Transcript LC-MS/MS

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
• Biomolecular Ion Accelerator
• Particle Mass Spectrometer
• Portable Mass Spectrometer
• MS for Proteomic Analysis
• Biomarker Discovery
• Future Perspective
Major Categories for Nobel Prize
(1) Hypothesis & Theory (25%):
Relativity; Quantum Theory; Evolution
(2) Breakthrough Discovery (35%):
Structure of DNA, protein, ribosome; microRNA; 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
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?
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
Desorption (solid) → Ionization
↓
Mass-to-charge ratio Analyzer
↓
Detection
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
ESI Spectra
+12 +11
714.6 779.5
Ubiquitin:1pmole/uL
+10
857.3
MW:8564.47
+13
659.8
+9
952.4
+8
1071.4 +7
1224.3
+16 +15
773.3 824.8
+17
727.9
+18
687.5
+19
651.4
+20
618.8
Cytochrome C:1pmole/uL
+14
883.6
MW:12,384
+13
951.4
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%
Large DNA Detection
Low Secondary ejection efficiency
for ions with low velocity
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.
Charge Detector for Large Biomolecule Detection
1.95V
1.38V
Left
He 30mtorr
Faraday
Plate
Right
Signal Ratio ≒ 0.7
355nm
RF
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
Charge detector
(MALDI TOF)
(MALDI Ion Trap)
Cyto+
Cyto+
BSA+
BSA+
Secondary Ion Measurements
Schematic diagram
10mm
－
－
－
－
＋ ＋
＋
＋
＋ ＋
＋
＋
＋ ＋
＋
＋
＋
－
－
－
10
＋
mm
－
3
－
mm
－
Faraday charge detector
－
＋
－
－
10mm
Stainless steel
Faraday charge detector
Ion Trap
(+10~30KV)
Secondary positive ion ejecting ratio
Secondary positive ions
Trapping negative ions
Secondary Ion Ejection Coefficients for
different Compounds at various Energies
Charge Amplification Detector for
Large Biomolecular Ions
Approach: Secondary ion production
RF shielding
High voltage
Single IgG+ (M/Z: 350,000) Detection
Single ion
laser power=1.2μJ
Resistance=2kΩ
laser power=1.2μJ
Average of 11 shots
Accumulation of 15 shots
Resistance=2kΩ
laser power=3.8μJ
Resistance=1MΩ
Single IgM (980 KDa) Ion Detection
Biomolecular Ion Accelerator
Experimental scheme
Ion flying
Z-gap MCP
detector
175kV
145kV
115kV
85kV
55kV
25kV
Time sequence of pulses
Typical function/waveform
(output delay 260 ns)
Function generator
input
Switch voltage
output
Biomolecular Ion Accelerator for
Lactoferrin Detection
Acceleration for Fibrinogen (MW: 350 kDa)
Accelerator Mass Spectrometer for efficient Collision-induceddissociation for large biomolecules
Z-gap MCP
detector
Secondary
electrons and
ions
Conversion
dynode
Acceleration stage
Photo of
Biomolecular
accelerator
Ion trap
MALDI source
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
Review of mass range in mass spectrometry
Mass Range
Our MALDI ion trap mass spectrometer
(1K~1000K Da)
Cell mass spectrometer
Commercial mass spectrometer
(500G~10P Da)
(10 ~100K Da)
10
1K
peptide
protein
100K
10M
1G
100G
10T
1000T 10P
immunoglobin
Huge glycoprotein
virus
cell
Da
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.
Charge Monitoring Laser Induced
Acoustic Desorption Mass Spectrometer
A high speed MS from atom to cell
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.
Mass and charge distributions for various
sizes of polystyrene microparticles
Mass distributions of lymphocyte (CD3+ cells), CEM and
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.
(Huan Chang at IAMS)
Cellular uptake of nanoparticles
HeLa cell uptake of 30nm gold
nanoparticles with Cell-MS and ICP-MS
Raw264.7 cell uptake of several
NIST polystyrene with Cell-MS
60nm polystyrene
(Number= 135,000)
100nm polystyrene
(Number= 28,000)
300nm polystyrene
(Number= 1,200)
1 μ m polystyrene
(Number= 30)
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
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
• 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
Dual Polarity Mass Spectrometer (Y. S. Wang)
Duplex MALDI ion source
h
An
io
nt
Anion MCP
detector
Anion
flight tube
raj
ec
tor
y
Extraction
electrodes
Fli
gh
tt
ub
e
t
nle
i
ple
m
Sa
Sample electrode
Fli
gh
tt
ub
e
MALDI
Extraction
electrodes
Ca
ti
Cation
flight tube
on
tra
jec
tor
y
Cation MCP
detector
ESI
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
Flow chart for proteomic analysis
Tissue
Total protein extraction
Serum
Supernatant fraction Removal of major proteins
PAGE separation
In-Solution digestion
In-Gel digestion
IEF separation
Mass analysis
MASCOT search
Quantitative analysis
No treatment
Collision induced Dissociation (CID)
Other Fragmentation Methods:
IRMPD; ECD; ETD; VUV and etc
Major Processes for Comparative Liver Cancer Stem Cell Analysis
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
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:
1.Significance threshold: P < 0.01
2.Individual ions scores > 40
3.Require bold red: These hits
represent the highest scoring protein
that contains one or more top
ranking peptide matches.
