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UROPPoster2014

Remi Gai
Remi Gai

The goal of the project was to test a new methodology called C-terminal peptide analysis to identify antigens in immune complexes that cause vasculitis. Precipitation of immune complexes was optimized using keyhole limpet hemocyanin (KLH) with an antibody to KLH ratio of 1000/300 μg/ml at 4°C. Addition of rheumatoid factor increased antigen precipitation and an optimal ratio of 1000/300/300 μg/ml was found. Key steps of C-terminal peptide analysis including reduction, alkylation, and amine/carboxyl protection were tested on gels and shown to be successful, establishing the technique for identifying antigens in immune complexes and blood. This could help determine the specific causes of vas

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Remi GAI, Paul MONACH
School of Medicine
Study of the C-terminal Peptide Analysis Technique on Artificial immune Complexes
Vasculitis is a group of diseases that involve inflammatory destruction of blood vessels.
Several forms of vasculitis occur when immune complexes (binding of antibodies to
their target antigens) are not adequately cleared by immune cells, giving rise to an
inflammatory response and attraction of leukocytes (immune system cells), which
attack the blood vessels and surrounding tissues even in the absence of infection at the
site.
The first part of the research was to optimize the creation of artificial immune
complexes, by testing different factors on SDS-gels : the ratio of antibody/antigen, the
temperature and the addition of rheumatoid factor (anti-rabbit-IgG). Then, a
methodology originally developed for another context, called C-terminal peptide
analysis, will be used in this case to identify antigens in immune complexes. If the C-
terminal peptide analysis technique is found to be efficient for identifying antigens in
blood, finding the cause of vasculitis on patients would be a lot more easier and
specific.
Precipitation of KLH by anti-KLH antiserum was optimized at a ratio of 1000/300 µg/ml
(respectively antibody/antigen), and better at 4oC than at room temperature. Addition
of anti-rabbit-IgG allowed precipitation at lower concentrations, and also worked better
in the cold than in the room temperature. Success of individual steps from the C-
terminal protocol, including reduction and alkylation, amine protection, and carboxyl
protection, have been tested using ovalbumin and SDS and non-SDS gels.
Abstract :
a) Optimization of immune complexes : (tested on SDS gels)
1. Solubilize antigens into water.
2. Addition of antibodies.
3. Addition of Rheumatoid factors
4. Incubation at 4oC for 12h.
5. Centrifugation at 10,000 x rpm for 5 minutes. Keep pellet, discard supernatant
and wash with 2oo µl of PBS.
6. Repeat centrifugation and washing process two more times.
b) C-peptide enrichment : (each step was tested on SDS, Non SDS gels)
1. Reduction of disulfides and alkylation of cysteine residues, using dithiothreitol
(20mM) and iodoacetamide (5 – 10 mM ).
2. Protection of primary amines (N-terminus, lysine residues), using formaldehyde
(20mM) and NaCNBH3 (20mM).
3. Protection of Carboxyls (C-terminus, aspartate and glutamate residues), using
MES (200mM), ethanolamine (1.0 M), NHS (10mM) and EDC (20mM).
Methods :
Vasculitis is a condition that involves inflammation of the blood vessels. Inflammation
is a body’s response to injury, and may involve pain, redness, warmth, swelling and
loss of function in the affected tissues. Vasculitis, type III hypersensitivity, occurs
when immune complexes (binding of an antibody to an antigen) are not adequately
cleared by immune cells, giving rise to an inflammatory response and attraction of
leukocytes (immune system cells), which attack the blood vessels by mistake.
Vasculitis is divided into many types (affecting different part of skin and organs) and
can be the result of an infection, a medicine, another disease or condition. The
treatments are generally directed toward stopping the inflammation and suppressing
the immune system, but they are not always effective when the cause is not
determined. In order to find an efficient treatment, the specific immune complexes
causing the disease should be first identified, so that a medication can be directly
matched the cause.
The goal of this project is to test out a new methodology for identifying antigens in
immune complexes, called C-terminal peptide analysis. The technique will first be
established with standard protein, in order to determine the fidelity of the
identification. If the C-terminal peptide analysis is found to be efficient for identifying
antigens in blood, finding the cause of vasculitis on patients would be a lot more
easier and specific. With the identification of the immune complexes that cause
