New Developments on Mass Spectrometry and Their Applications 質譜儀的新發展和其應用...
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![Page 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.](https://reader037.fdocuments.net/reader037/viewer/2022102714/56649e0c5503460f94af556a/html5/thumbnails/1.jpg)
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
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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
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Major Categories for Nobel Prize(1) 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
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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.
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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.
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Schematic of Mass Spectrometry
Ionization
↓Mass-to-charge ratio Analyzer
↓Detection
Desorption (solid)
→
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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
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779.5714.6
659.8
857.3
952.4
1071.41224.3
Ubiquitin:1pmole/uL
618.8
651.4
687.5
727.9
773.3 824.8
883.6
951.4
Cytochrome C:1pmole/uL
MW:12,384
ESI Spectra
MW:8564.47
+11
+10
+9
+8+7
+12
+13
+16
+17
+18
+19
+20
+15
+14
+13
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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%
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Large DNA Detection
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Low Secondary ejection efficiency for ions with low velocity
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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.
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1.95V
1.38V
RF
355nm
Yag laser
Charge Detector for Large Biomolecule Detection
He 30mtorrLeft
Right
Signal Ratio 0.7≒
Advantage: M/Z independent; Quantitative; Pressure resistence and Inexpensive
Disadvantage: Detection limit: ~100 ions
Faraday Plate
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Quantification
Cyto+
BSA+
Cyto+ BSA+
Electron multiplier detector(MALDI TOF)
Charge detector(MALDI Ion Trap)
Comparison of Multiplier & Charge Detector
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Secondary Ion Measurements
Faraday charge detector
Faraday charge detector
Stainless steel
(+10~30KV) Ion Trap
---
--
--
-
-
-
-- -
++++
++ ++
+
+
+
+ ++
+1
0m
m
3m
m
10mm
10mm
Schematic diagram
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Secondary positive ion ejecting ratio
Trapping negative ions
Secondary positive ions
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Secondary Ion Ejection Coefficients for different Compounds at various Energies
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Charge Amplification Detector for Large Biomolecular Ions
Approach: Secondary ion production
High voltage
RF shielding
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laser power=1.2μJ
Resistance=2kΩ
laser power=1.2μJ
Resistance=2kΩ
Single ion
Average of 11 shots
Accumulation of 15 shots laser power=3.8μJ
Resistance=1MΩ
Single IgG+ (M/Z: 350,000) Detection
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Single IgM (980 KDa) Ion Detection
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Biomolecular Ion AcceleratorExperimental scheme
Ion flying
Z-gap MCP detector
25kV55kV85kV115kV145kV175kV
Time sequence of pulsesFunction generator Function generator inputinput
Switch voltage Switch voltage outputoutput
Typical function/waveform (output delay 260 ns)
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Biomolecular Ion Accelerator for Lactoferrin Detection
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Acceleration for Fibrinogen (MW: 350 kDa)
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Accelerator Mass Spectrometer for efficient Collision-induced-dissociation for large biomolecules
Z-gap MCP detector
Conversion dynode
Acceleration stage
Secondary Secondary electrons and electrons and ionsions
Ion trap MALDI source
Photo of Biomolecular accelerator
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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
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10 1K 100K 10M 1G 100G 10T 1000T Da10P
Commercial mass spectrometer
(10 ~100K Da)
Our MALDI ion trap mass spectrometer
(1K~1000K Da)
Cell mass spectrometer
(500G~10P Da)
immunoglobin proteinpeptide
Huge glycoprotein virus
cell
Review of mass range in mass spectrometry Mass Range
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Single Cell Light Scattering Detector Mass Spectrometer
(Huan Chang at IAMS in Sinica)M/Z = 4Ve/(qr0
2ω2)
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.
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Charge Monitoring Laser Induced Acoustic Desorption Mass Spectrometer
A high speed MS from atom to cell
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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.
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Mass and charge distributions for various
sizes of polystyrene microparticles
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Mass distributions of lymphocyte (CD3+ cells), CEM and mixtures of lymphocyte and CEM
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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)
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Cellular uptake of nanoparticlesCellular uptake of nanoparticles
60nm polystyrene60nm polystyrene(Number= 135,000135,000)
100nm polystyrene100nm polystyrene(Number= 28,00028,000)
300nm polystyrene300nm polystyrene(Number= 1,2001,200)
11 μ μ m polystyrenem polystyrene(Number= 3030)
HeLa cell uptake of 30nm gold HeLa cell uptake of 30nm gold nanoparticles with Cell-MS and ICP-MSnanoparticles with Cell-MS and ICP-MS
Raw264.7 cell uptake of several Raw264.7 cell uptake of several NIST polystyrene with Cell-MSNIST polystyrene with Cell-MS
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A Portable Multiple Function MS
Size: 26 cm x 24 cm x 20 cm; Weight: 16KgFunction: MALDI; ESI; LIADMass Range: atom to Cell
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Portable MS
BRUKERUltraflex II (TOF/TOF)
Size comparison of PMFMS to a commercial MALDI-TOF (Jung-Lee Lin, Ming-Lee Chu)
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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.
