Proteome screening

for macromolecular complexes

in molecular medicine

DETECTprotein binding pair-

Analytical detection by high throughput screen

Conditions can be varied simply, cheaply

CONFIRMmolecular interaction at the structural level-

Biochemical characterization by IP / MS

ESTABLISH biological significance-

Confirmed by the identification of intracellular interactions

that are crosslinked in vivo,

then characterized as above

DETECTprotein binding pair-

Analytical detection by high throughput screen

Conditions can be varied simply, cheaply

Part I

C1qC1r2C1s2

IgG

Schematic of C1 binding to an

immune complex

C1

C1=C1q+2C1s+2C1r

EM of C1q v. C1 reveals

coronal viewsagittal view

C1q

C1

C1

C1q

Suggestion– there is a modification to a well-established

Q-ELISA format, that would allow for a screen for interacting

proteins. First, recall a Quantitative ELISA format -

Trap protein of interest with a monoclonal AB, allow-

ingthe protein to be uniformly oriented and mostly free

Bathe the mAB with your physiological fluid or

culture isolate of choice

Detect protein of interest with a polyclonal AB, allowing the

protein to be bound with multiple AB molecules to optimize

the signal from biotin

Detect the protein of interest with sensitive laser and

~irreversible formation of signal

Suggestion–

using this same Q-ELISA based

format, with the monoclonal

capture antibody, and a 2nd

detection antibody, one can

change the detection antibody to

an AB (Layer 3) that recognizes

the interaction partner to identify

the macromolecular complex,

and characterize the conditions

under which the complex is

present or absent.

Uses standard clinical assay Q-ELISA based instrumentation,

developed for high throughput, minimal sample volume, has

been verified by numerous assays for several antigens, by

commercial kit or customized design

Kits already in use

Apoptosis

Signal transduction, AKT pathway

Acute phase response panel

Inflammation panel

Death receptor pathway

Growth factors

Lipoprotein panel, cardiac evaluation

•mAB to lipoproteins for the capture step

•pAB –biotin for the detection step

•Standardized control apolipoprotein (conc. known)

•Construct beads with mAB attached

•Run the assay using the standardized control

apolipoprotein to confirm that the reagents are working as

expected.

•Run the assay using unknown sample, and control sample

for the standard curve.

Detection of protein or protein complex is determined by

the selection of trapping and detecting antibody pairs

*Quantitative difference likely due to

(1) Change in C1q level due to loss associated with blood clotting

and clot removal from the serum, or

(2) an increase in accessibility of the C1q epitopes when the protein

is free (plasma) v. in complex, (serum), since C1s, C1r coat the 6

arms of the C1q molecule.

Quantization of C1s is similar for free v. complexed protein

C1s is free in plasma, in the C1 complex in serum

Single component detection

***Since the C1s concentration does not

change appreciably between serum and plasma, it is likely that

(2) is the reason for the change in C1q concentration.***

Examples for the components of C1 and the C1 complex of 3 proteins

Detection

Capture /

trapping

Information

Yield

Q S R

Q S R

Q QQ

C1q C1 C1

Examples for the components of C1 and the C1 complex of 3 proteins

Detection

Capture /trapping

Information Yield

Q S R

Q S R

S SS

C1 C1s C1r2C1s2

Part II

CONFIRMmolecular interaction at the structural level-

Biochemical characterization by IP / MS

Biochemical proteomics

Further molecular analysis at the biochemical level-

1. magnetic beads to capture the immune complex,

2. followed by further screening, including MS

AB-AG

Part III

ESTABLISH biological significance-

Confirmed by the identification of intracellular interactions that

are crosslinked in vivo, then characterized as above, Part I, II

Intracellular ID of complex, using biosynthetically labeled proteins with highly

reactive and nonspecific AA chemistry, either diazirine- M, or L, or acetylene-M.

Protein – protein interactions that change to initiate, or, as a result of, signal transduction

-using physiological conditions, screen for signal - specific interactions-

such as ligand binding, metal binding, oxidative stress, Calcium concentration,

low Oxygen tension, other pathway signals, that will change on signal transduction,

or with the onset of pathophysiology.

Document the correlation between signal and the protein – protein interaction in question.

Determine the physical factors that allow optimal complex formation, rapidly,

with a minimal amount of material, and under a range of conditions.

1. Screen recombinant proteins v. endogenous, native proteins.

2. Determine if an antibody that is generated to a peptide, can then

recognize the folded domain or protein.

3. Screen proteins that have been altered by engineering,

mutation, or chemical changes

4. Optimize conditions to study the interaction of interest.

It is possible to use a tag antibody, such as anti-His, to produce

a general screen for protein – protein interactions where one

partner is not known. Furthermore, it is possible to use an

antibody that targets a specific chemical modification, such as

biotin, thereby allowing for screening of a mixture of unknown

proteins for either the capture or detection sample.

Part I1. Assay format (96 well plate, complex formation on beads, streptavidin-biotin detection, laser

detection, single bead analysis) is well established. Adequate technical support is available.

2. Assay design has been validated on a complex that is well understood, from both a physiologic

standpoint, and a biophysical standpoint.

3. Assay format is suitable for complex mixtures, in a physiologic background.

4. Assay design is viable for proteins that are minor components of the protein milieu.

Part IIThis aspect of the program is in place to allow biochemical and biophysical confirmation of

complex formation. It capitalizes on the optimization screening that is carried out in Part I, by

using a similar format (immunocapture on beads), but in the larger scales required for

biochemical and biophysical analysis, in comparison to laser based fluorescence detection.

Part III1. Familiarity with the nuances of crosslink formation in proteins is in place from previous work.

2. Cell biology is in place from previous work.

3. Assays from Part I can be used to screen for crosslink formation, again requiring small

amounts of material for reliable screening, to compensate for possible low yields @ the

crosslink and harvesting steps.

4. Methods do not require radioactivity.

Acknowledgements

Solomon Murphy, chief cook and bead washer

Seok J Hong, Gregory M Landes, Moral support

Arthur Sands, CEO, Lexicon Pharmaceuticals