Improvements in Mass
Spectrometry for Life
Science Research –
Does Agilent Have the
Answer?
Ashley Sage PhD
What is Mass Spectrometry?
A powerful analytical chemistry technique that is used to:
– elucidate the structure and chemical properties of molecules
– quantify trace levels of compounds in complex matrices
Detection of compounds can be accomplished with very
minute quantities
A mass spectrometer does not actually measure the
molecular mass directly, but rather the mass-to-charge
ratio (m/z) of the ions formed from the molecules
Mass Spectrometry – could it ever be routine?
Aston’s Spectrograph
Circa 1937
Mid 70s onwards
Circa 1954
Optimizing all Analytical Dimensions
- consider the variables
Signal Response
• Sensitivity
• Dynamic Range
• Linearity
• Separation Speed
• Peak Capacity
Mass Spectrum
Software
• Data Mining
• Analysis
• Differential Profiling
• Mass Accuracy
• Resolving Power
• Acquisition Rate
MS today – it’s come a long way, but what to use?
Single Quad
Triple Quad
TOF/Q-TOF
Data Output – its only software!
1980’s data
output
Obtaining Meaningful Data.....but what do you want
to find?
MassHunter Compound-Centric Data Processing
Acquire Data
LC/MS+GC/MS
Find
Compounds
Compare
Compounds
Find Compounds
MFE
Find Compounds
LMFE (Proteins)
TOF MS
Q-TOF MS/MS
QQQ MS/MS
Single Q MS
Trap MSn
GC/MS
GC/MS/MS
Protein DB Search
Compare compounds
between samples
• Metabolite ID
• Mass Profiler
Find Compounds
Auto MS/MS
Find Compounds
Targeted MS/MS
Identify
Compounds
Compare compounds
between 2 sample sets
• Mass Profiler
• Mass Profiler Pro
• Spectrum Mill
• Mascot
• Others (via mzXML, mzData)
Accurate Mass and RT
(AMRT) Database Search
• Endogenous Metabolites (METLIN)
• Food, Forensics, Environmental
Spectral Library Search
Find Compounds
By Formula
Compare compounds
between >2 sample sets
• Mass Profiler Pro
Find Compounds
(GC/MS)
Fully automated
• LC/MS/MS Library Search
• NIST GC/EI-MS Library
• Fiehn GC/EI-MS Library
Molecular Formula Generation
(MFG)
• Via accurate mass MS and MS/MS
Typical Applications for Mass Spectrometry
Small Molecule Analysis
– Mass confirmation
– Structural elucidation by MSMS
– Impurity Profiling
Metabolism Studies
– Metabolite ID
Metabolomics
– Sample comparison
– Metabolism pathways
– Biomarker discovery
Proteomics
– Intact protein analysis
– Peptide analysis and quant
– Biomarker discovery
Clinical Research & Diagnostics
– Inborn errors
– Therapeutic drugs etc
Food Safety & Environmental
– Water quality testing
– Residue analysis
– Soil testing
Forensics
– drugs of abuse
– Sports testing
Pharmacokinetics
– Drug discovery & delivery
And the list goes on........