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Molecular Imaging by MS
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Dual Polarity Mass Spectrometer (Y. S. Wang)
Anion MCP Anion MCP detectordetector
Cation MCP Cation MCP detectordetector
hh
Sample electrode
Extraction electrodes
Extraction electrodes
Anion flight tube
Cation flight tube
hh
Duplex MALDI ion sourceDuplex MALDI ion source
hh
Sample electrode
Extraction electrodes
Extraction electrodes
Anion flight tube
Cation flight tube
hh
Duplex MALDI ion sourceDuplex MALDI ion source
hh
Sample electrode
Extraction electrodes
Extraction electrodes
Anion flight tube
Cation flight tube
hh
Duplex MALDI ion sourceDuplex MALDI ion source
MALDI ESI
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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
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Flow chart for proteomic analysis
Tissue Serum
Removal of major proteins
In-Solution digestion
No treatment
Mass analysis
Supernatant fraction
PAGE separation
In-Gel digestion
MASCOT search
Total protein extraction
IEF separation
Quantitative analysis
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Collision induced Dissociation (CID)
Other Fragmentation Methods:
IRMPD; ECD; ETD; VUV and etc
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Major Processes for Comparative Liver Cancer Stem Cell Analysis
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2 X 105 cell lysate
12% SDS-PAGE
16 sections
In-gel digestion (reduction, alkylation)
LC / LTQ-FT MS
IPI Human database
2. Determination of proteome of CD133+/--Huh7 cells by SDS-PAGE and MS analysis
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Database search criteria:1. IPI Human database2. Peptide tolerant:30 ppm3. Fragment tolerant: 0.8 Da4. Modification:Carbamidomethyl (C), Deamidated (NQ), Oxidation (M)5. Missed cleavages:2
Protein validation criteria:1.Significance threshold: P < 0.012.Individual ions scores > 403.Require bold red: These hits represent the highest scoring protein that contains one or more top ranking peptide matches.
3. Identification of the proteome by Mascot and International Protein Index (IPI) database
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Peptidomic Analysis
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Biomarker Search
MS for Gastric Cancer Marker Search
Stomach Cancer Profile: Pepsinogen (↓); α 1-antitripsin (↑); Albumin (↑); Leucine zipper protein (↓)
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Peptidomic Analysis of Gastric Juices
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Stomach Biomarker Study
No. of peptide
1 2 3 4 5
Sensitivity (%)
91 82 79 79 35
Specificity (%)
69 81 92 92 94
Sensitivity and specificity based on the number of peptides meet the prediction of up-and down-regulation.
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Lung Cancer Biomarker Search with Exhaled Air Sample
Sensitivity : < 10 attomole; Disease Threshold: 190 attomole
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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.
8
Dermcidin peptide E-R11 showed differential expression
Tandem mass spectrum of E-R11
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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).
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Glycoprotein enrich(Lectin enrich)
Glycopeptide
Glycopeptidematch
Deglycan(PNGase F
release)
Glycan profile(MALDI-TOF)
Glycosylation sites Identified(LC-MS/MS)
Glycopeptide(LC-MS/MS)
Glycopeptide enrich(Lectin enrich)
Glycoprotein Identified(LC-MS/MS)(Non-glycopeptide)
Glycan sequence(MALDI-TOFTOF;ESI-MSn)
Glyco-proteomics
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Platform of Biomics for Biomarker Search
Samples from Tissue, Cell and Body Fluid (Urine, Gastric juice, Saliva, Serum, Plasma, Sweat) Biomolecule & Metabolite Separation→
↙ ↘
Metabolites
↙ ↘
GC/MS LC/MS
↘ ↙
Metabolomics
FTICR or Orbitrap MS
↓
Salts
↓
Element Analysis by
ICP-MS
Biomolecules
↙ ↘
mRNA/micro RNA
DNA
↓
SNP/Genome Sequencing
↓
Micro-array
← ---Protein
↘
Peptidomics
↓
MALDI TOF/TOF
↙
MALDI Marker
Screening
↓ESI/FTICR Protein ID
Protein Markers
← ---PTM Protein
↓Glycosylation
↓
MALDI Screening
↘
Protein ID
↙Glycan
Sequencing
↘ ↙
Glycoprotein Biomarkers
↓
Metabolite Markers
↓
Element Markers↓
Genomic Mutation
& Markers
↓
Transcriptomics
↓
Peptide Markers
↙
Phosphorylation
↙
MALDI Screening
↓ESI Protein ID Phosphorylate
d Markers
←Other PTM Proteins
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Single Cell Proteomic Analyzer
MCP detector
Conversion dynode
Acceleration stage
Secondary Secondary electrons and electrons and ionsions
Ion trapNd-YAG Laser
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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???
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Genomics Research Center (GRC)Genomics Research Center (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
GRC Incubation Center
NangGang Software Industry Park Focus of GRC Incubator: New Technologies and Drug DiscoveryWorking area: 88,000 ft2
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Acknowledgement
• 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
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Thank You Very Much for Your Attention!
Please come to visit us!
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Single Cell Proteomics
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Huh7 cells
-Hepatocellular carcinoma cells
-A large population of Huh7 cells express progenitor characteristics.
CD133+ cells were detected in 64.9% of Huh7 cells
1. Separation of the CD133+/- cells from hepatoma cell line (Huh7) by fluorescence activated cell sorting (FACS)
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Table 1. The up-regulated protein candidates in CD133+-Huh7 cells
Identification of the up-regulation 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
IPI00022202Isoform A of Phosphate carrier protein
7 2.1
IPI00015476 Neutral amino acid transporter A 2 1.9
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Proteomic Analysis of Gastric Juice from Normal & Cancer Patients
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13
Validation of DCD expression (I)
(A)
(B)
(C)
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
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Phosphoproteomics
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Glycoprotein
Glycopepteide
Glycopeptidematch
Deglycan(PNGase F
release)
Glycan profile(MALDI-TOF)
Peptide Identified(LC-MS/MS)
Glycopeptide(LC-MS/MS)
Glycan sequence(MALDI-TOFTOF;ESI-MSn)
Single glycoprotein
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Glycoproteomics & Glycomics
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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.
(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.
Quantitative calibration by using synthetic E-R1112
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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