LYMPH-CARRIED SELF- PEPTIDOME DERIVES FROM A VARIETY OF PROCESSING ENZYMES

AND CONTRIBUTES TO THE DENDRITIC CELLS MHC II PEPTIDOME

Cristina C. Clement Aniuska Becerra Scott Shafer Lawrence J. Stern and Laura Santambrogio

June 4th 2015

“Peptidome Pattern Diagnostic”

healthy vs.cancer; autoimmne diseases

Peptidome/Degradome in Biological Fluids

“Peptidome Atlas”

RA JRA OA

PROTEOME PROTEASES

and DEGRADOME SUBSTRATES

Activation Inhibition

Degradation PEPTIDOME

MW>8-10 kDa MW<8-10 kDa

Lymph Lymph node

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.

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.

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

COLLECTION OF HUMAN PRE- NODAL LYMPH

For Peptidomics and Proteomics Forty-six lymph vessel cannulations were carried out under full sterile conditions. An horizontal incision 15;–20 mm wide was made 6–10 cm above the ankle. With the aid of an operating microscope (model M650; Wild Heerbrugg, Glattbrugg, Switzerland), the local subcutaneous lymph vessels were dissected, and one was selected for cannulation. The lymph vessel was ligated with silk (Mersilk, 5/0), and then opened distal to the ligature. The cannula was inserted into the vessel for 5–10 mm (toward the foot), and secured with a silk ligature (Mersilk, 5/0). The other (untapered) end of the cannula was passed through a hole in the top of a graduated 2-mL screw-topped

polypropylene cryovial containing 2 mg of Na2EDTA. Collection was carried out for 3-6 hours. Plasma was collected from the same individuals..

MHC-II are highly polymorphic

CD4+T cell repertoire is restricted to processed and MHC-II presented peptide

Immunologically relevant Peptides Epitopes

Enough antigen amount

(pM-µM)

Relevant MHC binding affinity <50 nM strong binding >50 nM weak binding

TCR repertoire available

TOLERANCE OR AUTOIMMUNITY?!

THE MHC-II bound peptidome DC (other APC)

CD4+T cell

Protein Autoimmune disease

Collagen I anterior uveitis, scleritis, thromboangiitis obliterans, thyroid-associated ophthalmopathy

Collagen II RA, otosclerosis, auricular chondritis Collagen III , thromboangiitis obliterans, thyroid-associated ophthalmopathy Collagen IV Goodpasture's (alpha3 chain), GN, systemic sclerosis, Alport syndrome (alpha5

chain), thyroid-associated ophthalmopathy Collagen V thyroid-associated ophthalmopathy Collagen VI Cogan's syndrome (high in neural CT) Collagen VII epidermolysis bullosa acquisita + bullous systemic lupus erythematosus Collagen IX otosclerosis Collagen XI RA Collagen XVII Pemphigoid gestationis, mucus membrane pemphigoid, bullous pemphigus Collagen XVIII Cleaved to endostatin, involved in regulating proliferation laminin thyroid-associated ophthalmopathy, SLE (alpha 1), diabetic microangiopathy Fibrosin Fxn = modulates expression of myofibroblasts (wound heal, fibrosis)

Alcohol-induced fibrosis, scleroderma Cartilage intermediate layer protein

OA, RA, lumbar disc disease

Junctophilin I disease, hypertrophic cardiomyopathy Cartilage derived morphogenic protein I

OA

Adlecan ECM remodeling in chronic CAD Mucin Asthma, allograph rejection Aggrecan RA, juvenile idiopathic arthritis Desmoglein 1 pemphigus foliaccus (Pemphigus vulgaris = desmoglein 3) Matrix GIA protein Dental caries (prevents mineralization)

Vit K dependent prevents vascular Ca2+ Fibronectin thyroid-associated ophthalmopathy, SLE, glomerular diseases, vasculitis

TOLERANCE OR AUTOIMMUNITY?!

DR1-haplotype

Lymph-carried self- peptidome Extraction, fractionation and top-down sequencing by nLC MS/MS on Orbitrap. Analysis of the molecular function and cellular pathways associated with the self-peptidome carried by the human lymph. Degradome analysis: antigen processing pathways. 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/fluorescence polarization assay).

