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adipocyte
Greasing the Wheels
of Lipidomics
Lipids have traditionally been among the hardest biomolecules
to study, but new technologies and techniques are gradually
revealing more of the lipidome. By Alan Dove
B
iology is swaddled in lipids. Fats, oils, and waxes enclose cells and organelles, mediate vast networks of
information flow, protect fragile tissues from hostile
environments, and store essential energy for all kinds of organisms. Unfortunately, lipid structures don’t rely on the sorts of
well-defined building blocks and simple rules that govern DNA,
RNA, and proteins. From the complete DNA sequences of the
biosynthetic genes in a cell, for example, one can reliably predict the RNA transcripts likely to arise from those genes and
the protein sequences they’ll encode; the lipids synthesized by
those proteins, though, will remain mostly mysterious.
This disjunction, between the relatively predictable patterns of
nucleic acids and proteins and the largely inscrutable world of
lipids, has skewed the progress of biochemistry for years. Genome sequencing technology and genomics quickly fed the rise
transcriptomics and proteomics. As those fields charged ahead,
amassing terabytes of data on the biology of RNA and proteins
respectively, lipid biochemists felt largely left out.
That began to change in 2003, as a major National Institutes
of Health (NIH)-funded effort set out to establish the field of
lipidomics. In the ensuing decade, researchers and equipment
makers have developed several new techniques and tools for
analyzing these challenging molecules.
The fat of the land
“In 2002 ... there were no publications using the word ‘lipidomics,’” says Edward Dennis, Ph.D., professor of chemistry,
biochemistry, and pharmacology at the University of California
in San Diego. That year, Dennis proposed a project to create
the field, and the NIH’s National Institute of General Medical
Sciences (NIGMS) agreed to fund it. The result was the 10-year,
$73 million Lipid Metabolites and Pathways Strategy (Lipid
MAPS) effort, which Dennis oversaw.
Dennis started by recruiting five other researchers at institutions across the country, each an expert on a particular type of
lipid. The scientists began developing techniques to isolate and
quantify their assigned lipid categories from biological samples,
but they ran into a major problem immediately. “We came to
realize that there was no classification for lipids that was suitable for the bioinformatics age,” says Dennis, adding that “you
need a taxonomy to classify them and ... store all the billions of
bits of information we were intending to get.”
To answer that need, the team invented a new classification
scheme, dividing all lipids into eight major categories, six of
which are found in mammals. The group also revised the nomenclature for lipid structures and developed a standardized
system for drawing them. The Lipid MAPS website now features
a searchable database with more than 40,000 structures, all
classified and drawn using the new system, which researchers
worldwide have also adopted. “I would say virtually everybody
in the world today who talks about a lipid uses this structural
representation,” says Dennis.
With the taxonomy in place, the Lipid MAPS researchers focused on lipidomics protocols. All of the labs began with identical equipment, based on the AB Sciex QTRAP 4000 Liquid
Chromatography/Mass Spectrometry (LC/MS) system that was
then considered the state of the art. Each scientist optimized
fractionation methods to isolate one specific category of lipids,
then added radioactively labeled standard compounds before
performing mass spectrometry. The combination of fractionation and mass spectrometry identified the lipids in the sample,
and the internal standards allowed precise quantitation of them.
Combining the data from all six labs produced complete lipidomic profiles of some samples that answered specific biological
questions. Lipid MAPS also discovered a number of novel lipids, though that wasn’t their primary goal.
In an era when genomics tools are a mouse-click away and
most major universities run dedicated core labs for proteomics,
the scale and intensity of the six-facility Lipid MAPS approach
may seem out of reach for nonspecialists. It probably is. “It
would be very expensive to set it up from scratch,” Dennis concedes. With the conclusion of the Lipid MAPS project, though,
many of the individual participants have continued to keep their
doors open. Dennis’s group, for example, offers its lipidomics
services to other researchers through collaborative grants or
recharge funding.
The techniques and technologies for lipidomics both evolved
during the decade-long project. Dennis’s lab now uses an AB
Sciex QTRAP 6500M—the successor to the 4000—combined
with an Acquity ultrahigh-performance liquid chromatography
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(UPLC) system from Waters in Milford, Massachusetts. Other Lipid MAPS
groups, and the growing
population of lipidomics
researchers outside the
project, have adopted
other strategies and tools
depending on the types of
experiments they pursue.
increase the precision of
the data a lot,” says Welti.