Small Molecule
Accurate Mass – using
TOF MS with Database
Searching
The Advantage of Accurate Mass Measurement
- increased specificity
C33H40N2O9 has a protonated ion at 609.28066
Quadrupole MS reports mass to +/- 0.1Da = 165 ppm
High Resolution MS reports to <2ppm
Possible Formulas (C,H,N,O)
• 165 ppm
209
• 10 ppm
13
• 5 ppm
7
• 3 ppm
4
• 2 ppm
2
0.7Da FWHM
40,000 FWHM
Isotopic Interpretation – aid deconvolution
Correct Identity
C19H17N5S
Elements Used
to Calculate
Formulas
Metabolite Identification
Precursor Isotope Pattern and Fragment Mass Agreement
x10 1
7
402.2500
C21 H32 N5 O3
6
Buspirone monohydroxy metabolite
0.07 ppm
5
4
3
x10 2
2
121.0509
1
MS spectrum
403.2530
C21 H32 N5 O3
1
0.8
0
0.6
0.4
0.2
109.0762
135.0443
402.2500
C21 H32 N5 O3
404.2532
402
403
404
Counts (%) vs. Mass-to-Charge (m/z)
194.1171
224.1282
327.2009
0
122.0705
C6 H8 N3
-3.32 ppm
x10 2
1.2
1
0.8
150.1022
C8 H12 N3
0.6
-2.36 ppm
219.1604
C12 H19 N4
-0.54 ppm
402.2493
238.1424
C11 H18 N4 O2
-0.28 ppm
178.1217
C9 H14 N4
2.07 ppm
0.4
MS/MS spectrum
0.2
0
100
120
140
160
180
200
220
240
260
280
300
Counts (%) vs. Mass-to-Charge (m/z)
320
340
360
380
400
420
Metabolism – important part of Pharmacuetical
Development
O
N
Cl
N
N
N
H
N-Oxidation
Clozapine-N-oxide
N
HO
N
N
N
Hydroxylation
N
N
Cl
Thiomethylation
H3CS
N
H
N
H
3-Hydroxyclozapine
Demethylation
Parent: Clozapine
N
Cl
N
3-Thiomethylclozapine
Demethylation
NH
N
N
N
H
Demethylation
N
HO
N
N
Hydroxylation
Thiomethylation
N
H
N
H
3-Hydroxynorclozapine
Norclozapine
N
NH
H3CS
NH
N
N
H
3-Thiomethylnorclozapine
Accurate Mass MS/MS Database and Library
• Database of over 7500 compounds/
metabolites with MSMS spectra
• MS/MS spectra are collected in positive and
negative ion mode
• MS/MS spectra are produced from the
isolated mono-isotopic ion
• Fragmentation data is collected at three
collision energies: 10, 20 and 40eV
10, 20, 40 eV
• Matching is done used forward and reverse
database searches
• Match Quality score ranks search results
Case 353-10 – Unknowns Analysis (forensics)
• MS Database Search
– Isomers can’t be distinguished,
– Accurate mass and isotopic pattern allows for empirical formula confirmation
- Dexamisole?
- Tetramisole?
- ???
[M+H]+
- Benzoylecgonine?
- Roletamide?
- Norcocaine?
- ???
C11H12N2S
[M+H]+
C16H19NO4
[M+H]+
[M+Na]+
C17H21NO4
- Cocaine?
- Fenoterol?
- Hydromorphinol?
- Scopolamine?
- ???
Case 353-10 – Unknowns Analysis (forensics)
• MSMS Library search
– Isomers are identified
– MSMS spectra containing structural information
Measured
Comparison
Library
-- Benzoylecgonine?
Benzoylecgonine!
-- Roletamide?
Roletamide?
-- Norcocaine?
Norcocaine?
--Dexamisole?
Dexamisole?
--Tetramisole!
Tetramisole?
-- Cocaine!
Cocaine?
-- Fenoterol?
Fenoterol?
-- Hydromorphinol?
Hydromorphinol?
-- Scopolamine?
Scopolamine?
Measured
Comparison
Library
Agilent Solutions for
Metabolomics
Metabolomics Is …..
Metabolomics is the comparative
analysis of endogenous metabolites
found in biological samples:
• Compare two or more biological groups
• Find and identify potential biomarkers
• Look for biomarkers of toxicology
• Understand biological pathways
• Discover new metabolites
Metabolites are the by-products of
metabolism
• Range of physico-chemical properties
• Classes: Amino acids, Sugars, organic
acids, fatty acids, lipids…
• Molecular weights upto 1000Da
What are the chemical
differences that result in
the observable difference
Agilent Metabolomics Workflow
Separate &
Detect
Feature
Finding
Quantitate
Alignment &
Statistics
Identify
