Hermanson G. Bioconjugate Techniques, Second Edition

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separations in one or two dimensions can be done to fractionate the peptides in complex sam-

ples by charge, size, or hydrophobicity before being injected into the mass spec.

Another major technique to simplify the analysis of protein samples is to use mass tags. Mass

tags are modiﬁ cation reagents that contain a reactive group for coupling to biomolecules and

another component of known mass, which behaves predictably upon MS separation. The mass

tag also may contain a functional group for capture and separation on an afﬁ nity support,

which permits further fractionation of the proteome. MS analysis of mass tagged peptides can

be done by focusing only on those peptides that contain an additional mass component rep-

resenting the tag ’s known mass contribution. Thus, all other peaks on the MS spectrum can

be ignored, which greatly reduces the complexity of the sample. Mass tag reagents have been

developed with reactive groups to modify speciﬁ cally only certain low frequency amino acids

within proteins. For instance, a thiol-reactive iodoacetyl group on a mass tag can be used to

modify only those peptides having cysteine residues, thus removing from the analysis window

all other peptides not containing cysteine.

The design of mass tags also can be combined with stable isotope labels to create more than

one mass unit for each tag type (Schneider and Hall, 2005). For example, certain hydrogen

atoms on one mass tag can be replaced with deuterium atoms on another derivative. Everything

else on the tag is identical except for the isotope substitutions. Thus, the two mass tag analogs

will differ in molecular weight by exactly the mass difference represented by the isotopic substi-

tutions. Such tags can be used to modify a test sample with the stable isotope tag versus a con-

trol sample modiﬁ ed with the normal tag. If the two samples then are combined and analyzed

by mass spec, their signal peaks generated from the tagged peptides will differ in mass units by

the isotopic mass differences in the two tags. Identiﬁ cation of the peptides from both samples

is done by looking for peptide peak pairs differing by the characteristic mass amount, therefore

greatly reducing the complexity of sample analysis, and allowing simultaneous investigation of

two samples. In this way, a test sample ’s protein expression levels can be compared to a control

sample by measuring the different areas of the paired peptide peaks. The ability to analyze pro-

tein expression in two samples is vitally important to drug discovery and life science research

applications studying the proteome.

Mass tags also can be broad spectrum in their modiﬁ cation properties to derivatize all pep-

tides as they are formed upon proteolysis. For instance, one of the simplest mass tagging sys-

tems is to use the oxygen isotope

O in the water used during the enzymatic digestion of a

protein sample (Miyagi and Rao, 2007). Upon hydrolysis by trypsin, the resultant C-terminal

carboxylates that are formed each incorporate two

O atoms. Thus, peptides formed from

O digestion will be four mass units heavier than peptides formed by proteolysis using normal

water. Mass spec analysis of this difference can identify the peptide pairs resulting from a con-

trol sample and a test sample run simultaneously.

Other broad-spectrum mass tag modiﬁ cation agents are designed to modify all amine groups

and yield tags on every peptide at their N-terminal amines. For instance, small molecule tags

using deuterium labeled forms and regular hydrogen labeled ones, such as the use of isotopi-

cally labeled propionic anhydride (Zappacosta and Annan, 2004), provide differentiation in

the mass spec signals of peptides from test samples and controls. To eliminate interference,

side chain lysine amines are blocked by guanidination with O-methylisourea hemisulfate and

cysteine thiols are blocked with iodoacetamide (Leitner and Lindner, 2004). Some mass tag

reagents of this type are able to differentiate peptides from 6 to 10 samples analyzed at the

same time (see section on isobaric tags, this chapter).

650 16. Mass Tags and Isotope Tags

The following sections describe some of the major mass tag types and discuss the general

protocols for their use.

1. ICAT Reagents

Isotope coded afﬁ nity tags (ICAT) are bifunctional mass tagging agents containing a reactive

group on one end of the molecule and an afﬁ nity capture group on the other end (Gygi et al .,

1999; Aebersold, 2003) ( Figure 16.1 ). In addition, a portion of the tag can contain stable iso-

tope substitutions, usually designed to be in the cross-bridge between the reactive group and

the biotin handle. The original ICAT reagent contained eight deuterium atom substitutions on

the outer ends of an ethylene oxide spacer. The isotope tagged version thus differs from its nor-

mal atom analog by exactly eight mass units.

Most ICAT style compounds contain a thiol-reactive iodoacetyl group on one end and a

biotin handle on the other end of a spacer arm ( Figure 16.2 ). Reagents of this type are highly

speciﬁ c for reacting with cysteine thiols in proteins to result in stable thioether modiﬁ cations

containing a terminal biotin group ( Figure 16.3 ). After enzymatic digestion, modiﬁ ed peptides

then can be isolated using immobilized (strept)avidin, which speciﬁ cally binds only to those

peptides containing the biotin tag and allows the other peptides to be discarded. Thus, the sam-

ple complexity can be reduced to analyze only peptides that contain a cysteine residue, which

in the human proteome represents about 26.6 percent of the total tryptic peptides in a sample.