Lipidomics in general,
and direct infusion in particular, can also be a messy
business. “There isn’t any
instrument that doesn’t
need to be cleaned a lot if
you’re [studying] lipids by
direct infusion,” says Welti.
Despite using different
“Even if you’re not an expert in lipid
Wax on, wax off
approaches, Welti and
analysis, the way lipids fragment and the
“There isn’t one method
Dennis agree on the need
that people are using
for specialized lipidomics
way that you measure them and detect
everywhere,” says Ruth
facilities. The combination
them is quite defined.”
Welti, Ph.D., director of
of technical complexity and
the lipidomics core faciloften high instrument mainity at Kansas State Unitenance causes many core
versity in Manhattan, Kansas. One reason for the diversity of
facilities to avoid lipidomics. As a result, Welti estimates that her
techniques is that lipidomics researchers fall into two separate
group has collaborated with about 400 other laboratories in the
camps: high precision and high throughput. High-precision projpast decade. “It’s a big need and people don’t have this kind of
ects, most notably, focus on obtaining accurate measurements
thing available to them,” she says.
of the levels of specific types of lipids. High throughput experiIn addition to academic facilities such as Welti’s, at least one
ments, in contrast, tend to process large numbers of samples
company, Avanti Polar Lipids in Alabaster, Alabama, also offers
looking for relative changes in lipid levels, “sort of more like a
lipidomics services to the scientific community. Avanti worked
gene expression analysis, where the absolute amounts aren’t so
on the Lipid MAPS project and now makes its facilities available
important but the comparison across the samples is important,”
for a fee.
says Welti.
Welti is a practitioner of the high throughput approach. InDon’t fear the hydrophobes
stead of fractionating samples and looking for specific types
As more researchers discover the potential of lipidomics,
of lipids in each fraction, she and her colleagues infuse crude
though, some are starting to do the work themselves rather
samples directly into a Waters triple-quadrupole mass specthan collaborate with a dedicated lipid facility. “It’s not only the
trometer. That allows the team to analyze thousands of samples
Ed Dennises of the world who are doing lipidomics today,” says
for a single experiment, a useful capability for Welti’s large-scale
Fadi Abdi, Ph.D., senior global market manager for lipidomics,
studies on agriculturally important plant phenotypes.
metabolomics, and imaging at AB Sciex in Framingham, MasBesides enabling higher throughput, direct infusion also mitisachusetts. Abdi adds that “there’s a transition in the market
gates one of the drawbacks of LC-coupled MS. Lipids in a samfrom the proteomics side to the metabolomics and lipidomics
ple can affect one another’s ionization in the mass spectrometer. side.”
By separating the sample into distinct groups of lipids, LC
Indeed, all of the major mass spectrometer makers now offer
sends particular subsets of molecules into the mass spectromproducts specifically for lipidomics. At AB Sciex, that means
eter in distinct pulses. “If you use LC, then at some points the
catering to both the high throughput and high-precision markets
percentage of particular lipids that are ionized might relate to
with different mass spectrometry systems. For high throughput
what other lipids they’re going in with,” says Welti. By sending
survey experiments, the company sells triple time-of-flight mass
the entire sample through without fractionation, the concentraspectrometers that provide accurate mass information very
tions of all of the lipids remain constant relative to each other.
quickly. “That allows us to do discovery type of work for lipids.
Direct infusion has its own weaknesses, of course, and reAt the same time, we have another set of products, which are
searchers using the technique have to take steps to address
the QTRAP platforms, which allows us to do more targeted
them. For example, most high throughput experiments need
types of work,” says Abdi.
quality control samples made from a pool of all of the samples
A few miles away in Waltham, Massachusetts, Thermo
being analyzed. The pooled sample contains all of the lipids
Scientific now offers several lipidomics systems built around
that could show up in any experimental sample. “A big problem
the company’s Orbitrap mass spectrometry technology.
with mass spec in general is normalization of data across time
Meanwhile Bruker, in Billerica, Massachusetts, caters to
on the same mass spec, across different machines, across difthe lipidomics market with a unique combination of mass
ferent labs, so having reference standards that contain all of the
spectrometers, thin layer chromatography, and nuclear
compounds that are in your samples is really helpful and can
magnetic resonance (NMR) instruments. continued>
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LIPIDOMICS
The diversity of technolFeatured Participants
ogy on the market reflects
AB Sciex
the unusual challenges of
www.absciex.com
lipidomics. Unlike proteins
Bruker
and nucleic acids, lipids in
www.bruker.com
cells often come in different
Kansas Lipidomics
isomeric forms, with identiResearch Center
cal compositions and mowww.k-state.edu/lipid/
lecular weights but different
lipidomics
structures. Even a perfectly
Premier Biosoft
accurate mass measurewww.premierbiosoft.com
ment can’t distinguish
Thermo Scientific
between isomers, so highwww.thermoscientific.com
precision lipidomics studies
University of California,
often have to rely on addiSan Diego
tional analytical techniques.
www.ucsd.edu
Separating the samples
by UPLC before the mass
spectrometer, or analyzing
them with methods such as
NMR afterward, may help fill gaps in the data.
