lipidomics Flashcards
what does “ome” mean
is a suffix that refers to the
collective objects of study within a field
– “ome” was derived from the Greek “ομα”,
which itself is not a Greek suffix
what does genome mean
the complete collection of
genes within a species
what is genomics
the study of
genomes within a species
what are some of the other omic subfields of genomics
– Cognitive genomics
– Comparative genomics
– Functional genomics
– Transcriptomics
– Epigenomics
– Nutrigenomics
proteome
the complete collection of
proteins and modified proteins produced
by a species
peoteomics
the study of proteomes within
a species
* Proteomics typically requires the use of
mass spectrometry methods
what are the subfield omics of proteomics
– Structural proteomics
– Nutriproteomics
– Immunoproteomics
lipidome
the complete collection of lipids
and modified lipids within a species
lipidomics
the study of lipidomes within a
species
Advances in mass spectrometry (in 1991)
allowed for its use to detect different
classes of lipids
emergence of lipidomics
Lipidomics first emerged in 2003 whereby
differing multiple species and classes of
lipids from animal models of various
metabolic disorders could be quantified
simultaneously within minutes
why care about the lipidome
understanding cellular functions
deciphering metabolic changes
identifying markers of sudden physiological events
food: are you really getting what you think you are purhcasing
understanding cellular function
identifying the specific lipid species that
modify protein functions, membrane
localization of lipid species, specific lipid
species involved in intracellular signalling
deciphering metabolic changes
specific
changes to the lipidome can reflect
specific metabolic disorders associated
with lipid metabolism
identifying markers of sudden physiological events
levels of specific
species of lipids can abruptly change in
the bloodstream or in sections of tissues in response to a pending event or already
occurred event
what are the basic steps prior to lipidomics analases
tissue or cells
homogenization and membrane fractionation
//optional: addition of antioxidants
lipid extraction
//optional: addition of internal standards
storage/ concentration of lipid extracts
basic steps for lipidomics analyses
storage concentration of lipid extracts
direct infusion or front end separation
(branches into two sections)
1- survey scan
provide information on molecule ion mass
- tandem mass spectrometry/ product ion scan/ precursor ion scan/ neutral ion scan
provide information on polar headgroups and fatty acyl constituents
how many primary mass spectrometry
methods are typically used for identifying
and quantifying lipids and what are they
three
– Gas chromatography mass spectrometry
(GC-MS)
– Electrospray ionization mass spectrometry
(ESI-MS)
– Matrix assisted laser desorption ionization
mass spectrometry (MALDI-MS)
what does mass spectrometry involve
Mass spectrometry involves the charging
of a particle which then passes through a
magnetic field to deflect the charged
particle along a circular path on a radius
that is proportional to the ratio of mass to
charge (or m/z)
mass spectrometry steps
sample goes into ion source then into mass analyzer then to a detector and finally we analyze the data
what does EI-MS produce
it is the simplest mass spectrometry method and produces cations
that undergo fragmentatio
EI-MS is considered
a ‘hard ionization’
method because radical-mediated
fragmentation of a molecule continues
until a stable fragment is achieve
what is CI-MS
method of mass spectrometry
that introduces a gas to act as a reagent
for ion generation at a lower voltage
what is the voltage used for CI-MS
20-40 eV
what is the voltage used for EI-MS
70 eV
what does the ion generated in CI-MS do
The ion generated can react with the
compounds injected into the mass
spectrometer, and in turn the ion transfers
its charge to the compounds
charged compounds of CI-MS
The charged compounds exhibit little
fragmentation (‘soft ionization’)
Ion production in CI-MS using methane as the reagent gas
The CH 5+ and C 2H5+ ions collide with the
compound M to form the following ions:
what is GC-MS
GC-MS combines the chromatographic
separation of gas vapours based on time
and sample temperature, together with
either EI-MS or CI-MS
Why is GC-MS useful
GC-MS is useful for the detection of fatty
acids, fatty alcohols, and cholesterol
generated ions in GC-MS
Generated ions are up to m/z of 400
how can we detect lipids in GC-MS
The lipids cannot normally be detected by
GC-MS because they do not vaporize due
to their associated –OH groups
– However, the –OH group on lipids can be
esterified with other molecules to make them
capable of vaporizing
What does PFB-Br stand for
Pentafluorobenzyl bromide
why is Pentafluorobenzyl bromide useful in GC-MS
Pentafluorobenzyl bromide (PFB-Br) can
readily esterify with the –OH group of most
lipids
* PFB esters readily vaporize in a GC-MS
PFB in GC-MS
In the MS, PFB readily dissociates from
the lipid through ‘neutral loss’
– during detection, you would look for the lipid of interest at an m/z corresponding to [M-H] - (ie. MW palmitate is 256… m/z will be 255)
– detection will be using ‘negative ion mode’ for anions (which is unlike ‘positive ion mode’ for detecting cations generated by EI or CI)
positive ion mode
a detection mode that
identifies only positively charged ions
negative ion mode
a detection mode that
identifies only negatively charged ions
neutral loss
(NL): the loss of an
uncharged fragment of an ion during MS
collision induced dissociation
CID
the
fragmentation of an ion via inert gas (i.e.,
argon) bombardment
NL scan
the filtering for all ions that
exhibit a specific NL
survey scanning (also seen as MS, MS1,
and Q1MS):
a scan of all ions detected
without any filtering
precursor ion
charged ion that is
derived from an ion during fragmentation
precursor ion scanning
the filtering for all
ions that produce a specific precursor ion
ESI-MS generates what
ESI-MS generates charged molecules
from a liquid stream (provided by direct
infusion/injection or from chromatographic
separation)
what voltage is used for ESI-MS
2000-6000 V
What temperature is used for ESI-MS
200-500 °C
what happens to molecules in ESI-MS
In the presence of voltage (2000-6000 V)
and temperature (200-500 °C), the
molecules in the stream ionize; during this
process, an extremely fine spray of
droplets is formed
what happens to the electrospray in ESI-MS
The ‘electrospray’ loses solvent, and the
ions enter the quadrupoles - (region with
four sets of magnets)
what m/z ions can be detected in ESI-MS
Ions with m/z between 50-3000 can be
detected
what lipids can ESI-MS detect
ESI-MS can be used to detect all classes
of lipids without chemical modification,
except cholesterol (which poorly ionizes
with this method)
