Xianlin Han
Diabetes and Obesity Research Center
Sanford-Burnham Medical Research Institute – Lake Nona
Orlando, FL 32827
Principles of Shotgun Lipidomics
What is shotgun lipidomics?
Original definition (in analogous to shotgun proteomics):
Han & Gross, Mass Spectrom. Rev. 24 (2005), 367
What is shotgun lipidomics?
Original definition (in analogous to shotgun proteomics):
Han & Gross, Mass Spectrom. Rev. 24 (2005), 367
ASMS 2004
What is shotgun lipidomics?
Original definition (in analogous to shotgun proteomics):
Han & Gross, Mass Spectrom. Rev. 24 (2005), 367
Currently it is generally referred to any approach/platform
after direct infusion (but not include loop injection)
Unique features: (1) High throughput; (2) MS analysis under a constant
concentration of lipid solution without any time constraints; (3)
Molecular species of a lipid class are commonly displayed in an
identical mass spectrum
Classification of shotgun lipidomics
• Lipid class-specific tandem MS-based shotgun
lipidomics (Brugger et al., PNAS 94 (1997), 2339; Welti et al., JBC
277 (2002), 31994; etc)
• High mass resolution-based shotgun lipidomics (the
platform developed by Dr. Shevchenko’s group)
• Multi-dimensional MS-based shotgun lipidomics (the platform developed by our group; Mass Spectrom. Rev. 31 (2012),
134)
• Total ion mapping by using high mass
accurate/resolution mass spectrometers (AB Sciex
platform)
• Ion-mobility based shotgun lipidomics (Waters
Platform)
J. Lipid Res. 46 (2005) 839
“We divide lipids into eight categories (fatty acyls,
glycerolipids, glycerophospholipids, sphingolipids, sterol lipids,
prenol lipids, saccharolipids, and polyketides) containing distinct
classes and subclasses of molecules.”
Cellular lipids are very complex
Glycerophospholipids:
Classes of Glycerophospholipids:
(14 to 22 C, 0
to 6 DBs, 3
linkages)
~ 100
~ 30
~ 10
Principle of shotgun lipidomics (In general)
To maximally exploit the differences in the
physical and chemical properties between the
classes of lipids and the uniqueness of a cellular
lipid class in combination with the relationship
between the tandem MS techniques after direct
infusion
Han, Yang, & Gross, Mass Spectrom. Rev. 31 (2012), 134
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping
Principles of shotgun lipidomics (In specific)
Han, Yang, & Gross, Mass Spectrom. Rev. 31 (2012), 134
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping
Principles of shotgun lipidomics (In specific)
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
Principles of shotgun lipidomics (In specific)
Anionic lipids (AL) which intrinsically carry net
negative charge(s) under weakly acidic
conditions (e.g., pH < 5):
PG, PS, PI, PA, cardiolipin, sulfatide, acyl-CoA,
……
Weak anionic lipids which do not carry net a
negative charge under acidic conditions, but
become anionic under physiological conditions
(i.e., pH ~ 7):
PE, free fatty acid, ceramide ……
Charge neutral, but polar lipids:
PC, SM, cerebroside, acylcarnitine, MAG, DAG,
TAG,
PG
PE
PC
15:0-15:0 PG
22:6-22:6 PG
15:0-15:0 PE
20:4-20:4 PE
14:1-14:1 PC
18:1-18:1 PC
ESI/MS analyses of a lipid mixture in the
negative- and positive-ion modes
JASMS 17 (2006), 264
Anionic lipids (1)
Weak anionic lipids (15)
Charge neutral but
polar lipids (10)
Negative-ion mode Positive-ion mode
Before addition
of LiOH
After addition of LiOH
PG:PE:PC, 1:15:10
ESI/MS analyses of a lipid mixture in the
negative- and positive-ion modes
JASMS 17 (2006), 264
-
PC TAG
+ -
AP PE PC TAG FA
-
PC TAG
+
FA/PE/AP
+
AP/PE/FA
pI = 7
Imaginary analysis of
lipids by electrophoresis
-
PC TAG
+ -
AP PE PC TAG FA
-
PC TAG
+
FA/PE/AP
+
AP/PE/FA
pI = 7
Imaginary analysis of
lipids by electrophoresis
A lipid
extract of a
biological
sample
Negative-
ion mode Anionic lipid
molecular
species
Addition
of LiOH
A mildly
basic lipid
extract
Weak anionic
lipid molecular
species
Charge neutral
but polar lipid
molecular
species
Intrasource separation
of cellular lipid classes
Mass spectra of a
cellular lipidome
A lipid
extract of a
biological
sample
Negative-
ion mode Anionic lipid
molecular
species
Addition
of LiOH
A mildly
basic lipid
extract
Weak anionic
lipid molecular
species
Charge neutral
but polar lipid
molecular
species
Intrasource separation
