Hermanson G. Bioconjugate Techniques, Second Edition

Подождите немного. Документ загружается.

1010 28. Bioconjugation in the Study of Protein Interactions

Protocol

1. Dissolve the protein(s) to be studied in 20 mM HEPES buffer, pH 7.5, at a concentration

of 5–10  M.

2. Dissolve together in one solution the desired heavy and light crosslinker analogs in dry

DMSO at an equal concentration of up to 100 mM. Use either the pair BS

G-d

/BS

G-d

or BS

G-d

/BS

G-d

, but do not mix the different sized crosslinkers together. The heavy

and light analogs of the same type should always be dissolved at equivalent concentra-

tions in DMSO to prepare the stock solution.

3. Add a quantity of the crosslinker solution to the protein solution to obtain at least a

10-fold molar excess of the crosslinkers over the concentration of the protein. Studies

should be done at several levels of crosslinker addition to determine the optimal conjuga-

tion conditions (i.e., 10-, 50-, 100-, and 200-fold excess).

4. Quench the reaction by the addition of NH

HCO

to a ﬁ nal concentration of 20 mM.

The optimal time course for the reaction should be determined by removing portions

of the solution at different points, starting at about 5–10 minutes and extending out to

2 hours in length.

5. Analyze the quenched reaction by SDS electrophoresis, Western blotting, and mass spec

analysis.

1.3. Formaldehyde

By Far the simplest bifunctional crosslinking agent is formaldehyde. Although structurally a

mono-functional aldehyde compound, formaldehyde reacts with proteins via a two-step reaction

Figure 28.4 Formaldehyde can be used to capture protein interactions if it is used at low concentrations. The

reaction proceeds through modiﬁ cation of a protein to create an intermediate immonium cation, which then

goes on to react with a neighboring protein to form the crosslinked product via secondary amine bonds.

to yield a methylene bridge crosslink between two amines on proteins and other molecules, thus

it behaves as though it is bifunctional in nature ( Figure 28.4 ). The reaction of formaldehyde

with proteins is rapid and efﬁ cient, and it has long been used to ﬁ x cells and tissue samples

for preservation, staining, and probing. Formaldehyde quickly penetrates cells and at the right

concentration, results in locking biomolecules in place, preventing diffusion, morphological

changes, or loss of proteins through solubilization .

Formaldehyde also can be used at limiting concentrations to crosslink interacting proteins,

while avoiding the extensive global crosslinking typically obtained when ﬁ xing cells. At rela-

tively low concentrations, formaldehyde will link together only those proteins and other amine-

containing molecules within proximity to one another and presumably, therefore, undergoing

speciﬁ c biological interactions (Prossnitz et al., 1988; Skare et al., 1993; Derouiche et al., 1995;

Orlando et al ., 1997; Orlando, 2000; Hall and Struhl, 2002; Vasilescu et al ., 2004).

Guerrero et al. (2006) used this technique along with the quantitative mass spec strategy

called SILAC (stable isotope labeling of amino acids in cell culture; Ong et al., 2002) to iden-

tify the yeast proteins that interact with the 26 S proteasome.

The following protocol is a generalized method that summarizes the publications on the use

of formaldehyde for capturing interaction proteins. The ranges indicated for concentrations of

reactants and time of the reaction need to be optimized for each protein interaction studied.

Protocol

1. Grow cells and wash into ice-cold 10 mM PBS buffer, pH 6.8.

2. Add formaldehyde to a ﬁ nal concentration of 0.125–1 percent (w/w) (optimize to ﬁ nd

the best concentration level for the particular protein being studied).

3. React at room temperature to 37°C for 5–60 minutes (optimize to ﬁ nd the best reaction

time for the protein being studied).

4. Wash cells and solubilize pellet in SDS-PAGE sample buffer.

5. Heat at 37°C for 10 minutes to fully solubilize and maintain crosslinked proteins, and

then enrich speciﬁ c complexes by immunoprecipitation using an immobilized antibody

speciﬁ c for the bait protein that was used. Alternatively, heat at 96°C for 20 minutes to

solubilize and break all crosslinks (this may be used as a control).

