1. -
Integrated Omics for the global
mapping of biomolecules in LDL
Ana Reis1, Corinne M Spickett1 and Alisa Rudnitskaya2
1 School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK;
2 CESAM and Department of Chemistry, Universidade de Aveiro, 3810-193 Aveiro, Portugal
Results
Overview
Knowledge of LDL biomolecule composition (lipids, protein
and glycans) is scarce, limiting our understanding of the
structural motifs that lie behind oxLDL atherogenicity.
 Mass spectrometry was used to profile lipids, protein
modifications and glycan chains in LDL.
Biomolecule profiles can be used to map modifications in
OxLDL, identifying disease markers and the architectural
features responsible for mediating receptor recognition and
macrophage uptake.
This research was supported by a Marie Curie Intra-European Fellowship within the
7th European Community Framework Programme (proposal 255076) and the
Fundacao para a Ciencia e Tecnologia (FCT) through the European Social Fund (ESF)
and Programa Operacional Potencial Humano (POPH).
Acknowledgements
Concluding Remarks
 Mass Spectrometry is revealing the high molecular complexity of LDL particles.
 Integration of different Omic approaches provides a much more comprehensive
knowledge of LDL particles architecture and identify the structural motifs as potential
biomarkers of diseases.
Fig. 3: Extracted Ion Chromatogram (XIC) of fragment @ m/z 204.08 (HexNAc) of trypsin
digested ApoB-100 protein with a 2ppm window (0.0003 Da).
Type of modification Site of modification in ApoB-100 sequence
Oxidation (HWY) W1981, H2316, W3563, Y3958, W3970
Oxidation (C) Sulfinic: C3194, Sulfonic: C1112, C3194, C4353
LysineAllysine K1121,K1852, K3451, K4187
Amino (Y) Y1603, Y4425
HNE (CHK) H2272
Myristoylation (C) C3194, C4217
Phosphorylation (STY) S1838, S1840, T1845
2. PROTEOMICS
…>70% sequence coverage enabling the identification
of many new modifications of ApoB-100.
Table 2. Examples of modifications mapped in ApoB-100 sequence.
Fig. 2: nanoLC-MS chromatogram of LDL protein (ApoB-100) enzymatic tryptic digest under
reverse phase conditions.
Experimental
Folch
extraction
plasma Salt gradient
Ultracentrifugation
(2hr, 70,000rpm)
LDL
NP-LCMS
Orbitrap
Enz. Digestion
(trypsin)
RP-LCMS
TripleToF (protein)
Qtrap (sugars)
Database
search
(MASCOT)
Tinn-R processing
(MzMatchR)
MASS SPECTROMETRY ANALYSIS . Experiments were performed on: 1) Lipidomics:
Exactive Orbitrap MS (ThermoFisher Scientific Inc., Hemel Hempstead, UK) operated in
dual polarity mode; 2) Proteomics: 5600TripleTof (ABSciex, Warrington, UK), +ve ion
mode, 3) Proteoglycomics: 5600TripleTof (ABSciex, Warrington, UK), +ve ion mode, 4)
Adductomics: 5600TripleTof (ABSciex, Warrington, UK), +ve ion mode.
3. GLYCOPROTEOMICS
Fig. 4: MS spectrum of non-sialylated glycan linked to Asn3384 located near the receptor-
binding site (FVEGSHNSTVSLTTK) at retention time 17.6 min (dotted box fig. 3).
Hex
Hex
…28 N-glycan chains found distributed through
8 glycosylation sites in ApoB-100.
www.caymanchem.com XIC @ m/z 184.07
4. ADDUCTOMICS
Fig.5: Extracted Ion Chromatogram of fragment @ m/z 184.07 (phosphocholine –
[MH]+) in ApoB-100 trypsin digest with a 2ppm window (0.0003 Da).
… sites of oxidized lipid adduction to ApoB-100
are located in both -helix and -sheet regions.
Peptides
(false positives)
Phosphocholines
OxPC-peptide
adducts
1
(58-795)
2
(2045-2587)
3
(4017-4515)
1
(827-2001)
2
(2571-4037)
OxPC – H2366
OxPC - H3184
Fig.6: Tandem MS of OXPC-peptide adduct in ApoB-100 trypsin digest.
1. LIPIDOMICS
Fig.1: LC-MS chromatogram of LDL lipid extract
acquired in positive (+) and negative (-) ion mode.
…more than 350 lipid molecular species
from 11 different classes support and
interact with ApoB-100.
Table 1. Lipid classes and number of individual
molecular species identified in LDL extract .
RT: 0.00 - 29.97
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
2.88
2.73
21.18
3.25
13.03
10.09
12.77
20.685.72
9.76
17.7913.37
22.63
NL:
1.94E8
TIC F: FTMS
{1,1} + p ESI
Full ms
[100.00-
1200.00] MS
1-1-2
RT: 0.00 - 29.97
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
2.85
2.70
21.20
2.29 3.45
20.71 27.132.01 26.99 27.3322.66
24.521.86
20.0612.815.29
NL:
4.31E7
TIC F: FTMS
{1,2} - p ESI Full
lock ms
[100.00-1200.00]
MS 1-1-2
(+) ion mode
(-) ion mode
Lipid Classes Number of
Mol. Ions
Glycerolipids 69
Triacylglycerols 60
Diacylglycerols 9
Sterols and steroids 12
Cholesterol 1
Cholesterol sulphates 3
Cholesteryl esters 8
Steroid conjugates 3
Fatty acids and conjugates 30
Free fatty acids 23
Fatty acid conjugates 5
Eicosanoids 2
Sphingolipids 106
Sphingomyelins 38
Ceramides 13
Ceramide-inositols 8
Hexosyl-Ceramides 11
Lactosyl-Ceramides 6
Acidic Glycosphingolipids (Sulfatides) 29
Glycerophospholipids 135
Phosphatidylinositols 19
Phosphatidylglycerols 6
Phosphatidylserines -
Phosphatidylethanolamines 48
Diacyl-PE 16
Plasmenyl-PE 32
Lyso-phosphatidylethanolamines 5
Phosphatidylcholines 53
Diacyl-PC 33
Plasmenyl/plasmanyl-PC 20
Lyso-phosphatidylcholines 4
Lipid-related compounds 11
Prenols (tocotrienol+carotene) 5
Secosteroids (vit derivatives) 3
Lipoaminoacids 3
TOTAL 361