vasculitis, more targeted drug can be prescribed or created for the patients. This
methodology would also be very helpful for the study other diseases involving
immune complexes. If C-terminal peptide analysis is found to be ineffective, at least
the inefficiency of this new methodology would be proven. The technique could then
be modified or discarded in the future.
Introduction :
Results :
Acknowledgements :
Money were donated by patients, friends and relatives.
References :
Schilling O, Barré O, Huesgen PF, Overall CM. Proteome-wide analysis of protein
carboxy termini: C terminomics, Nature Methods 2010 ; 7:508
Schilling Oliver , Barré Olivier , Huesgen Pitter F. & Overall Christopher M.
ENRICHMENT OF NATURAL AND PROTEOLYTICALLY GENERATED
PROTEIN C–TERMINI ON A PROTEOME–WIDE SCALE, Protocol Exchange (2010)
doi:10.1038/nprot.2010.100
Since it has been proven that the addition of rheumatoid factor can increase
precipitation, it has the potential be used in blood samples as a way to purify some
specific immune complexes.
The protocol of C peptide technique is not complete, due to lack of equipment,
such as mass spectrometry. Future directions would be to go through the rest of
the protocol with trypsin digestion, second amine protection, polymer coupling,
filtration and analysis of C-terminal peptides through mass spectrometry.
The short-term goal of this project would be to improve the ability to analyze
circulating immune complexes, by selectively precipitating them from the blood
and by applying C-terminal peptidomic technique. The long-term goal would be to
discover causes of vasculitis by identifying antigens in immune complexes.
Future directions :
Precipitation of KLH immune complexes was optimized at a ratio of 1000/300/300
µg/ml (respectively antibody/antigen/rheumatoid factor) at 4oC. The addition of
rheumatoid factor has been proven useful to increase precipitations.
Different steps from the C-terminal protocol, including reduction and alkylation,
amine protection, and carboxyl protection, have been proven successful with SDS
and non-SDS gels.
Conclusions :
The results from figure 1 showed that addition of anti-rabbit-IgG allowed
precipitation at lower concentrations. With the presence of rheumatoid factor,
there was a significant increase in the amount of antigen, which can be seen from
the lower part of the gel. Indeed, rheumatoid factor would bind to the Fc portion of
the target antigen, thus improving it’s precipitation.
Results from the first part of the research showed that precipitation of KLH by
anti-KLH antiserum was optimized at a ratio of 1000/300 µg/ml (respectively
antibody/antigen), and better at 4oC than at room temperature.
From the optimal antibody/antigen ratio, rheumatoid factor was added in variable
amounts. Figure 2 demonstrated that the amount of antigen increased as the
amount of rheumatoid factor increased. The optimal ratio was found to be
1000/300/300 µg/ml (respectively antibody/antigen).
The success of C-peptide enrichment has been proven through SDS and non SDS
gels :
Reducing and alkylation (Figure 3.a) : The non SDS gel shows that the initial (non
reduced) band is on a lower position compared to the reduced ones in non SDS
and SDS gels, that are at the same level. Indeed, the initial KLH contains six
disulfide bonds, and has a more compact structure, therefore will run faster on the
gel. The reduced KLH are less compact without their disulfide bonds, therefore will
run slower on gel.
Protection of amines (Figure 3.b): The non SDS gel shows that KLH with protected
amines is on a lower position compared to the non protected band. This is
explained by the fact that KLH was more negatively charged because of the amine
protection process, therefore ran through the gel faster, towards the positive end.
The SDS gel didn’t show any relevant information since SDS denatures everything
then added uniform negative charges to all the proteins. Therefore it was expected
that amines protected or non protected would look the same on the SDS gel.
Protection of carboxyls (Figure 3.c) : The non SDS gel shows that KLH with
protected carboxyls is on a higher position compared to the non protected band.
This is explained by the fact that KLH was more positively charged because of the
carboxyls protection process, therefore ran slower since the other end of the gel
was also positive. The differences of the two bands on SDS gel could be the result
of chemical side reactions that either cleaved or combined some proteins after the
denaturation.
Figure 1 : SDS gel showing an increase of antigens with
the addition of RF.
Figure 2 : SDS gel showing the amount of antigens
through the addition of different ratio of RF.
Figure 3 : gels from different steps
of the C-peptide enrichment.
Figure 4 : C-peptide enrichment steps.
a)
b)
c)

