Poster presented at the 8th Lipidomics Forum - A Conference of the International Lipidomics Society and Partners, Vienna, Austria (August 27-30th, 2023).

1. 1Quality control samples (QC) were experimental replicates of a pool of
all serum samples (one replicate for every group f 3 samples)
Exosome
Parent serum
QCs1
PCA 2D Scores plot
Significant separation
=
different composition
Tightly clustered
• Five exosome samples isolated
from serum
• Five parent serum samples
Serum EVs
1
Cell EVs
2
Transfection
3
• Three sEV samples isolated
from cell cultures
• Three sEV samples isolated
from non-transfected cells
• Three sEV samples isolated
from cells transfected with an
RNA transfection reagent
nanoLC-QToF-MS UHPLC-QToF-MS/MS UHPLC-QToF-MS/MS
CHARACTERIZING THE COMPLEX LIPIDIC COMPOSITION OF
SMALL EXTRACELLULAR VESICLES (sEVs)
THE LIPIDOME OF CELLULAR sEVs
THE LIPIDOME OF SERUM EXOSOMES
THE EFFECT OF RNA TRANSFECTION COMPLEXES ON sEVs
ADRIANA ZARDINI BUZATTO 1,3, LIANG LI 2,3
1. UNIVERSITY OF CALGARY • 2. UNIVERSITY OF ALBERTA • 3. THE METABOLOMICS INNOVATION CENTRE (TMIC) - CANADA
SAMPLE
1 2 3 4 5
6 7
DCM
MeOH
Internal
standards
Dry
Centrifuge
Resuspension
LC-MS
Vortex
Organic
layer
1
2
3
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, Canadian
Institutes of Health Research, the Canadian Foundation for Innovations, Genome Canada and Alberta Innovates.
adriana.zardinibuzat@ucalgary.ca • https://www.buzattoresearchgroup.com
lysosomal
degradation
early
endosome
multivesicular
body microvesicles
Endosome
target cell
exosomes
Genetic material
Metabolites
Lipids
Lipid membrane
Proteins
Although small (50-150 nm), EVs can influence physiological and pathological processes, being an ideal
target for biomarker research. While the interest in sEVs has grown in recent years, their complex
composition has not yet been fully defined. Their small size, heterogeneity, and difficult isolation
remain an analytical challenge.
SMALL
EXTRACELLULAR
VESICLES (sEV):
membranous structures
secreted by eukaryotic
cells (exosomes and
microvesicles)
investigation of the lipid composition of sEVs from serum and cell samples
GOAL
Collaboration with Jie Zhang, Dr. Jianhui Zhu, and Dr. David Lubman
(University of Michigan)
0
1
2
3
4
5
6
7
8
9
10
-12 -8 -4 0 4 8 12
-log
10
(p)
log2(fold-change)
Volcano plot: Exosome / Serum2
Not significant Significantly increased Significantly decreased
419 (16.1%)
increased
520 (20.2%)
decreased
602
404
1118
Exosomes
Serum
Untargeted lipidomics of exosomes isolated from serum and the parent serum samples using nanoLC-MS: 1878 lipids identified for serum exosomes
(dataset processed with Bruker Metaboscape® combined with the in-house developed software package LipidScreener)
2Significance criteria: fold-change (FC) ≥1.4 or ≤0.71, raw p-value <0.05, Storey’s q-value <0.25
0 20 40 60 80
CAR
FA
FAHFA, WE
NA, NAE, NAT
Other FAs
MG
DG
TG
Other GLs
LPA
LPC
LPE
LPG
LPI
LPS
PA
PC
PE
PG
PI
PIP
PS, PS-Nac
Other GP
Cer
GlcCer, HexCer
PhosphoSL
SphingoL Sulfate
SM, LSM
SPB, HexSPB,…
CE
ST
PK
PR
Number of significantly altered lipids
Subclass distribution for significantly altered lipids
(Exosome / Serum)
Significantly increased
Significantly decreased
2
Exosome samples showed higher proportions of carnitines,
mono- and diacylglycerols, phosphatidic acids, and
glycerophosphates when compared to their parent serum.
The 602 lipids detected only for exosomes could be potential
biomarkers to characterize these structures (although
validation is required).
Collaboration with Jenna McCann, Dr. Derrick J. Gibbings and Dr. James A. Taylor (University of Ottawa)
Untargeted lipidomics of sEVs isolated from culture media of mammalian cells using UHPLC-MS/MS: 2256 lipids identified for cell sEVs
(dataset processed with the in-house developed software package LipidScreener)
304
Serum
exosomes
Cell sEVs
0 2 4 6 8 10 12 14 16 18 Time [min]
0.0
0.2
0.4
0.6
0.8
1.0
7
x10
Intens.
P_QC04_01_G-B1_1_20564.d: BPC +All MS
0 5 10 15 20 25 30 35 40 Time [min]
0.0
0.5
1.0
1.5
7
x10
Intens.