of cellular lipid classes
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping
Principles of shotgun lipidomics (In specific)
Product-ion analysis, neutral loss scanning, and
precursor-ion scanning are interchangeable
Suppose:
1. M1-m1a=M2-m2a=M3-
m3a =constant (i.e.,
ma)
2. M1c=m2c=m3c=constan
t (i.e., mc)
3. m2b=m3b
Product-
ion
analysis
Product-ion analysis, neutral loss scanning, and
precursor-ion scanning are interchangeable
Suppose:
1. M1-m1a=M2-m2a=M3-
m3a =constant (i.e.,
ma)
2. M1c=m2c=m3c=constan
t (i.e., mc)
3. m2b=m3b
Product-
ion
analysis
Neutral-loss
scanning (NLS)
Product-ion analysis, neutral loss scanning, and
precursor-ion scanning are interchangeable
Suppose:
1. M1-m1a=M2-m2a=M3-
m3a =constant (i.e.,
ma)
2. M1c=m2c=m3c=constan
t (i.e., mc)
3. m2b=m3b
Product-
ion
analysis
Neutral-loss
scanning (NLS)
Product-ion analysis, neutral loss scanning, and
precursor-ion scanning are interchangeable
Han, Yang, & Gross,
Mass Spectrom. Rev. 31
(2012), 134
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping
Principles of shotgun lipidomics (In specific)
Building blocks of glycerol-based lipids
General structure
of glycerol-based
lipids
General
structure of
sphingolipids
Building blocks of sphingolipids
Identification of lipid building blocks by PIS and
NLS to identify molecular ion peaks
Analysis of lyso-choline glycerophospholipids
Analysis of lyso-choline glycerophospholipids
Structure of lysoPC
1-acyl lysophosphatidylcholine
(sn-2 lysoPC)
2-acyl lysophosphatidylcholine
(sn-1 lysoPC)
JACS 118 (1996), 451
Analysis of lyso-choline glycerophospholipids
JACS 118 (1996), 451
Analysis of lyso-choline glycerophospholipids
J. Chromatogr. B 877 (2009), 2924
LysoPC molecular species in the lipid extracts of rat myocardium*
*pmol/mg of protein 13 ion peaks and 19 species
Analysis of lyso-choline glycerophospholipids
J. Chromatogr. B 877 (2009), 2924
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping
Principles of shotgun lipidomics (In specific)
Lipids can go aggregation in a manner depending on concentration, solvent,
lipid classes, etc. due to the nature of hydrophobicity. The aggregates cannot be
well ionized and most of the lipids in the aggregated states go to waste
Therefore, ionization efficiency becomes molecular species dependent (i.e., the
numbers of carbon atoms and double bonds present in acyl chains) in the
aggregated states
The lipid concentration at which aggregation occurs in CHCl3/MeOH: ~ 200
pmol/ml in 2:1, 50 pmol/ml in 1:1, and 10 pmol/ml in 1:2
Lipids go aggregation!!!
An easy method to test whether we are
analyzing lipids in an aggregate state
y = ax + b
An easy method to test whether we are
analyzing lipids in an aggregate state
log(y - b) = log(a) + log(x)
y = ax + b
r2 = ???
J. Lipid Res. 50 (2009), 162
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping (Key: no aggregation)
Principles of shotgun lipidomics (In specific)
1. There is no absolute quantification in mass spectrometry
as in optical spectroscopy where the Beer-Lambert law (A
= ξl[c]) can be followed
2. Only relative quantification in comparison to an internal
standard is possible under certain conditions
Quantification by mass spectrometry
I = ax + b or I-b = ax where I is ion intensity, b is spectral background, a is the
response factor, and x is the molar concentration
Is/Iu = xs/xu
Where I >> b and as ≈ au
Ideally isotope-labeled internal std, as = au
So what’s in reality?
Yang & Han, Metabolites 1 (2011), 21
The response factor “a”
The “a” should include all possible factors,
affecting the quantification, from ionization to
detection, a = a1.a2.a3.a4. …
a1, ionization efficiency;
a2, concentration factor (in ideal solution or not);
a3, tandem MS factor;
a4, matrix factor if ionization occurs under different
conditions;
……
Quantification in shotgun lipidomics
The linear dynamic range of concentration (intensity vs.
concentration) (The upper limit of a linear dynamic range for lipid
analysis is the aggregated state of a lipid solution)
One internal standard of a lipid class for quantitation of the
abundant molecular species in the class (Is/Iu = xs/xu where I
>> b and as ≈ au)
Dynamic range of relative intensities in comparison to a
selected internal standard (low abundance species??)