Pathways
GCMS
Mass Profiler
(MP)
MassHunter Qual
GC/MSD
GC-QQQ
AMDIS or Find by
chromatographic
deconvolution
ID Browser
LCMS
Mass Profiler
Professional
(MPP)
MassHunter Qual
LC-TOF/QTOF
LC-QQQ
MFE,
Find by Formula,
Find by Ion
Pathway module
Cytoscape
Differential Analysis & Visualization - Software
Mass Profiler
–
–
–
–
Performs pair wise differential analysis
Designed for TOF data only
Simple t-test
METLIN Personal database is integrated
Mass Profiler Professional
– Simple or complex data sets
– Performs many types of statistical
analyses
– Numerous visualizations
– Import and process data from Agilent:
GC/MS, LC/TOF, LC/Q-TOF, LC/QQQ
data)
– Identify metabolites using integrated ID
browser
25,157 unique mass features
59 up-regulated unique mass features
Statistics
PCA analysis of all Red Blood Cell samples reveals
separation based on pH of extraction solvent
pH
2
7
9
Pathway analysis in MPP showing differential
abundances for three compounds in the urea cycle
L-Arg (Arginine)
Ornithine
Citrulline
12000
10000
8000
6000
[IRBC_pH9] avg :Raw
[NRBC_pH9] avg :Raw
4000
2000
0
L-arginine
Infected
Blood cells
2500
2000
1500
20
1000
[IRBC_pH9] avg :Raw
18
[NRBC_pH9] avg :Raw
16
14
500
12
10
0
[IRBC_pH9] avg :Raw
8
L-ornithine
[NRBC_pH9] avg :Raw
6
4
2
0
citrulline
2500
2000
1500
[IRBC_pH9] avg :Raw
1000
[NRBC_pH9] avg :Raw
Non infected
500
0
L-ornithine
Intact Protein Analysis
Intact Protein Analysis
Check QC of manufacture
• Molecular weight confirmation
• Check impurities
Determine protein modifications
• Glycosylation etc
Measurement of therapeutic
antibodies (monoclonal
antibodies)
MassHunter BioConfirm – Intact Protein Analysis
Configurable
Workflow for
Easy Data
Interpretation
Chromatogram
Spectrum
Deconvolution Results
Compound List
based on Protein
Sequence
Typical LCMS Analytical Conditions
LC Conditions
1200 Binary SL pump + Degasser
1200 SL Autosampler
1200 SL Diode Array
Thermostatted Column Compartment
Column : Poroshell 300SB – C8, 1.0x75mm, 5µ
Mobile Phase :
(A) Water + 0.05% TFA
(B) Acetonitrile + 0.05% TFA
Flow Rate : 0.25mL/min
Injection Volume : 10µL
LC Gradient : 95% A to 95%B over 10 mins
Myoglobin – Mass Spectrum
Horse Heart Myoglobin – MaxEnt Deconvolution
0.51ppm mass
measurement
MW = 16951Da
Structure and Modifications of Antibodies
• Analyze intact antibody
Heavy Chain
Antigen
binding
Pyroglutamate
Disulfide
shuffling
• Analyze deglycosylated
antibody (enzymatic)
Light Chain
• Analyze reduced antibody
Fab
Deamidation/oxidation
• Analyze Fab and Fc
Hinge
Fc
Fucose – 146 Da
Mannose – 162 Da
N-Acetylglucosamine – 203 Da
Galactose – 162 Da
(light and heavy chains)
Truncation
(lysine)
Glycosylation
site
regions (papain cleavage)
Mass Spectrum of a mAb – HPLC-Chip/MS of 10 ng
On-column
Poroshell SB300-C18
Mwt = 149,500Da
Intact mAb Analysis - Intact and Deglycosylated
G0F/G
0F
Intact mAb
Δ2890.81
Δ1444.87
4
x10
8
145924.41
7
6
Deglycosylated
(sugar groups removed enzymatically)
5
4
3
2
1
0
Page 32
146272.08
No glycanattached
species
144500 145000 145500 146000 146500 147000 147500 148000 148500 149000 149500
Counts vs. Deconvoluted Mass (amu)
Comparison of Two Antibody Batches
Batch Comparison
60
Batch 1
Relative Percent
50
40
30
Batch 1
20
Batch 2
Man8
G2-fuc
G1-fuc +sialic acid NGNA
Man5
G1-fuc
G2+sialic acid NGNA
G0-fuc
G2
G1+sialic acid NGNA
Batch 2
G0
G1
0
G1-fuc-GlcNac +sialic acid…
10
Glycan Type
2000
Results:
• Profiles for batch 1 and 2 look similar.
• Batch 2 has a higher percentage and greater
variety of sialylated glycan forms.