Two PEPTIDOMES

Dendritic cells

The peptidome displayed by MHC-II DR1 Elution of peptides from MHC-II-DR1, fractionation and top-down sequencing by nLC MS/MS on Orbitrap/Q-exactive. Analysis of the molecular function and cellular pathways characterizing the self-peptidome displayed by MHC-II DR-1. Degradome analysis: antigen processing pathways for the peptidome displayed by MHC-II DR-1 molecules. Functional validation of the immunological relevant peptidome displayed by MHC-II by assessing its affinity for MHC-II DR1-molecules.

Clement CC et al. L. Santambrogio lab Aniuska Becerra Lawrence J. Stern lab

Lymph Self-Peptidome Sequenced at low resolution by nLC-LTQ ion trap

Clement CC et al. PLoS One 2010

LII_8 #8633 RT: 50.32 AV: 1 NL: 1.31E6T: ITMS + c NSI Full ms [ 300.00-1800.00]

400 600 800 1000 1200 1400 1600 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

585.02

602.30

679.86

548.18

311.34

741.29

875.54455.48

820.45

935.39368.10 1321.741033.601168.65 1458.64 1614.92 1749.44959.19

A (2

80 n

m) Peptidome

FPLC

Lymph Self-Peptidome Sequenced at low resolution by nLC-LTQ ion trap

Molecular and cellular functions

Clement et al. PLoS One 2010

Analysis of more then 300 sequences from the healthy human lymph identified self-peptides derived from both intracellular and extracellular proteins. The peptidome of the human lymph reflects the variety of catabolic products

transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration.

Empty HLA-DR1

Analysis of HLA-DR1 and HLA-DR4 binding affinity

peptide loaded HLA-DR1

competition ELISA displacement assays with recombinant MHC-II molecules

Immunologically relevant Peptides Epitopes

Enough antigen amount

Relevant MHC binding affinity

TCR repertoire

The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome displayed by MHC-II, generated in endosomal compartment. The peptidome carried by the lymph expands the tissue-specific self-repertoire available for the maintenance of immunological tolerance.

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

A (

28

0 n

m)

Mice (DR1+)

Transgenic mice (humanized) for MHC-II-DR-1

Expand the number of conventional DC (cDC: CD11c+)

HLA-DR1 molecules purification by immuno-affinity chromatography using anti-MHC-II (DR1) antibodies.

Protein A/G

Purified human MHC-II-DR1 with mouse displayed peptides

Acid elution of the peptides with TFA 0.1 % (5 minutes, RT)

Nano LC –MS/MS LTQ/Orbitrap/Q exactive

MS/MS-searched against Mouse database (Swiss Prot) MASCOT and PEAKS 7.0 (searching engines)

“NO ENZYME” restriction

Human MHC-II DR1

SEQUENCING THE MHC-II PEPTIDOME high energy collision dissociation (HCD), nLC-MS/MS on Orbitrap/Qexactive

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 8Time (min)

64.13

60.47

72.22

59.78

59.06

45.86 67.55

41.9557.84

53.68

73.2041.5148.96 76.0738.75

80.2935.990.49 13.48 26.3022.5519.6811.51 28.547.07

Lymph FPLC Fraction 2

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 8Time (min)

71.80

71.91

44.76

72.3772.98

46.54

63.76

73.0863.5773.29

42.99 73.49

62.5473.78

59.5940.64 47.94 64.99 74.4656.2374.8955.02 79.97

40.1435.9211.900.43 13.987.63 22.88 26.31

Lymph FPLC Fraction 1

0 90 min

Rela

tive

abun

danc

e

0

100%

100%

Representative MS1 base peaks chromatograms: Lymph peptidome

90 min

Representative MS1 base peaks chromatograms: eluted peptidome from MHC-II DR1

minutes 0 190

DE novo sequencing and database search analysis of the lymph and MHC-II eluted peptidomes

The PEAKS Studio workflow including de novo sequencing of endogenous peptides

Representative 2D-map m/z vs time for a lymph peptidome fraction

identified features (m/z vs RT) PEAKS DB identified MS/MS

m/z

RT

(m

in)

Representative 2D-map m/z vs time for MHC-II eluted peptidome fraction

60

50

40

30

200

150

100

50

0 200 1500 200 1500 m/z

Novel peptide? PTM? Mutations?