Besides learning about the strengths and limitations of the
many equipment configurations, researchers considering taking up lipidomics should ask companies about training and
user-friendliness. Most are happy to guide newcomers. “Even
if you’re not an expert in lipid analysis, the way lipids fragment
and the way that you measure them and detect them is quite
defined,” says Baljit Ubhi, Ph.D., staff scientist in metabolomics
and lipidomics applications at AB Sciex. Ubhi adds that “we
have predefined methods [and] application specialists who are
experts in that field who can get a user up and running fairly
quickly.”
Mass spectrometers inevitably come with software as well.
In addition to the applications that run the machine, most
manufacturers include data analysis packages with varying
degrees of sophistication and flexibility. Software optimized for
proteomics or metabolomics may be useless for lipidomics, so
scientists who want a complete system should look for lipidomics-specific data handling options. Advanced systems either
include their own databases of lipids, or tie into the Lipid MAPS
database to identify specific lipid species in a sample.
Apps for fats
Many lipidomics researchers use the software that came with
their mass spectrometers only for initial data collection, preferring to export the data to another program for analysis. For
some experimenters, that means transferring it into a common
spreadsheet program such as Microsoft Excel. Dedicated lipidomics specialists, however, often prefer to develop their own
software.
Christer Ejsing, Ph.D., associate professor in the department
of biochemistry and molecular biology at the University of
Southern Denmark in Odense, Denmark, is one of the scientists who chose to write his own data analysis program. “If you
have several hundreds of injections on your instrument and you
790
want to organize the data,
you don’t want to do that in
University of Southern
Excel,” says Ejsing.
Denmark
www.sdu.dk/en
As a result, Ejsing and
his colleagues created
Waters
the Analysis of Lipid Exwww.waters.com
periments (ALEX) software
package, which is specifiAdditional Resources
cally designed for the type
Lipid MAPS
of high-precision lipidomics
www.lipidmaps.org
Ejsing’s lab pursues. EjsMetaboLights
ing uses a small script to
www.ebi.ac.uk/metabolights/
export data from his mass
about
spectrometry software into
Plant/Eukaryotic
a database format, then
and Microbial Systems
Resource
uses Tableau, a data visumetnetdb.org/pmr
alization language originally
developed for the banking
industry, to create graphs.
Typical of most software
from academic researchers, ALEX is open source, free for other
scientists to download, use, and modify.
For those pursuing high throughput lipidomics, Welti’s software project, LipidomeDB, may be more useful. LipidomeDB is
a web-based system that takes Microsoft Excel files of mass
spectrometry data as inputs and exports data in the same format. The freely accessible system has been quite popular with
Welti’s collaborators. “A lot of people use it, if we run samples
for people, [they] have the option of processing it themselves or
having us process it for them,” she says.
Several other lipidomics labs have developed their own analysis software as well. Each program is optimized for the lab that
created it, but most are freely available to other researchers. At
least one company, Premier Biosoft in Palo Alto, California,
has also released a commercial stand-alone lipidomics data
analysis application.
The wide selection of data analysis tools is both a blessing
and a curse. While researchers can likely find a ready-built application that will fit their own needs, there’s no standardization
across the systems to ensure that they’ll handle data consistently. As a result, scientists working in lipidomics often like to
export their raw mass spectrometer data to outlets such as the
European Molecular Biology Laboratory’s MetaboLights database or Iowa State University’s Plant/Eukaryotic and Microbial
Systems Resource. That allows other scientists to reanalyze the
data with their own applications to make comparisons across
systems.
Analyzing lipids may never be as straightforward as studying
nucleic acids or proteins, but lipidomics is clearly off to a strong
start. A recent search of PubMed revealed nearly a thousand
publications using the term—not bad for a field that didn’t exist
12 years ago.
Alan Dove is a science writer and editor based in Massachusetts.
DOI: 10.1126/science.opms.p1500091
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