Principles of Shotgun Lipidomics - avcr.czmab.uochb.cas.cz/iochb/ELM2013/han.pdf · concentration of lipid solution without any time ... Interchangeable relationship among product-ion

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




ASMS 2004




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)









of NLS and PIS










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



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








of NLS and PIS







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



Suppose:

1. M1-m1a=M2-m2a=M3-


ma)


t (i.e., mc)

3. m2b=m3b

Product-

ion

analysis

Neutral-loss

scanning (NLS)



Suppose:

1. M1-m1a=M2-m2a=M3-


ma)


t (i.e., mc)

3. m2b=m3b

Product-

ion

analysis

Neutral-loss

scanning (NLS)



Han, Yang, & Gross,

Mass Spectrom. Rev. 31

(2012), 134








of NLS and PIS







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


Structure of lysoPC

1-acyl lysophosphatidylcholine

(sn-2 lysoPC)

2-acyl lysophosphatidylcholine

(sn-1 lysoPC)

JACS 118 (1996), 451


JACS 118 (1996), 451


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


J. Chromatogr. B 877 (2009), 2924








of NLS and PIS







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








of NLS and PIS





in the full MS scan after de-isotoping (Key: no aggregation)


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



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) + ……)


M

M+1

M+2


0.16 pmol/μl

0.8 pmol/μl

4 pmol/μl 16 pmol/μl


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.


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


JASMS 22 (2011), 2090

Quantification of low abundance species

(a two step procedure)


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.


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








of NLS and PIS





in the full MS scan after de-isotoping (Key: no aggregation)



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Principles of Shotgun Lipidomics - avcr.czmab.uochb.cas.cz/iochb/ELM2013/han.pdf · concentration of lipid solution without any time ... Interchangeable relationship among product-ion