LCMS Analysis of Therapeutic
Oligonucleotides
DMTO
HO
B2
O
O OR1
O P O
O
N
3% DCA in DCM
B1
O
B2
O
DMTO
O OR1
O P O
Detritylation
O
N
OR1
O
OR1
O
activator
O
N
N
S
DMTO
CPG solid phase support
DMTO
O
OR1
P O
O
O
HO
B2
O P O
O
N
O
O
O
B2
O
OR1
O
O P
O
O OR1
Agilent 6520 Q-TOF (-ve ion)
O
O
N
B1
O
Product, ready for
another cycle
DMTO
HO
O
B1
O
O
OR1
N
B1
OR1
O
Coupled product
OP
O
O
O
O
O
OR1
O
N
+
O
FLP
B2
N
OR1
Where B = rG(ibu), rA(bz), rC(acetyl)
R1=TBDMS,
I2
Oxidation
OR1
P O
O
O
B2
O
O
Coupling failure
N-6
B2
B3
O
O
ACN, 2,6 lutidine,
acetic anhydride
N-5 & N-4
O
Coupling failure
OR1
Capping
N-2
O
O
OR1
O
N-1
+
O
Capped Coupling failure
Column: C18, at 35oC
Mobile Phase: HFIP/TEA/ Methanol
N
O
O
B1
N +
O P O
O
O
P
O OR1
O P O
OR1
O
O
OR1
N
B2
O
B3
O
O
B3
O
O
N-7
CN
N
N
O
OR1
P O
N
B1
O
B3
O
O
Coupling
B1
OR1
N
O P O
B1
O
O
O
OR1
O
Oxidized product
High Resolution & Accuracy is Key
Me-phosphonate-DNA PS
CsAvGvTsCvAsGsTvAsCvGsT
S: phosphorothioate
V: methylphosphonate thioate
Peptide Biomarker
Analysis
Biomarker ID and Quant Workflow
Step-2
Spectrum Mill
• Run samples on QTOF for protein ID in
data-dependent
MS/MS mode.
Step-1
Q-TOF
• Search QTOF data
using Spectrum Mill
• Use Spectrum Mill
MRM Selector to
create a list of MRM
transitions with RT
• Import the MRM list
into QQQ Acquisition
software
• Run samples on
QQQ in Dynamic
MRM mode
Step-3
QQQ
Step-4
Mass Profiler Pro
• Integrate the MRM
chromatograms
• Import quantitation
results into MPP to
perform statistical
analysis
Q-TOF LCMS for Peptide Mapping
• Chromatographic peak
width (half-height) 0.30.8 seconds
• MS acquisition (3003000) at 10 spectra/sec
in high resolution mode
• 98.8% sequence
coverage
LC/MS/MS Analysis of Serotransferrin Digest
81% sequence coverage and 93 unique peptides
• Red indicates matched peptides
• For transferrin, the first 19 amino acids are the signal peptide
6490 vs 6460 for Peptide Quant, 2 mm id Column
Human Serum Albumin Peptide (LVNEVTEFAK, 575.5  937.5)
Quantifier
Qualifiers
Calibration Curves
6460 + AJS
1 fmol on-column
1 fmol 25 pmol
6490-iFunnel + AJS
100 amol on-column
100 amol 25 pmol
Poroshell 120 2.1 x 150 mm column at 0.5 mL/min
6490 – Technology Enhancements
• iFunnel Technology
– Agilent Jet Stream Ion Generation
– Hexabore capillary
– Dual ion funnel (DIF) technology
• Two stages for ion focusing and gas removal
• Improvements for wide m/z range transmission
• Collision Cell
– Hexapole field axial focusing curved collision cell
• Tapered cell structure for increased ion acceptance at entrance
• Reduction of ionizer generated noise
• Improved Quad Drive Electronics
– Improved Quad DC frequency response
– Higher RF power capability
– Quad drive frequency increased to 1.4 MHz
High and Low Pressure Funnels
Steroid Analysis – using ESI
HPLC Method
Column:
Poroshell 120EC 2.1 x 150 mm, 2.7 um
Injection volume:
10 μl
Column Temp:
50oC
Mobile Phases:
A:0.05mM Ammonia soln: 10% Methanol
B:Methanol
Flow rate:
0.4 ml/min
Gradient:
Time
%A
%B
0.0
95
5
0.5
95
5
10.5
10
90
12
10
90
(mins)
15 minute run
Steroids Chromatogram, Std 8
17b Estradiol
17a Estradiol
β-Estradiol lowest std: 0.02 ng/ml
T2 s/n 165.3
T1 s/n 236.9
Food Safety - 300 Pesticides in 15 min.
600 MRM Transitions acquired
Agilent and the Future.....?
Continued LC, MS hardware and software development
– QTOF development for increased sensitivity for biomarker coverage
Increased productivity with software workflow
– Study Manager concept for multi-user environment
– Structure related visual aids for mass spec interpretation
Next Generation Mass spectrometer......what ever that may be?
The future of mass spectrometry.........
Thank You