Negative-ion mode Positive-ion mode
Before addition
of LiOH
After addition
of LiOH
PG:PE:PC, 1:15:10
ESI/MS analyses of a lipid mixture in the
negative- and positive-ion modes
JASMS 17 (2006), 264
Isotopologues Isotope Ratio Peak Intensity (area)
M 1 IM
M+1 0.0109n 0.0109nIM
M+2 0.01092n(n-1)/2 0.00594n(n-1)IM
M+3 0.01093n(n-1)(n-2)/6 2.16x10-7n(n-1)(n-2)IM
…… …… ……
De-isotoping of 13C isotopologues based on monoisotopic
ion peak intensity (IM):
Itotal = IM(1 + 0.0109n + 0.00594n(n-1) + ……)
Quantification in shotgun lipidomics
M
M+1
M+2
Quantification in shotgun lipidomics
0.16 pmol/μl
0.8 pmol/μl
4 pmol/μl 16 pmol/μl
Han, Yang, & Gross, Mass Spectrom. Rev. 31 (2012), 134
Koivusalo et al., J. Lipid Res. 42 (2001), 663
An equimolar mixture of 14
PC species including 6
diunsaturated ones, ranging
from C34 to C56 of total
carbon atoms.
Quantification in shotgun lipidomics
0.1 pmol/µl
0.4 pmol/µl
The internal standard of a lipid
class is spiked during lipid
extraction
Analysis should be conducted at
a low concentration
Correction for differential 13C
isotopologue distributions
Quantitation of abundant species
(S/N > 5) after correction for
chemical background and
baseline drift.
Full MS scan for quantitation of
abundant and non-overlap species
Quantification in shotgun lipidomics
JASMS 22 (2011), 2090
Quantification of low abundance species
(a two step procedure)
Quantification in shotgun lipidomics
Facts:
The dynamic range is very limited in
quantitation with full MS scan; the
overlapped and/or low abundance species
can not be determined.
Solution:
Use the NLS and/or PIS for building blocks,
which extends the dynamic range
substantially, detects the low abundance
species, and resolves the overlap ones.
Concerns:
Any NLS or PIS analysis of a lipid class
depends on the fragmentation pathways of
individual species, thereby depending on
the physical properties of individual
species.
Solution:
1. Use endogenously determined species in
the full MS (i.e., first step) as standards to
quantify those structurally similar species
(i.e., second step of quantification)
2. Use multiple PIS/NLS to refine the
accuracy in quantitation.
Quantification in shotgun lipidomics
NLS of 183 u Full MS spectrum
Classification of Shotgun Lipidomics
• Lipid class-specific tandem MS-based shotgun
lipidomics (Brugger et al., PNAS 94 (1997), 2339; Welti et al., JBC
277 (2002), 31994; etc)
• High mass resolution-based shotgun lipidomics (the
platform developed by Dr. Shevchenko’s group)
• Multi-dimensional MS-based shotgun lipidomics (the platform developed by our group; Mass Spectrom. Rev. 31 (2012),
134)
• Total ion mapping by using high mass
accurate/resolution mass spectrometers (AB Sciex
platform)
• Ion-mobility based shotgun lipidomics (Waters
Platform)
1. Isomers possessing identical fragmentation
patterns (e.g., chiral isomers, GluCer and GalCer
in the positive-ion mode) can’t be separately
analyzed at the current stage;
2. This technology needs pre-characterization of a
lipid class of interest by product-ion analysis for
the identification of a novel lipid class.
3. Ion suppression???
Drawbacks of shotgun lipidomics
1. Different charge properties of lipid classes can be used to selectively
ionize different lipid categories
2. Interchangeable relationship among product-ion analysis, neutral-loss
scanning (NLS), and precursor-ion scanning (PIS) can be exploited to
effectively identify lipid species
3. Unique structural construction of the majority of lipids (i.e., building
blocks) can be employed to efficiently identify lipid species in combination
of NLS and PIS
4. Unique physical property of lipids (i.e., hydrophobicity) should be kept in
mind for quantification of lipids
5. Quantitation of lipid species can be achieved through direct ratio
comparison (ratiometrics) of their ion peaks to a selected internal standard
in the full MS scan after de-isotoping (Key: no aggregation)
Principles of shotgun lipidomics (In specific)
Han, Yang, & Gross, Mass Spectrom. Rev. 31 (2012), 134