Fragmentation Methods:

IRMPD; ECD; ETD; VUV and etc



Analysis the proteome of liver cancer stem cells Analysis

  • 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


Analysis the proteome of liver cancer stem cells Analysis

  • 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.



Biomarker search
Biomarker Search Analysis

MS for Gastric Cancer Marker Search

Stomach Cancer Profile: Pepsinogen (↓); α 1-antitripsin (↑); Albumin (↑); Leucine zipper protein (↓)



Stomach biomarker study
Stomach Biomarker Study Analysis

Sensitivity and specificity based on the number of peptides meet the prediction of up-and down-regulation.


Lung cancer biomarker search with exhaled air sample
Lung Cancer Biomarker Search with Exhaled Air Sample Analysis

Sensitivity : < 10 attomole; Disease Threshold: 190 attomole


8 Analysis

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.


Summary of lung cancer biomarker studies
Summary of Lung Cancer Biomarker Studies Analysis

* 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.

  • Samples are from 12 healthy subjects, 14 pneumonia patients, 11 chronic obstructive pulmonary disease (COPD) patients, 10 squamous carcinoma patients, 32 adenocarcinoma patients, and 5 small cell carcinoma patients.

  • The identified “marker” is E-R11 in DCD with its sequence to be ENAGEDPGLAR.

  • E-R11 shows its sensitivity and specificity as 60% and 92%, respectively. Nevertheless, E-R11 is not suitable for detection of small cell lung cancer (SCLC).


Glycoprotein enrich Analysis

(Lectin enrich)

Glyco-proteomics

Glycopeptide enrich

(Lectin enrich)

Glycopeptide

Glycoprotein Identified

(LC-MS/MS)(Non-glycopeptide)

Deglycan

(PNGase F release)

Glycopeptide

(LC-MS/MS)

Glycan profile

(MALDI-TOF)

Glycosylation sites Identified

(LC-MS/MS)

Glycopeptide

match

Glycan sequence

(MALDI-TOFTOF;ESI-MSn)



Conclusion
Conclusion Analysis

  • MS is an important tool for biomedical applications

  • Biomics will play a critical role in disease diagnosis and drug development

  • Novel Technology Development will continue to play an important role in MS applications to biomedical research especially Single Cell Proteomics

  • Solution MS for invivo analysis???


Genomics research center grc
Genomics Research Center (GRC) Analysis

GRC

Incubation Center

  • 10 floors including 2 basement floors for parking

  • Lab space: ~ 8000 ft2 each floor for 6 floors

  • Office Space: ~4000 ft2 each floor for 6 floors

NangGang Software Industry Park

Focus of GRC Incubator: New Technologies and Drug Discovery

Working area: 88,000 ft2


Acknowledgement
Acknowledgement Analysis

  • Jung-Lee Lin *Huan Chang

  • Yuan T Lee * Yi-Sheng Wang Wen-Ping Peng * Chin-Chen Lin

  • Huan-Chang Lin * Ju Ru Chen

  • Quistin Wu * C. H. Wong

    * Ming-Lee Chu * Lori Jessomi

  • Valery Golovlev * I-Chung Lu

  • Steve Allman * Yuan C Lee

  • Nien Yeen Hsu * Tsun Ren Hsaw


Thank You Very Much for Your Attention! Analysis

Please come to visit us!



Analysis the proteome of liver cancer stem cells Analysis

  • 1. Separation of the CD133+/- cells from hepatoma cell line (Huh7) by

  • fluorescence activated cell sorting (FACS)

Huh7 cells

-Hepatocellular carcinoma cells

-A large population of Huh7 cells express progenitor characteristics.

CD133+ cells were detected in 64.9% of Huh7 cells


Quantitative determination of the proteome from liver cancer stem cells

  • Identification of the up-regulation protein candidates in CD133+-Huh7 cells

Table 1. The up-regulated protein candidates in CD133+-Huh7 cells



( PatientsA)

(C)

(B)

13

Validation of DCD expression (I)

  • Endogenous expression of DCD

  • detected by RT-PCR

  • Patients with lung squamous carcinoma

  • Patients with lung adenocarcinoma

  • Lung cancer cell lines:

  • WI-38: lung fibroblast

  • BEAS-2B: bronchial epithelial

  • H520: squamous

  • H1299: non-small cell lung cancer

  • PC13: adenocarcinoma



Single glycoprotein Patients

Glycoprotein

Glycopepteide

Deglycan

(PNGase F release)

Glycopeptide

(LC-MS/MS)

Glycan profile

(MALDI-TOF)

Peptide Identified

(LC-MS/MS)

Glycopeptide

match

Glycan sequence

(MALDI-TOFTOF;ESI-MSn)



12 Patients

Quantitative calibration by using synthetic E-R11

  • Quantitative calibration of E-R11.

  • Synthetic E-R11 peptides with the quantity of 10, 100, 1,000 and 10,000 attomoles were analyzed by nano-LC / LTQ-FTICR MS, and the peak-areas were measured.

  • The logarithm value of the peptide quantity and peak-areas were applied to construction of a linear function.

  • Linear equation obtained by using all four spots;

  • Linear equation obtained by using three spots with quantity equal to or more than 100 attomoles.

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.


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