Peptidomic Analysis
Biomarker Search
MS for Gastric Cancer Marker Search
Stomach Cancer Profile: Pepsinogen (↓); α 1-antitripsin (↑);
Albumin (↑); Leucine zipper protein (↓)
Peptidomic Analysis of Gastric Juices
Stomach Biomarker Study
Sensitivity and specificity based on the number of
peptides meet the prediction of up-and down-regulation.
No. of peptide
1
2
3
4
5
Sensitivity (%)
91
82
79
79
35
Specificity (%)
69
81
92
92
94
Lung Cancer Biomarker Search
with Exhaled Air Sample
Sensitivity : < 10 attomole;
Disease Threshold: 190 attomole
8
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
* 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
(Lectin enrich)
Glyco-proteomics
Glycopeptide enrich
(Lectin enrich)
Glycoprotein Identified
(LC-MS/MS)(Non-glycopeptide)
Glycopeptide
Deglycan
(PNGase F release)
Glycan profile
(MALDI-TOF)
Glycan sequence
(MALDI-TOFTOF;ESI-MSn)
Glycosylation sites Identified
(LC-MS/MS)
Glycopeptide
(LC-MS/MS)
Glycopeptide
match
Platform of Biomics for Biomarker Search
Samples from Tissue, Cell and Body Fluid (Urine,
Gastric juice, Saliva, Serum, Plasma, Sweat)
→
Biomolecule & Metabolite Separation
↙
Other PTM
Proteins
←
PTM ←--Protein
↙
Phosph
orylation
↙
↙
Glycos
ylation
Peptido
mics
↓
↓
↓
Glycan
Sequencing
ESI Protein ID
Phosphorylate
d Markers
↘
Protein ID
↙
Glycoprotein
Biomarkers
Biomolecules
Salts
↙↘
↓
↘
MALDI
Marker
Screening
ESI/FTICR
MALDI
Protein ID
Screening Protein
↙ ↘
Markers
MALDI
Screening
↓
↓
Protein ←---
MALDI
TOF/TOF
↓
Peptide
Markers
↓
mRNA/mi
cro RNA
DNA
↓
↓
Microarray
↓
Transcri
ptomics
Element
Analysis by
ICP-MS
SNP/Genome
Sequencing
↓
Genomic
Mutation
&
Markers
↓
Element
Markers
↘
Metabolites
↙ ↘
GC/MS
LC/MS
↘
↙
Metabolomics
FTICR or
Orbitrap MS
↓
Metabolite
Markers
Single Cell Proteomic Analyzer
MCP
detector
Secondary
electrons and
ions
Conversion
dynode
Ion trap
Acceleration stage
Nd-YAG
Laser
Conclusion
• 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)
GRC
•
•
•
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
Incubation
Center
NangGang Software Industry Park
Focus of GRC Incubator: New Technologies
and Drug Discovery
Working area: 88,000 ft2
Acknowledgement
• Jung-Lee Lin
• Yuan T Lee
Wen-Ping Peng
• Huan-Chang Lin
• Quistin Wu
* Ming-Lee Chu
• Valery Golovlev
• Steve Allman
• Nien Yeen Hsu
*Huan Chang
* Yi-Sheng Wang
* Chin-Chen Lin
* Ju Ru Chen
* C. H. Wong
* Lori Jessomi
* I-Chung Lu
* Yuan C Lee
* Tsun Ren Hsaw
Thank You Very Much for Your Attention!
Please come to visit us!
Single Cell Proteomics
Analysis the proteome of liver cancer stem cells
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
Accession
Protein
Unique peptides
修正後 CD133p/ CD133n 倍
數
IPI00027547
Dermcidin
4
9999
IPI00012540
Prominin-1
2
35.5
IPI00745872
Isoform 1 of Serum albumin
7
12.9
IPI00290198
Interleukin-18
2
10.5
IPI00018953
Dipeptidyl peptidase 4
2
5.6
IPI00024095
Annexin A3
11
3.9
IPI00216138
Transgelin
16
3.2
IPI00220099
Isoform B of Syntaxin-3
2
2.4
IPI00028635
Ribophorin II
7
2.3
IPI00022202
Isoform A of Phosphate carrier
protein
7
2.1
IPI00015476
Neutral amino acid transporter A
2
1.9
Proteomic Analysis of Gastric Juice
from Normal & Cancer Patients
13
Validation of DCD expression (I)
(A)
(C)
(B)
■
Endogenous expression of DCD
detected by RT-PCR
(A) Patients with lung squamous carcinoma
(B) Patients with lung adenocarcinoma
(C) Lung cancer cell lines:
WI-38: lung fibroblast
BEAS-2B: bronchial epithelial
H520: squamous
H1299: non-small cell lung cancer
PC13: adenocarcinoma
Phosphoproteomics
Single glycoprotein
Glycoprotein
Glycopepteide
Deglycan
(PNGase F release)
Glycan profile
(MALDI-TOF)
Glycan sequence
(MALDI-TOFTOF;ESI-MSn)
Peptide Identified
(LC-MS/MS)
Glycopeptide
(LC-MS/MS)
Glycopeptide
match
Glycoproteomics & Glycomics
12
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 nanoLC / LTQ-FTICR MS, and the peakareas were measured.
The logarithm value of the peptide
quantity and peak-areas were
applied to construction of a linear
function.
(A) Linear equation obtained by using
all four spots;
(B) 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.