20200221P_QC_Ser_G4_6277.d: BPC +All MS
nanoLC-MS (50.0 min)
Serum 40X diluted
Serum 8X diluted
EVs from
transfected cells
EVs from
untransfected cells
Transfection complex
reagent
QCs3
PCA 2D Scores plot
EVs
(untransfected)
Transf.
complex
PE 18:1_18:1 (DOPE)
Transfected
Untransfected
MS/MS score >500, precursor
m/z error <5.0 mDa and 20 ppm
TIER 1
MS/MS score <500, precursor
m/z error <5.0 mDa and 20 ppm
TIER 2
m/z match with tolerance of 5.0
mDa and 20 ppm (lipid species)
TIER 3
1 incorrect match
(PC 15:0_18:1 → PE 18:0_18:1)
11 correct IDs
(species level)
79.7%
18.6%
47 correct IDs
(molecular species)
59
lipid standards
VALIDATION
OF IDs:
LipidScreener
MS/MS match, m/z match,
10-layer filtering and scoring system
NanoLC-MS offers high sensitivity and low
consumption of solvents, but it requires longer analysis
times. We also observed lower reproducibility and peak
resolution. The choice of best technique depends on the
goals and limitations of the project and the analyst.
RNA
Liposome
Endosome
Maturation
sEV
Main component: DOPE (PE 18:1_18:1)
Untargeted lipidomics of sEVs isolated from media of mammalian cells transfected with a liposome-based RNA transfection complex by UHPLC-MS/MS
There is a discussion in the scientific community regarding the presence of the intact or parts of liposome-based transfection complexes in sEV samples obtained from
transfected cells. We found some evidence of lipids from the transfection complex in the sEV samples, indicating that caution is necessary when evaluating sEVs after
transfection.
The investigation of the lipid composition of sEVs and exosomes is still evolving. Detailed studies can elucidate these small structures, allowing for further biomarker research. NanoLC-MS lipidomics can be
a great tool for extremely diluted samples, but we recommend tight quality control and previous technical experience. We found limited but clear evidence of the presence of the transfection complex
reagent in sEVs isolated from transfected cells. Although the annotation of lipids with our in-house developed software package LipidScreener was validated with standards, there is still a lack for practical
tools to ensure reliable lipid annotations. We are currently investigating the annotation of unexpected lipid species.
EVs from
transfected cells
EVs from untransfected
cells
Transfection complex
reagent
PLS-DA 2D Scores plot
p-value: 0.01 (100 perm.)
R2: 0.992 • Q2: 0.884
Significant
separation
3Quality control samples (QC) were experimental replicates of a pool of all samples
(one replicate for every group of 9 samples)
Although DOPE was elevated for EVs from transfected
cells, the difference was not deemed significant (criteria:
FC >1.4, p-value <0.05 and FDR-corrected p-value <0.25).
Exosome
Serum
different
composition
nanoLC-MS × UHPLC-MS
0
50
100
150
200
250
300
350
CAR
CoA
FA
FAHFA,
WE
NA,
NAE,
NAT
Other
FA
DG
MG
TG
Other
GL
BMP
LPA
LPC
LPE
LPG
LPI
LPS
PA
PC
PE
PG
PI
PIP
PS
Other
GP
Cer
GlycoSP
SM,
LSM
Other
PhosphoSP
SPB,
SPBP
CE
ST
PK,
PR
SL
FA GL GP SP ST Others
Number
of
annotations
Distribution of lipid annotation results: cell sEVs
Tier 1 Tier 2 Tier 3 Serum exosomes
FA GL GP SP ST Others
*Unexpected annotations are currently under investigation
0
50
100
150
200
Number
of
annotations
Identified lipids Lipids detected only for exosomes
Distribution of lipid annotation results: exosomes isolated from serum samples
Exosomes have a rich lipid variety, requiring high sensitivity methods for unbiased characterization.
*Unexpected annotations are currently under investigation
0
50
100
150
200
250
300
350
CAR
CoA
FA
FAHFA,
WE
NA,
NAE,
NAT
Other
FA
DG
MG
TG
Other
GL
BMP
LPA
LPC
LPE
LPG
LPI
LPS
PA
PC
PE
PG
PI
PIP
PS
Other
GP
Cer
GlycoSP
SM,
LSM
Other
PhosphoSP
SPB,
SPBP
CE
ST
PK,
PR
SL
FA GL GP SP ST Others
Number
of
annotations
Distribution of lipid annotation results: RNA transfection complex
Transfection complex reagent EVs from untransfected cells
The liposome-based transfection complex has a similar composition when compared to sEVs from
untransfected cells, but with lower lipid class variability.
*Unexpected annotations are currently under investigation
UHPLC-MS (21.5 min)
Collaboration with Jenna McCann, Dr. Derrick J. Gibbings and Dr. James A. Taylor (University of Ottawa) • McCann, J. et al., J Extracell Vesicles. 2022 Oct;11(10):e12220. DOI: 10.1002/jev2.12220