Novel peptide? PTM? Mutations?

FDR <0.5%

Representative PEAKS 7.0 output for a FPLC-lymph peptidome fraction

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

Representative processing of a self-antigen found in the human lymph peptidome: fibrinogen alpha derived endogenous peptides

Representative processing of a self-antigen found in the human lymph peptidome: complement C3 derived endogenous peptides

Oncostatin-M-specific Receptor subunit beta

Glyceraldehyde-3-phosphate dehydrogenase

HCD Human lymph peptidome

Elongation factor 1-alpha 1 HCD

Chromosomal protein HMG 17

Alpha-1-antitrypsin

Pigment epithelium-derived factor

ETD

Histone H1.4

Histone H2B type 1

HCD

ETD

Polymeric immunoglobulin receptor

Apolipoprotein A1

HCD

Fibrinogen alpha chain

Kininogen 1

HCD

Macrophage migration inhibitory factor

Triosephosphate isomerase

HCD

Proteoglycan 4

Collagen alpha-1 (I)

HCD

Cytochrome c oxidase subunit 5A

Malate dehydrogenase, mitochondrial

HCD

Splicing factor 3A subunit-2

Solute carrier organic anion transporter family member 6A1

HCD

Insulin-like growth factor 1 receptor

Transmembrane glycoprotein NMB

HCD

Collagen alpha-1(XV)

Complement C3

HCD

Acute phase response signaling

Major biochemical pathways: the human lymph peptidome

Lymph-filtrate of plasma

Coagulation system

Major biochemical pathways: the human lymph peptidome

Lymph-filtrate of plasma

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

Validation of the proteins generating the human lymph peptidome A proteomics approach

Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS-PAGE coupled with nanoLC-ESI-MS/MS bottom-up proteomics. Clement CC, Aphkhazava D, Nieves E, Callaway M, Olszewski W, Rotzschke O, Santambrogio L. J Proteomics. 2013 Jan 14;78:172-87.

MHC-II are highly polymorphic

CD4+T cell repertoire is restricted to processed and MHC-II presented peptide

Immunologically relevant Peptides Epitopes

Enough antigen amount

(pM-µM)

Relevant MHC binding affinity <50 nM strong binding >50 nM weak binding

TCR repertoire available

TOLERANCE OR AUTOIMMUNITY?!

THE MHC-II bound peptidome DC (other APC)

CD4+T cell

Representative PEAKS 7.0 output for a MHC-II eluted peptidome

FDR <0.5%

Representative processing of a mouse self-antigen displayed by the human MHC-II DR1 molecule

Deoxyribonuclease gamma processing in the mice DR1+

m/z=758.9308, z=2, RT=114.28, -lgP=33.69, ppm=-0.7 m/z=766.9274, z=2, RT=100.52, -lgP=32.31, ppm=-2.0

Complement C3 processing in mouse (DR1+)

m/z=705.3583, z=2, RT=109.95 -lgP=33.60, ppm=2.4 m/z=631.8220, z=2, RT=92.23 -lgP=27.81, ppm=-0.6

MHC-II antigen: alpha chain processing in mouse (DR1 (+)

cDC MHC-II Eluted Peptidome

3216 peptides, from 688 proteins FDR <0. 1% p 0.004

Muixi; Genes Immunol. 2011 Bozzacco; J. Prot. Res. 2011 Fissolo; Mol. Cell Prot. 2009 Rohn; Nat. Immunol. 2004 Adamopoulou; Nat. Comms 2013 Costantino; PlosOne 2012 Haeri; Immunogenetics 2005 Rudensky; Nature 1991 Vogt; J. Immunol. 1994 Strug; J. Prot. Res. 2009

cDC MHC-II Eluted Peptidome

Aniuska Becerra Lawrence J. Stern lab, this reported research

Complement system

Major biochemical pathways associated with the eluted peptidome from MHC-II-DR1 molecules