This translates into the ability to cover 96.1 percent of all the proteins in the human proteome

by targeting only cysteine-containing peptides (Zhang et al ., 2004; Yan and Chen, 2005).

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer

arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at par-

ticular functional groups and the biotin afﬁ nity tag is used to isolate labeled molecules using immobilized

(strept)avidin.

1. ICAT Reagents 651

652 16. Mass Tags and Isotope Tags

ICAT reagents can be used to compare two different samples by mass spec analysis. For

instance, one cell population can be treated with a drug candidate, while another one remains

untreated and acts as a control. Alternatively, one cell population can represent a disease

state and the control population is the normal cell line. After cell lysis, the proteins in each

Figure 16.3 The ICAT reagent reacts with cysteine-containing peptides to form a thioether bond.

Figure 16.2 The original design of the ICAT compound. The iodoacetyl group provides reactivity with thiol

groups. The isotopically labeled spacer arm typically is substituted with eight deuterium atoms.

sample are denatured and reduced to make available all of the cysteine thiols for modiﬁ ca-

tion. One sample then is reacted with the heavy atom ICAT reagent, while the other sample

is reacted with the normal isotope compound. The two samples next are combined and enzy-

matically digested with trypsin to generate peptide fragments, some of which will contain ICAT

labeled cysteine groups. This combined peptide sample is afﬁ nity separated on an immobilized

(strept)avidin column (or monomeric avidin column), which binds biotin labeled peptides from

both sample populations equally. After removal of the non-biotinylated peptides by washing

the column followed by elution of the ICAT labeled peptides, the sample is subjected to capil-

lary reverse phase chromatography leading into ESI or MALDI mass spec analysis. The ﬁ nal

HPLC separation again reduces the complexity of the sample set by further fractionating the

peptides based on comparative hydrophobicity. In the MS spectrum, the relative peptide con-

centrations are determined by comparing all peaks separated by exactly the mass unit differen-

tial between the heavy atom mass tag and the normal atom mass tag. Each peptide sequence

then is identiﬁ ed by fragmentation of the peptides into amino acid ions in a second dimen-

sion MS separation (MS/MS). Comparison of the amino acid sequence of each peptide peak

to known sequence databases can identify the protein from which it came. Thus, the resultant

peptide peak ratios are directly proportional to the relative amounts of the corresponding pro-

teins present in the cell population.

The original ICAT design was found to have a number of deﬁ ciencies that often prevent the

reagent from providing acceptable MS results. First, the deuterium isotope-labeled compound

has a tendency to behave differently than the normal hydrogen isotope during reverse phase

separation (Regnier et al., 2002). If the labeled peptides that are identical except for the pres-

ence or absence of a D

ICAT modiﬁ cation don ’t elute at precisely the same point in an HPLC

separation, then the MS analysis won ’t provide the peak pairs necessary for quantiﬁ cation. To

solve this problem, a second-generation ICAT compound was designed containing

C isotopes

instead of deuterium atoms. This type of reagent facilitates precise chromatographic separation

of the labeled peptides and thus gives far superior performance upon MS analysis.

A second problem in the original ICAT design relates to the presence of the biotin tag. The

biotinylated peptides often give undesirable fragmentation patterns during MS/MS analy-

sis, which interferes with the smooth identiﬁ cation of peaks. Removing the biotin tag before

mass spec analysis therefore would be beneﬁ cial to interpreting the MS results. Another issue

with using a biotin tag is the elution step from the immobilized (strept)avidin column. Only

under severely denaturing conditions is the interaction between biotin and (strept)avidin dis-

rupted. However, even when using such conditions, the bound peptides do not always get

released reproducibly from the column. The result is inefﬁ cient recovery of labeled peptides,

which directly translates into a lack of precision in the MS data. Even using an immobilized

monomeric avidin column does not completely solve this problem, because this afﬁ nity sup-

port sometimes has higher afﬁ nity binding sites or binds non-biotinylated peptides nonspe-

ciﬁ cally. To solve these issues, new cleavable ICAT designs were created that contain a bond

within the cross-bridge that can be chemically broken (Li et al., 2003). After binding to the

(strept)avidin column, elution can be accomplished by cleaving the biotin arm, not by breaking

the (strept)avidin–biotin interaction. The cleavage site can consist of a disulﬁ de group within the

cross-bridge (Turecek, 2002) that can be reduced for elution from the (strept)avidin column or

it can consist of an acid cleavable linker arm (e.g., a carbamate bond) within the ICAT structure

(Fauq et al., 2006) ( Figure 16.4 ). Either method dramatically improves the recovery of labeled

peptides from the afﬁ nity column and thus provides increased precision in the samples leading

1. ICAT Reagents 653

654 16. Mass Tags and Isotope Tags

into the LC–MS analysis. The

C labeled, acid cleavable ICAT reagent has been used to identify

successfully low-level protein expression in highly complex samples (Hansen et al., 2003).

