1. LYMPH-CARRIED SELF-PEPTIDOME DERIVES
FROM A VARIETY OF PROCESSING ENZYMES
AND CONTRIBUTES TO THE DENDRITIC CELLS
MHC II (DR1-0101) PEPTIDOME
Cristina C. Clement Aniuska Becerra Scott Shafer
Lawrence J. Stern and Laura Santambrogio
American Peptide Symposium
Orlando, Florida
June 21th 2015
L08, YI

2. Peptidome Pattern
Diagnostic”
healthy vs.cancer/autoimmne diseases
Peptidome/Degradome in Biological Fluids
All analyzed biological fluids contain a peptidome/ degradome composed of naturally
processed protein fragments and peptides.
Under physiological conditions the degradome/peptidome reflects the metabolic and
catabolic activities ongoing in the different organs from where the biological fluid is drained
During inflammatory or neoplastic conditions additional peptides are observed, which reflect
ongoing organ-specific pathological processes.
Peptidome
Atlas
RA
JRA
OA
PROTEOME
PROTEASES
and
DEGRADOME SUBSTRATES
Activation
Inhibition
Degradation
PEPTIDOME
MW>8-10 kDa MW<8-10 kDa
Human Lymph
Peptidome
nLC-LTQ
MS/MS
Improving collision induced dissociation (CID), high energy collision dissociation (HCD), and electron
transfer dissociation (ETD) fourier transform MS/MS degradome-peptidome identifications using high
accuracy mass information.
Shen Y, Tolić N, Purvine SO, Smith RD.
Blood peptidome-degradome profile of breast cancer.
Shen Y, Tolić N, Liu T, Zhao R, Petritis BO, Gritsenko MA, Camp DG, Moore RJ, Purvine SO, Esteva FJ,
Smith RD.
Strategy for degradomic-peptidomic analysis of human blood plasma.
Shen Y, Liu T, Tolić N, Petritis BO, Zhao R, Moore RJ, Purvine SO, Camp DG, Smith RD.
Clement CC et al.
PLoS One 2010
>6000 peptides in the plasma

3. Lymph
Biological fluid rich in biomarkers
Prenodal lymph is generated from the interstitial
fluid that surrounds organs, and thus contains
products of organ metabolism and catabolism.
 New proteomic analyses of lymph have
identified proteins and peptides that are
derived from capillary extravasation and tissue-
specific proteins.
 Proteins and processed peptides are filtered
from the lymph by circulating immature
dendritic cells (DCs) or non-activated nodal
antigen-presenting cells (APCs) (macrophages, B
cells and immature DCs.
 Hypothesis: Organ-specific self-antigens are
displayed to circulating and nodal APCs, thus
contributing to the maintenance of peripheral
tolerance.
 When the tolerance to self-antigens is broken
 autoimmune diseases
PEPTIDOME OF HUMAN LYMPH
Endogenously Peptides MW<8-10 kDa
NEW BIOMARKERS
The lymph as a pool of self-antigens.
Clement CC, Rotzschke O, Santambrogio L.
Trends Immunol. 2011 Jan;32(1):6-11
DCDC
DC
DC DC
Lymph
node

4. CD4+T cell repertoire is restricted to
processed and MHC-II presented peptide
biochemical studies have revealed that MHC II–peptide
complexes have half-lives of days to weeks
Immunologically relevant
Peptides Epitopes
1. Enough antigen amount
(pM-M; ng-mg???)
2. Relevant MHC binding affinity
<50 nM strong binding
50-500 nM weak binding
3. TCR repertoire available (clonal
selection)
TOLERANCE OR AUTOIMMUNITY?!
THE MHC-II bound peptidome
DC
(other APC)
CD4+T cell
Plasma membrane
Plasma membrane
MHC-II are highly polymorphic
DRB1*0101 haplotype:
autoimmune diseases: RA< JIA, OA

5. Lymph-carried self-peptidome
1. Extraction, fractionation and
middle-down sequencing by nLC MS/MS
on Orbitrap.
2. Analysis of the molecular function
and cellular pathways associated with
the self-peptidome carried by the
human lymph.
3. Degradome analysis: antigen
processing pathways.
4. Prediction and functional validation
of the immunological significance for the
human lymph peptidome by measuring
the affinity of lymph-derived
endogeneous peptides for the MHC-II
DR1 molecules (ELISA competitive
displacement)
The human Lymph Peptidome
Can be displayed on MHC-II on DC
Dendritic
cells
Clement CC et al. L. Santambrogio lab Aniuska Becerra Lawrence J. Stern lab
DRB1*0101 haplotype:
autoimmune diseases: RA< JIA, OA
Discovery of neo-peptide epitopes
SAR for MHC-II binding
Novel potential immnomodulators
of T cell activity