6. Analyze interacting proteins by electrophoresis, Western blotting, and mass spectrometry.

1.4. Protein Interaction Reporters

Standard homobifunctional crosslinkers, such as those described in the previous sections, can

capture protein interactions effectively through covalent linkages, but they create severe chal-

lenges for analyzing exactly what proteins have been conjugated. This is not just the result of

the large number of protein interacting partners that get crosslinked; it is also a result of the

wide variety of products that can form from the process, including side reactions, nonspeciﬁ c

conjugations, and crosslinkers that only reacted with one protein. Attempting to deconvolute

the identity of true protein interacting complexes created by large-scale crosslinking of complex

samples is the major problem of all conjugation techniques used to study protein interactions.

A new type of crosslinking strategy may overcome this problem, as it takes advantage of

deﬁ nitive mass spec identiﬁ cation after the chemical crosslinking of interacting proteins. The

1. Homobifunctional Crosslinking Agents 1011

1012 28. Bioconjugation in the Study of Protein Interactions

protein interaction reporter (PIR) reagent as described by Tang et al. (2005) is based on a

bifunctional crosslinker concept, but having the additional feature of containing two mass spec

cleavable bonds within a specially designed spacer arm. The cleavable parts of the molecule

consist of two RINK groups, which are amide-releasing, acid-cleavable components based

on the solid phase peptide synthesis resin ﬁ rst described by Rink (1987), and containing the

4-(2 ,4 -dimethoxyphenylaminomethyl)phenoxy linker on each side of a central mass reporter

tag ( Figure 28.5 ). On both ends of the PIR compound, NHS esters provide amine reactivity to

capture any interacting proteins in a complex sample, such as within a cell or lysate. In use,

the PIR crosslinker ﬁ rst is reacted with a sample to form conjugates and then the proteins are

subjected to proteolysis to create peptides. Some of the peptides that are formed will be conju-

gated together through the PIR crosslinks. This complex mixture of peptides then is subjected

to mass spec analysis using a mass spectrometer capable of MS

or MS

separations.

Second-generation PIR reagents have been designed to include an afﬁ nity handle branch-

ing off from the central reporter group ’s free carboxylate. In one such design, a biotin group

is built at the end of a hydrophilic PEG spacer ( Figure 28.6 ). The PEG arm provides increased

water-solubility to the overall reagent, which is otherwise rather hydrophobic. The biotin group

can be used to detect or capture crosslinked interacting proteins out of complex solutions. For

instance, immobilized streptavidin can be used to pull down any proteins modiﬁ ed with the

biotin–PIR reagent. Alternatively, a complex solution may be separated by electrophoresis and

the modiﬁ ed proteins identiﬁ ed after Western blotting through the use of a streptavidin conju-

gate detection complex.

The use of a crosslinker containing MS labile bonds along with a mass reporter group can sig-

niﬁ cantly reduce the complexity of ﬁ nding and identifying coupled peptides in a huge amount of

mass spec data. In the ﬁ rst dimension of a MS separation, the overall mass of the PIR-crosslinked

peptides can be accurately determined as a single peak in the MS spectrum. The resultant mass

correlates to the sum of the two crosslinked peptides plus the intervening PIR spacer which links

them together.

Figure 28.5 This PIR compound contains NHS esters at both ends to capture interacting proteins through

amide bond formation. It also contains MS cleavable bonds that release a central reporter group, which can be

used to identify crosslinked peptides by mass spec.

Figure 28.6 A trifunctional PIR compound that contains two NHS esters to capture interacting pro-

teins through amide bond formation and a PEG–biotin arm to permit isolation of crosslinked proteins on

(strept)avidin supports.

With bifunctional NHS ester reagents, one of three modiﬁ cation products can occur with

proteins: (1) a dead-end linkage wherein one end of the crosslinker has attached to an amine

group within a protein and the other end has hydrolyzed and not formed an attachment, (2) an

intra-protein crosslink wherein the PIR reagent has been coupled at both ends to amines within

the same protein, or (3) an inter-protein crosslink wherein both ends of the PIR reagent have

been coupled with amines on two different protein molecules ( Figure 28.7 ).

The second stage of an MS

analysis can be done to bombard with more energy the mass

products of the ﬁ rst stage separation in order to fragment the proteolytically digested complexes.