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UROPPoster2014

  • 1. Remi GAI, Paul MONACH School of Medicine Study of the C-terminal Peptide Analysis Technique on Artificial immune Complexes Vasculitis is a group of diseases that involve inflammatory destruction of blood vessels. Several forms of vasculitis occur when immune complexes (binding of antibodies to their target antigens) are not adequately cleared by immune cells, giving rise to an inflammatory response and attraction of leukocytes (immune system cells), which attack the blood vessels and surrounding tissues even in the absence of infection at the site. The first part of the research was to optimize the creation of artificial immune complexes, by testing different factors on SDS-gels : the ratio of antibody/antigen, the temperature and the addition of rheumatoid factor (anti-rabbit-IgG). Then, a methodology originally developed for another context, called C-terminal peptide analysis, will be used in this case to identify antigens in immune complexes. If the C- terminal peptide analysis technique is found to be efficient for identifying antigens in blood, finding the cause of vasculitis on patients would be a lot more easier and specific. Precipitation of KLH by anti-KLH antiserum was optimized at a ratio of 1000/300 µg/ml (respectively antibody/antigen), and better at 4oC than at room temperature. Addition of anti-rabbit-IgG allowed precipitation at lower concentrations, and also worked better in the cold than in the room temperature. Success of individual steps from the C- terminal protocol, including reduction and alkylation, amine protection, and carboxyl protection, have been tested using ovalbumin and SDS and non-SDS gels. Abstract : a) Optimization of immune complexes : (tested on SDS gels) 1. Solubilize antigens into water. 2. Addition of antibodies. 3. Addition of Rheumatoid factors 4. Incubation at 4oC for 12h. 5. Centrifugation at 10,000 x rpm for 5 minutes. Keep pellet, discard supernatant and wash with 2oo µl of PBS. 6. Repeat centrifugation and washing process two more times. b) C-peptide enrichment : (each step was tested on SDS, Non SDS gels) 1. Reduction of disulfides and alkylation of cysteine residues, using dithiothreitol (20mM) and iodoacetamide (5 – 10 mM ). 2. Protection of primary amines (N-terminus, lysine residues), using formaldehyde (20mM) and NaCNBH3 (20mM). 3. Protection of Carboxyls (C-terminus, aspartate and glutamate residues), using MES (200mM), ethanolamine (1.0 M), NHS (10mM) and EDC (20mM). Methods : Vasculitis is a condition that involves inflammation of the blood vessels. Inflammation is a body’s response to injury, and may involve pain, redness, warmth, swelling and loss of function in the affected tissues. Vasculitis, type III hypersensitivity, occurs when immune complexes (binding of an antibody to an antigen) are not adequately cleared by immune cells, giving rise to an inflammatory response and attraction of leukocytes (immune system cells), which attack the blood vessels by mistake. Vasculitis is divided into many types (affecting different part of skin and organs) and can be the result of an infection, a medicine, another disease or condition. The treatments are generally directed toward stopping the inflammation and suppressing the immune system, but they are not always effective when the cause is not determined. In order to find an efficient treatment, the specific immune complexes causing the disease should be first identified, so that a medication can be directly matched the cause. The goal of this project is to test out a new methodology for identifying antigens in immune complexes, called C-terminal peptide analysis. The technique will first be established with standard protein, in order to determine the fidelity of the identification. If the C-terminal peptide analysis is found to be efficient for identifying antigens in blood, finding the cause of vasculitis on patients would be a lot more easier and specific. With the identification of the immune complexes that cause vasculitis, more targeted drug can be prescribed or created for the patients. This methodology would also be very helpful for the study other diseases involving immune complexes. If C-terminal peptide analysis is found to be ineffective, at least the inefficiency of this new methodology would be proven. The technique could then be modified or discarded in the future. Introduction : Results : Acknowledgements : Money were donated by patients, friends and relatives. References : Schilling O, Barré O, Huesgen PF, Overall CM. Proteome-wide analysis of protein carboxy termini: C terminomics, Nature Methods 2010 ; 7:508 Schilling Oliver , Barré Olivier , Huesgen Pitter F. & Overall Christopher M. ENRICHMENT OF NATURAL AND PROTEOLYTICALLY GENERATED PROTEIN C–TERMINI ON A PROTEOME–WIDE SCALE, Protocol Exchange (2010) doi:10.1038/nprot.2010.100 Since