6. SEQUENCING THE LYMPH PEPTIDOME
collision induced dissociation (CID), high energy collision dissociation (HCD),
and electron transfer dissociation (ETD) nLC-MS/MS on Orbitrap
Human prenodal Lymph (18 healthy patients)
(+protease inhbitors)
Peptidome fractionation (10-15 mg total protein-pooled from 18 patients)
Ultrafiltration through Centriplus-devices
(MWCO 10,000 Da) (3x 60 min)
Filtrates (Peptides with MW <10kDa)
FPLC fractionation
Acid elution of the peptides
with TFA 0.1 % (5 minutes, RT)
(elute chaperones and albumin/Ig bound peptides)
FPLC fractions: Solid phase
extraction and fractionation on
C18 columns (RPC)
Nano LC –MS/MS
LTQ/Orbitrap (Velos)
MS/MS-searched against Human database (Swiss Prot)
MASCOT and PEAKS 7.0 (searching engines) (“NO ENZYME”) restriction
HCD
ETD
CID
0 2 5 0 0 5 0 0 0 7 5 0 0
0 .0 0 0
0 .0 2 5
0 .0 5 0
0 .0 7 5
0 .1 0 0
C o l u m n :S u p e r d e x P e p tid e 1 0 /3 0 0 G L ( T r ic o r n )
S a m p le : 5 0 0 u L o f ly m p h filtr a te ( 5 0 0 0 D a c u to ff)
F lo w r a t e : 0 .5 m L /m in a t r o o m te m p e r a tu r e
B u f f e r : 3 0 % a c e to n itr ile in 0 .1 % T F A
F r a c t io n s c o lle c te d a t 1 m L .
T im e
A(280nm)

7. FDR <0.5%; p<0.05
Representative PEAKS 7.0 output
for a FPLC-lymph peptidome fraction
552 PSM
>300 peptides/FPLC fraction

8. We sequenced over 900 peptides, each assigned with <0.5% false
discovery rate, which corresponds to a p-value between 0.01-0.05.
This analysis mapped naturally processed peptides deriving from over 480
proteins with different intra and extra-cellular locations.
The human lymph peptidomes sequenced at high resolution on
Orbitrap:CID/HCD/ETD-nLC-MS/MS

9. Oncostatin-M-specific Receptor subunit beta
Glyceraldehyde-3-phosphate dehydrogenase
HCD
Human lymph peptidome-novel epitopes
Elongation factor 1-alpha 1
Chromosomal protein HMG 17

10. Cytochrome c oxidase subunit 5A Malate dehydrogenase, mitochondrial
HCD
Splicing factor 3A subunit-2 Solute carrier
organic anion
transporter
family
member 6A1

11. Insulin-like growth factor 1
receptor
Transmembrane
glycoprotein NMB
HCD
Collagen alpha-1(XV)
Complement C3

12. Alpha-1-antitrypsin
Pigment epithelium-derived factor
ETD
Histone H1.4
Histone H2B type 1
ETD
ETD
HCD

13. Polymeric immunoglobulin receptor
Apolipoprotein A1
HCD
Fibrinogen alpha chain
Kininogen 1

14. Macrophage migration inhibitory
factor
Triosephosphate isomerase
HCD
Proteoglycan 4
Collagen alpha-1 (I)

15. Molecular functions and cellular distribution of the
human lymph peptidome
The endogenous peptides from human lymph derived from mitochondria, ER, Golgi enzymes,
cytosolic proteins, nuclear proteins and peptides from extracellular matrix proteins; playing
important roles as chaperones and in innate immune responses.
Several additional naturally-processed peptides derived from plasma membrane proteins and
surface receptors, in addition to the proteins found in the filtrate of the plasma (fibrinogen
and other acute-phase response proteins).
Lymph-is not only a filtrate of plasma

16. Empty
HLA-DR1 +/- DM
helper for peptide
loading)
Analysis of MHC-II-DR1
binding affinity
Competition ELISA displacement assays with
recombinant MHC-II molecules
IPMYSIITPNVLRL
DIC50= 0
IC50= 0.03 ± 0.01 M
SSKITHRIHWESASLLR
DIC50= 0.06 M
IC50= 0.07 ± 0.01 M
EEFGRFASFEAQGALA
DIC50= 0.07 M
IC50= 0.14 ± 0.04 M
ASFEAQGALANIAVDKA
DIC50= 3.40 M
IC50= 1.71 ± 0.28 M
Complement Protein C3 MHC-II a chain
LymphMHC-II-eluted LymphMHC-II-eluted
Peptide loaded
HLA-DR1 +/- DM
(helper for
peptide loading)
The immunological significance of the human lymph peptidomeIn VITRO