At this point, the mass reporter group within the PIR crosslink is released due to breakdown of

the two labile RINK bonds within the reagent structure (see Figure 28.5 ). Mass spec cleavage of

these groups and release of the reporter results in one of two potential mass signatures depend-

ing on if both ends of the PIR reagent have reacted (which gives a reporter mass  m/z 711) or

if only one end has reacted (a dead-end; reporter mass  m/z 828). The resulting mass spectrum

then shows either two or three peaks, depending on the type of modiﬁ cation formed from the

initial PIR reaction. This process also releases the crosslinked peptides without disrupting the

1. Homobifunctional Crosslinking Agents 1013

1014 28. Bioconjugation in the Study of Protein Interactions

Figure 28.7 Reaction of a PIR compound with a protein sample can result in several products, all of which

can be identiﬁ ed by mass spec analysis. A true inter-protein crosslink can occur that links two interacting pro-

teins together, which is the desired product. However, the crosslinking process also may result in intra-protein

crosslinks or dead ends wherein only one end of the PIR reagent has coupled to a protein.

peptide backbone and thus leaves a small part of the PIR compound attached to each peptide

fragment, minus the reporter group.

The speciﬁ c fragmentation pattern observed at this stage distinguishes the kind of crosslink

or modiﬁ cation that was initially formed. In this regard, a single modiﬁ ed peptide peak plus a

reporter peak of mass m/z 828 indicates a dead-end modiﬁ cation with no value in determining pro-

tein interactions. Alternatively, a single peptide fragment peak plus a reporter group peak of mass

m/z 711 indicates an intra-molecular crosslink made between regions of the same protein, which

also is not of interest. However, a fragmentation pattern containing two labeled peptide peaks plus

a reporter peak of mass m/z 711 indicates a successful conjugation between two protein molecules,

which may be indicative of a true protein–protein interaction. Note that alternative designs of a

PIR-type reagent containing other reporter structures, including those with a biotin handle and a

PEG spacer, will result in different reporter fragmentation mass values than those stated here.

The use of PIR compounds to study protein interactions is a signiﬁ cant advance over the use of

standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates decon-

volution of putative protein interaction complexes through a simpliﬁ ed mass spec analysis. The

software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide

peaks, which accompany the reporter signal of appropriate mass. This greatly simpliﬁ es the bio-

informatics of data analysis and provides deﬁ nitive conformation of protein–protein crosslinks.

Finally, knowledge of the peptide masses that resulted from the PIR conjugation provides

information to identify the parent proteins from which they originated. Peptide mass and

sequence databases now are sufﬁ ciently developed to provide rapid conﬁ rmation of protein–

protein interaction partners.

The following protocol is designed for treating cells with the PIR reagent to study protein

interactions in vivo. It is based on the method of Tang et al. (2005). The use of the PIR com-

pound to treat intact cells results in the crosslinking of proteins both on the cell surface and

within the cell, which indicates that the reagent is able to cross the cell membrane.

Protocol

1. Dissolve the PIR compound in dry DMSO to make a 100 mM stock solution.

2. Grow cells in media to a density of about OD

600

 1.2 and harvest in mid-log phase.

Centrifuge cells at 3,200 rpm to pellet them and wash 3 times with ice-cold PBS (150 mM

sodium phosphate, 100 mM NaCl, pH 7.5).

3. Suspend the cells in 1 ml of PBS and add an aliquot of the dissolved PIR compound to

bring the ﬁ nal concentration to 1 mM.

4. React at room temperature with gentle shaking for 5 minutes.

5. Quench the reaction by the addition of 50  l of 1 M Tris, pH 7.5.

6. Wash the cells 5 times by centrifugation with cold PBS to remove excess PIR reagent and

any secreted proteins.

7. Lyse the cells using a detergent lysis buffer suitable for the cell type being treated.

Centrifuge the lysate at 15,000 rpm for 40 minutes at 4°C to remove insoluble material.

Collect the supernatant and discard the pellet. At this point, the soluble protein fraction

may be analyzed by electrophoresis, if desired.

8. Remove unreacted PIR reagent and reaction by-products by gel ﬁ ltration or dialysis.

9. Precipitate the protein with TCA to further remove any remaining salts and detergent.

Centrifuge to pellet the precipitated protein, wash the pellet with TCA, and centrifuge again.

Redissolve the washed pellet in 100  l of 100 mM NH

HCO

, pH 7.8, containing 8 M urea.