Major biochemical pathways associated with the eluted peptidome from MHC-II-DR1 molecules

Antigen-Presentation Pathway

Clathrin-mediated endocytosis signaling

Major biochemical pathways associated with the eluted peptidome from MHC-II-DR1 molecules

Processing Pathways of Lymph and HLA-DR1-eluted peptidome

(The degradomes)

Experimentally-based databases

Enzymes involved in Endosomal Processing (The degradome) Thiol-reductase: GILT Cathepsins: S L F B D Aminopeptidases

Antigen processing within the endosomal compartments of DCs

MVB

MVB

Brian Scharf, Dr. Santambrogio lab

Generate the MHC-II bound peptidome

Human Lymph Peptidome/degradome

O60814|H2B1K_HUMAN (100%), 13,890.6 DaO60814|H2B1K_HUMAN5 exclusive unique peptides, 6 exclusive unique spectra, 43 total spectra, 42/126 amino acids (33% coverage)

M P E P A K S A P A P K K G S K K A V T K A Q K K D G K K R K R S R K E S Y S V Y V Y K V L K Q V H P D T G I S S K A MG I M N S F V N D I F E R I A G E A S R L A H Y N K R S T I T S R E I Q T A V R L L L P G E L A K H A V S E G T K A V TK Y T S A K

Q93079|H2B1H_HUMAN (100%), 13,892.6 DaQ93079|H2B1H_HUMAN2 exclusive unique peptides, 3 exclusive unique spectra, 24 total spectra, 54/126 amino acids (43% coverage)

M P D P A K S A P A P K K G S K K A V T K A Q K K D G K K R K R S R K E S Y S V Y V Y K V L K Q V H P D T G I S S K A MG I M N S F V N D I F E R I A G E A S R L A H Y N K R S T I T S R E I Q T A V R L L L P G E L A K H A V S E G T K A V TK Y T S S K

P10853|H2B1F_MOUSE (100%), 13,936.6 DaP10853|H2B1F_MOUSE3 exclusive unique peptides, 3 exclusive unique spectra, 7 total spectra, 15/126 amino acids (12% coverage)

M P E P A K S A P A P K K G S K K A V T K A Q K K D G K K R K R S R K E S Y S V Y V Y K V L K Q V H P D T G I S S K A MG I M N S F V N D I F E R I A S E A S R L A H Y N K R S T I T S R E I Q T A V R L L L P G E L A K H A V S E G T K A V TK Y T S S K

P10854|H2B1M_MOUSE (100%), 13,936.6 DaP10854|H2B1M_MOUSE3 exclusive unique peptides, 3 exclusive unique spectra, 6 total spectra, 14/126 amino acids (11% coverage)

M P E P T K S A P A P K K G S K K A V T K A Q K K D G K K R K R S R K E S Y S V Y V Y K V L K Q V H P D T G I S S K A MG I M N S F V N D I F E R I A G E A S R L A H Y N K R S T I T S R E I Q T A V R L L L P G E L A K H A V S E G T K A V TK Y T S S K

Human H2B histone

Mouse H2B histone

Differential processing of self antigens Comparison of human lymph peptidome with MHC-II eluted peptidome

P02647|APOA1_HUMAN (100%), 30,778.5 DaP02647|APOA1_HUMAN12 exclusive unique peptides, 18 exclusive unique spectra, 29 total spectra, 18/267 amino acids (7% coverage)

M K A A V L T L A V L F L T G S Q A R H F W Q Q D E P P Q S P W D R V K D L A T V Y V D V L K D S G R D Y V S Q F E G SA L G K Q L N L K L L D N W D S V T S T F S K L R E Q L G P V T Q E F W D N L E K E T E G L R Q E M S K D L E E V K A KV Q P Y L D D F Q K K W Q E E M E L Y R Q K V E P L R A E L Q E G A R Q K L H E L Q E K L S P L G E E M R D R A R A H VD A L R T H L A P Y S D E L R Q R L A A R L E A L K E N G G A R L A E Y H A K A T E H L S T L S E K A K P A L E D L R QG L L P V L E S F K V S F L S A L E E Y T K K L N T Q