it has been proven that the addition of rheumatoid factor can increase precipitation, it has the potential be used in blood samples as a way to purify some specific immune complexes. The protocol of C peptide technique is not complete, due to lack of equipment, such as mass spectrometry. Future directions would be to go through the rest of the protocol with trypsin digestion, second amine protection, polymer coupling, filtration and analysis of C-terminal peptides through mass spectrometry. The short-term goal of this project would be to improve the ability to analyze circulating immune complexes, by selectively precipitating them from the blood and by applying C-terminal peptidomic technique. The long-term goal would be to discover causes of vasculitis by identifying antigens in immune complexes. Future directions : Precipitation of KLH immune complexes was optimized at a ratio of 1000/300/300 µg/ml (respectively antibody/antigen/rheumatoid factor) at 4oC. The addition of rheumatoid factor has been proven useful to increase precipitations. Different steps from the C-terminal protocol, including reduction and alkylation, amine protection, and carboxyl protection, have been proven successful with SDS and non-SDS gels. Conclusions : The results from figure 1 showed that addition of anti-rabbit-IgG allowed precipitation at lower concentrations. With the presence of rheumatoid factor, there was a significant increase in the amount of antigen, which can be seen from the lower part of the gel. Indeed, rheumatoid factor would bind to the Fc portion of the target antigen, thus improving it’s precipitation. Results from the first part of the research showed that precipitation of KLH by anti-KLH antiserum was optimized at a ratio of 1000/300 µg/ml (respectively antibody/antigen), and better at 4oC than at room temperature. From the optimal antibody/antigen ratio, rheumatoid factor was added in variable amounts. Figure 2 demonstrated that the amount of antigen increased as the amount of rheumatoid factor increased. The optimal ratio was found to be 1000/300/300 µg/ml (respectively antibody/antigen). The success of C-peptide enrichment has been proven through SDS and non SDS gels : Reducing and alkylation (Figure 3.a) : The non SDS gel shows that the initial (non reduced) band is on a lower position compared to the reduced ones in non SDS and SDS gels, that are at the same level. Indeed, the initial KLH contains six disulfide bonds, and has a more compact structure, therefore will run faster on the gel. The reduced KLH are less compact without their disulfide bonds, therefore will run slower on gel. Protection of amines (Figure 3.b): The non SDS gel shows that KLH with protected amines is on a lower position compared to the non protected band. This is explained by the fact that KLH was more negatively charged because of the amine protection process, therefore ran through the gel faster, towards the positive end. The SDS gel didn’t show any relevant information since SDS denatures everything then added uniform negative charges to all the proteins. Therefore it was expected that amines protected or non protected would look the same on the SDS gel. Protection of carboxyls (Figure 3.c) : The non SDS gel shows that KLH with protected carboxyls is on a higher position compared to the non protected band. This is explained by the fact that KLH was more positively charged because of the carboxyls protection process, therefore ran slower since the other end of the gel was also positive. The differences of the two bands on SDS gel could be the result of chemical side reactions that either cleaved or combined some proteins after the denaturation. Figure 1 : SDS gel showing an increase of antigens with the addition of RF. Figure 2 : SDS gel showing the amount of antigens through the addition of different ratio of RF. Figure 3 : gels from different steps of the C-peptide enrichment. Figure 4 : C-peptide enrichment steps. a) b) c)

Editor's Notes

  1. References-St. Johnston 2002 for old GAL4 image 1) Pritchett, T., Tanner, E., McCall, K. (2009) Cracking open cell death in the Drosophila ovary. Apoptosis 14:969-979 2) Spradling, A.C. Developmental genetics of oogenesis. in The Development of Drosophila melanogaster, Vol. I (eds. Bate, M. & Martinez Arias, A.) 1-70 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993). 3) Logan MA, Freeman MR. 2007. The scoop on the fly brain: glial engulfment functions in Drosophila. Neuron Glia Biol.1:63-74. 4) St. Johnston D. (2002) The art and design of genetic screens: Drosophila melanogaster. Nature Reviews Genetics 3: 176-188. Spradling images Image info from from Beckman lunch talk Brightened greyscale c and d and pg150 GFP on the printing desktop-should do on this version as well.-did 6% each Now has correct BU logo from Pete Lyso images: Confocal 091312lysoPG150MCD8_11 in St12, 091812lysoPG150MCD8_05 in St13