1. Homobifunctional Crosslinking Agents 1015

1016 28. Bioconjugation in the Study of Protein Interactions

10. Reduce protein disulﬁ des by adding dithiothreitol (DTT) to a ﬁ nal concentration of

5 mM and incubate for 30 minutes at 60°C. Add iodoacetamide to a ﬁ nal concentration

of 25 mM to alkylate the thiols. React for 1 hour in the dark.

11. Dilute the solution 4-fold with 100 mM 25 mM NH

HCO

, and then add 20  g trypsin

to digest the proteins. Incubate at 37°C for 4 hours or overnight at 30°C with shaking.

12. Purify the tryptic peptides by chromatography on a C18 column to remove salts (follow

the manufacturer ’s directions for peptide puriﬁ cation). Dry the eluent and redissolve the

peptides in 20  l 0.1 percent formic acid.

13. Analyze the puriﬁ ed peptides using two-dimensional LC–MS/MS.

2. Use of Photoreactive Crosslinkers to Study Protein Interactions

The use of crosslinking agents containing at least one photoreactive group provides reagents

that can be activated at a desired point after bait proteins have been allowed to interact and

bind to prey proteins. The availability of the ﬁ rst photoreactive homobifunctional or hetero-

bifunctional compounds permitted protein interactions to be studied at a new level of speciﬁ -

city. Unlike the use of spontaneously reactive homobifunctional reagents, the incorporation of

a photoreactive group helps to prevent nonselective crosslinking and uncontrolled polymeriza-

tion of proteins that may or may not be speciﬁ cally interacting.

In use, a bait protein ﬁ rst is modiﬁ ed with the spontaneously reactive (thermoreactive) end

of a photoreactive heterobifunctional compound, while protecting the solution from light expo-

sure. The modiﬁ ed bait protein then is allowed to interact with a sample containing other pro-

teins and biomolecules, which may contain prey proteins able to interact with it. Finally, the

sample mixture is exposed to UV light to activate the photoreactive end of the crosslinker, caus-

ing conjugation of this group with any nearby interacting molecules. The rapid reaction rate of

the activated photoreactive intermediates assures that they don ’t survive long enough to cause

much conjugation to proteins just due to random collisions. Interacting proteins, however, that

are in proximity to the modiﬁ ed bait protein are more likely to be captured through reaction

with the activated photoreactive group.

The following sections discuss the application of several photoreactive heterobifunctional

crosslinkers to the study of protein interactions.

2.1. Sulfo-SAND, SANPAH, and Sulfo-SANPAH

These three photoreactive crosslinkers are described in terms of their properties and reactivi-

ties in the section on Amine-Reactive and Photoreactive Crosslinkers in Chapter 5, Section 3.

They represent early examples of the use of standard phenyl azide photoreactive compounds

for the study of protein interactions. All of them either contain an amine-reactive NHS ester or

the charged and water-soluble analog, a sulfo-NHS ester ( Figure 28.8 ). SANPAH is uncharged

and hydrophobic and thus provides membrane permeability for studying intracellular protein

interactions in whole cells (Mikhailov et al., 2001; Kota and Ljungdahl, 2005). Sulfo-SAND

and Sulfo-SANPAH, however, possess a negatively charged sulfonate group, which prevents

them from passing through cell membranes, and thus are membrane impermeable (Uckun et al.,

1995; Gaudet, et al., 2003). The sulfonated compounds may be used with whole cells to study

Figure 28.8 The heterobifunctional crosslinkers sulfo-SAND, SANPAH, and sulfo-SANPAH contain an amine-

reactive (sulfo)NHS ester on one end and a photoreactive phenyl azide group on the other end. Sulfo-SAND

allows release of conjugates by reduction of its internal disulﬁ de bridge.

cell-surface protein interactions, although in use, their concentration should be limited to pre-

vent internalization by active transport into cells.