Q00623|APOA1_MOUSE (100%), 30,587.4 DaQ00623|APOA1_MOUSE7 exclusive unique peptides, 7 exclusive unique spectra, 11 total spectra, 36/264 amino acids (14% coverage)

M K A V V L A V A L V F L T G S Q A W H V W Q Q D E P Q S Q W D K V K D F A N V Y V D A V K D S G R D Y V S Q F E S S SL G Q Q L N L N L L E N W D T L G S T V S Q L Q E R L G P L T R D F W D N L E K E T D W V R Q E M N K D L E E V K Q K VQ P Y L D E F Q K K W K E D V E L Y R Q K V A P L G A E L Q E S A R Q K L Q E L Q G R L S P V A E E F R D R M R T H V DS L R T Q L A P H S E Q M R E S L A Q R L A E L K S N P T L N E Y H T R A K T H L K T L G E K A R P A L E D L R H S L MP M L E T L K T K A Q S V I D K A S E T L T A Q

Human Apolipoprotein A1

Mouse Apolipoprotein A1

Differential processing of self antigens Comparison of human lymph peptidome with MHC-II eluted peptidome

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.

C3a C3b C3 convertase Kallikrein Factor I Factor I Factor I

LE rCathepsins

70 57 142

recombinant-Cathepsins B, D, L, S

C3 Processing: Enzymatic and cell free assays for validation of peptides epitopes derived from processing by late endosomes (LE) and cathepsins

RT: 0.00 - 85.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 8Time (min)

0

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35

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45

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65

70

75

80

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100

Relati

ve Ab

undanc

e

61.68

72.6772.98

60.8673.4138.87 72.2231.08

64.4538.72 73.71

33.0659.7339.24 45.2937.1025.64 74.3770.0445.5145.1426.07 59.42

75.1975.4175.7039.83 55.3323.72 46.03 81.612.10 1.97 22.61

7.68 14.24

LE+C3 (recombinant) 4 hours

RT: 0.00 - 85.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relativ

e Abun

dance

61.57

33.06

60.79

27.1073.1730.85 59.60

63.5772.69 73.60

64.0159.39 72.3739.77 59.13 74.5637.11 66.10

75.2339.99 43.9025.9923.55 75.45

58.6554.76 75.7548.5620.732.16 76.41 82.804.89 9.80 13.30

Cathepsins+C3 (recombinant) 4 hours

C3 peptides

Comparison between the cDC, MHC II-eluted, and lymph peptidome

,B, D

P9

scor

e

nM

DC-Eluted

P9

scor

e

nM

Lymph

Affinity of peptides for DRB1*0101 MHC-II-prediction DC-eluted peptides and lymph-identified peptides

P9 Algorithm Net MHCII PanServer

The immunological significance of the human lymph peptidome

NetMHCIIpan 3.0 server can produce predictions for peptides of 9 - 19 amino acids in length.

http://www.cbs.dtu.dk/services/NetMHCIIpan/

Empty HLA-DR1

Analysis of HLA-DR1 and HLA-DR4 binding affinity

peptide loaded HLA-DR1

Competition ELISA displacement assays with recombinant MHC-II molecules

Immunologically relevant Peptides Epitopes

Enough antigen amount

Relevant MHC binding affinity

TCR repertoire

IPMYSIITPNVLRL

∆IC50= 0 IC50= 0.03 ± 0.01 µM

SSKITHRIHWESASLLR

∆IC50= 0.06 µM IC50= 0.07 ± 0.01 µM

EEFGRFASFEAQGALA

∆IC50= 0.07 µM IC50= 0.14 ± 0.04 µM

ASFEAQGALANIAVDKA

∆IC50= 3.40 µM IC50= 1.71 ± 0.28 µM

Complement Protein C3 MHC-II a chain Lymph MHC-II-eluted Lymph MHC-II-eluted

Dr. Lawrence Stern, UMASS Clement CC et al. PLoS One 2010

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).

Development of a Label Free Quantitation (LFQ) Platform for quantifying peptides epitopes

eluted from MHC-II-DR molecules under different physiological states

3 independent peptidome elutions from MHC-II

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 reproted 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: 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.

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