17. Structure-Activity Relationship (SAR) for MHC-II DR1 peptides binders
The immunological significance of the human lymph peptidomeIn VITRO
Protein Sequence
Peptide
source IC50 (DR1)(nM)
Actin_ cytoplasmic 1 ALDFEQEMATAASSS DC 116 ± 47
Annexin A2 RDALNIETAVKTKG DC 283 ± 119
Cathepsin S TGKLISLSAQNLVD DC 18 ± 1
Complement factor B KGGSFQLLQGGQALE DC 42 ± 8
C-type lectin domain family 12 member A SEKMFLSEESERSTD DC 896 ± 735
Dihydropyrimidinase-related protein 2 DENQFVAVTSTNAAK DC 48 ± 9
Endoplasmic reticulum resident protein 29 GEFIKASSIEARQ DC 158 ± 52
Integrin beta-2 NSNQFQTEVGKQLIS DC 96 ± 21
Ras-related protein Rab-5C SPNIVIALAGNKAD DC 123 ± 34
Syntenin-1 KVIQAQTAYSANPA DC 53 ± 27
Transforming growth factor-beta-induced protein ig-h3 EAFQAMPPEELNK DC 292 ± 38
HLA class II histocompatibility antigen_ DR alpha chain EEFGRFASFEAQGALA DC 50 ± 9
Complement C3
IPMYSIITPNVLRL DC
17 ± 1
HLA class II histocompatibility antigen_ DR alpha chain
ASFEAQGALANIAVDKA Lymph
740 ± 109
Complement C3 SSKITHRIHWESASLLR Lymph 53 ± 4
Collagen II alpha-1 AGFKGEQGPKGEP Lymph 4399 ± 243
Netrin G2 MLHLLALFLH Lymph 7558 ± 3617
Fibrosin PGALLGAPPPLVPAP Lymph 2121 ± 152
Cartilage-oligomeric protein AWLDLEFISTVLGAP Lymph 141 ± 7
Collagen VI alpha-2 RNMTLFSDLVAEKFI Lymph 2542 ± 203
Adlican EDTFAHLTPTPT Lymph 221 ± 9
Collagen alpha-1(XII) KNFASVQGVSLESG Lymph 242 ± 55

18. In vitro T cells proliferation assay
Immunological relevance of the human lymph peptidome
Mice (DR1+)
Transgenic mice (humanized) for MHC-II-DR-1
Immunization of peptides
+/- CD4+CD25+ (regulatory Tcells)

19. Acute phase response signaling
Major biochemical pathways: the human lymph peptidome
Lymph-filtrate of plasma

20. Coagulation system
Major biochemical pathways: the human lymph peptidome
Lymph-filtrate of plasma

21. Development of a Label Free Quantitation (LFQ) Platform
for quantifying peptides epitopes
from human lymph and other biological fluids
(Translational Research)
P1 P2 P3 P4 P5 P6 P7 Lymph peptidome: healthy
individual patients samples
LFQ (MS1-based area ratios)
PEAKS 7.0-analysis of equal amounts
of the extracted lymph peptidome (average of
3 replicates/patient).
Healthy Patients

22. Human Lymph Peptidome
????Degradome
Processing Pathways
of lymph peptidome
THE DEGRADOME
Experimentally-based
databases

23. cDC MHC-II Eluted Peptidome
degradome
Human Lymph Peptidome
degradome
Overall a wide variety of proteases were
implicated in the generation of the lymph-bound
peptidome, with matrix metalloproteases (MMPs)
and cathepsins being highly represented, followed
by caspases and enzymes involved in the innate
immune responses (angiotensin converting enzyme,
complement factor I, granzymes) and enzymes of
the coagulation cascade (thrombin,/plasmin,
kallikreins).
Roughly one third of the eluted peptides
from MHC-II were processed by cathepsins at
either the N or C terminus; another third were
cleaved at the N terminus by a signal peptidase or
another S26 homolog; with the remainder cleaved
by a metalloprotease, calpain, caspase, or other
protease.
Thus, it appears that many proteases besides
endosomal cathepsins can contribute to the
formation of the MHC II-bound proteome.

24. CONCLUSIONS
 Selected peptides from the human lymph were shown to have low nM binding affinity
for MHC-II DR1 molecules.
Extracellular protein processing can contribute to the overall MHC II presented
peptidome, expanding the peptide repertoire available for the induction of self-
tolerance.
The first correlated analysis of the human lymph and MHC-II peptidomes employing nanoLC
Orbitrap-ESI-MS/MS and bioinformatic analysis of corresponding degradomes.
Immunological significance of the self-peptidome
carried by the human lymph
 This analysis enabled the characterization of human lymph peptidome at high resolution and
reveals new peptides epitopes never characterized before this study.
 The bioinformatics analysis of the reported peptidomes highlights the diverse processing
pathways involved in the generation of the MHC II peptidome.
 The first reported degradome analysis of the human lymph peptidome: Several categories
of protease/peptidase in additional to those present in endosomal/lysosomal compartments
are involved in the formation of the lymph peptidome.
 More than 3000 MHC-II bound mouse peptides with over 1000 core epitopes identified.
 And many proteases from various non-lysosomal intracellular and extracellular compartments
contribute to the shaping of the MHC II peptidome.

25. Acknowledgements
Albert Einstein College of Medicine (AECOM)
Laura Santambrogio lab
Cristina C. Clement
Valerio Zolla
Lawrence Stern Lab UMASS
Aniuska Becerra
Scott Shafer
$$$ NIH
Mass spectrometry
Collection of data and “Raw files”
Edward Nieves (AECOM)
Fa-Yun Che (AECOM)
MS Bioworks