Sulfo-SAND, SANPAH, and Sulfo-SANPAH all contain a nitrated phenyl azide photoreac-

tive group. The presence of the nitro group shifts the optimal wavelength for photoactivation

2. Use of Photoreactive Crosslinkers to Study Protein Interactions 1017

1018 28. Bioconjugation in the Study of Protein Interactions

to higher wavelengths, thus avoiding the potential for biomolecule damage due to UV

irradiation (photoactivation occurs at 320 –350 nm). Sulfo-SAND provides one further option

for studying interacting proteins. It has a cleavable disulﬁ de containing cross-bridge that per-

mits recovery of any prey proteins that have been captured by the photo-coupling process

(McMahan and Burgess, 1994; Uchiyama et al., 2002). Finally, each of these photoreactive

crosslinkers has phenyl azide rings that undergo ring expansion to the 7-membered ring, dehy-

droazepine intermediate, which reacts primarily with amines on target molecules. Thus, the

proteins that are captured by these crosslinkers typically are coupled through secondary amine

linkages to the photoreactive end of the reagents. A general protocol for the use of these com-

pounds to study protein interactions is given at the end of the next section.

2.2. Sulfo-SFAD

Sulfo-SFAD is sulfosuccinimidyl-[perﬂ uoroazidobenzamido]-ethyl- 1,3 -dithiopropionate, an

amine reactive and photoreactive crosslinker with more advanced properties than the photore-

active reagents discussed above. This reagent contains a sulfo-NHS ester to provide increased

water-solubility prior to conjugation, and this group will react with an amine on proteins and

other biomolecules to form an amide linkage. Sulfo-SFAD also contains a perﬂ uorophenyl

azide group that has better photo-insertion capability than the original unsubstituted phenyl

azide group. The reason for this enhanced yield is that after photoactivation, the nitrene inter-

mediate can ’t react with the phenyl ring itself and undergo ring expansion to a dehydroazepine

as happens with typical unsubstituted phenyl azides. The result is that the nitrene survives

long enough to react with neighboring molecules in the immediate molecular vicinity, such as

proteins that are interacting with the modiﬁ ed bait protein. Perﬂ uorophenyl azides thus have

higher yields of conjugation, and they can react by insertion into C  H bonds, N  H, unsatu-

rated carbon–carbon bonds, and other structures in target molecules. By contrast, unsubsti-

tuted phenyl azides ring expand and react mainly with amines, and even then at low yield. The

perﬂ uorophenyl azide may be activated with UV light at 320 nm.

Sulfo-SFAD also contains a disulﬁ de bond in its cross-bridge, which provides subsequent

cleavability of any crosslinks formed. Thus, prey proteins can be recovered from complexes for

analysis by simple reduction of the conjugate with DTT. Once reduced, the perﬂ uorophenyl ring

group of Sulfo-SFAD is transferred to the interacting prey protein. This group can be identiﬁ ed

F NMR, and it also provides a traceable label for identiﬁ cation of peptide fragments by

mass spec. Figure 28.9 illustrates the reactions of Sulfo-SFAD in capturing a bait–prey complex.

The following references provide further information on the use of Sulfo-SFAD and per-

ﬂ uorophenyl azide photoreactive groups: Pandurangi et al. (1995a, b, 1996, 1997a, b, 1998);

Yan et al . (1994).

A general protocol that can serve as a guide for the use of heterobifunctional crosslinkers in

the study of protein interactions is given below. Some optimization of concentrations may have

to be done depending on the particular type and properties of proteins being studied.

Protocol

1. Dissolve the photoreactive crosslinker in DMF or DMSO at a concentration of

10–25 mM (protect from light). Water-soluble crosslinkers can be added to water or

buffer, but should be used immediately.

2. Modify a puriﬁ ed bait protein by adding an aliquot of crosslinker to achieve a molar

excess of 2–10 moles per mole of protein (protect from light). For NHS ester-containing

reagents, react in 0.1 M sodium phosphate buffer at pH 7.2–7.4. Avoid using any other

amine-containing buffer components, such as Tris or imidazole, which will interfere with

the reaction.

3. React for 30–60 minutes in the dark (room temperature or 4  C).

4. Remove excess crosslinker from the modiﬁ ed bait protein by gel ﬁ ltration or dialysis in

the dark.

Figure 28.9 Sulfo-SFAD is an advanced heterobifunctional photoreactive crosslinker that contains an amine-

reactive sulfo-NHS ester on one end and a tetraﬂ uorophenyl azide group on the other end. The ﬂ uorine sub-

stitutions on the phenyl ring prevent the photoactivated nitrene from reacting with the ring, thereby providing

greater yields in capturing interacting proteins. The disulﬁ de-containing cross-bridge allows cleavage of conju-

gated molecules by reduction with DTT.

2. Use of Photoreactive Crosslinkers to Study Protein Interactions 1019