Another new ICAT design, termed a “catch-and-release” tag, contains a constrained, steri-

cally hindered disulﬁ de linkage with bulky alkyl groups on both sides. The hindered nature

of the disulﬁ de makes it stable to standard protein reduction procedures, but it can be speciﬁ -

cally reduced upon the addition of tris(2-carboxyethyl)phosphine(TCEP) (Gartner et al., 2007)

(Figure 16.5 ). This allows proteins to be labeled with the catch-and-release ICAT compound

that have undergone reduction using dithiothreitol (DTT) to cleave protein disulﬁ des but not

affect the disulﬁ de group in the reagent cross-bridge. Only after capture of labeled peptides on

Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on

either side of a disulﬁ de bond within its spacer arm. The hindered disulﬁ de permits the use of standard reducing

gel electrophoresis conditions using DTT without reduction. After puriﬁ cation on a (strept)avidin afﬁ nity col-

umn, however, the disulﬁ de group can be cleaved with TCEP, which provides recovery of the labeled peptides

prior to mass spec separation.

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled

peptides from a (strept)avidin afﬁ nity column. The use of

C isotopes instead of deuterium labels permits

precise reverse phase separations prior to mass spec that show no elution peak time differences between

isotope-labeled and normal atom-labeled peptides.

a (strept)avidin column is the cross-bridge cleaved by the addition of TCEP and the labeled

peptide recovered.

A variation on the ICAT mass tag concept was made by immobilizing the label on a solid

phase (Zhou et al., 2002). Using this design, cysteine-containing peptides are modiﬁ ed directly

on a beaded insoluble support. After washing away non-cysteine peptides, the linked peptides

can be cleaved from the matrix by use of a photo-cleavable group and eluting off the peptides

with an isotope tag modiﬁ cation. This approach results in cleaner and more efﬁ cient isolation

of tagged peptides and simpliﬁ es the ICAT labeling process ( Figure 16.6 ).

Another novel mass tag design involves a spectrally visible ICAT variant developed to

include a ﬂ uorescent group for detection purposes (Lu et al., 2004). Like the original ICAT

reagent, the VICAT compound includes a thiol-reactive iodoacetyl group, a cleavable cross-

bridge, an isotopically labeled portion, and a biotin handle. It also has another arm, however,

that contains the chromogenic label, which is detectable by absorption at 493 nm and emission

at 503 nm.This group allows detection of peptides in samples separated by chromatographic

or electrophoretic means. Quantiﬁ cation of the ﬂ uorescent tag in isolated peptides can provide

absolute information regarding the level of proteins present in a cell.

ICAT type compounds are designed to enrich for peptides containing one particular amino

acid residue, usually cysteine. The afﬁ nity capture step removes other non-cysteine peptides

and thus reduces the complexity of the MS data set. ICAT reagents also can be designed with

a different reactive group that is able to covalently couple to other amino acid groups (or even

sites of post-translation modiﬁ cation) to change the selectivity of the peptide population being

analyzed. However, it is best to target amino acids or functional groups present in limited

amounts within proteins, otherwise the tag may capture more peptides than could be conve-

niently measured by mass spec. Han et al. (2007) developed a hydrazide-ICAT compound to

identify proteins modiﬁ ed by 2-alkenals derived from lipid peroxidation (LPO). This type of

mass tag should be useful for the study of other oxidative changes on proteins, such as those

resulting in aldehyde or ketone modiﬁ cations (see Chapter 1, Section 1).

The following protocol describes the use of an acid-cleavable ICAT reagent, currently avail-

able from Applied Biosystems. This is not meant to be a detailed method describing every

aspect concerning the use of mass spectrometry, but only to describe the modiﬁ cation reaction

of the ICAT compound with proteins.

Protocol

1. Grow cells to 70–80 percent conﬂ uence and harvest by scraping the cells into 5 ml PBS,

5 mM EDTA, pH 7.4. Aliquot cell counts of approximately 2.5–5  10

for processing.

Lyse cells using a detergent lysis buffer (e.g., Poppers, Thermo Fisher) or by mechan-

ical means. Centrifuge and discard the cellular debris. Measure the total protein con-

centration using the BCA assay (Thermo Fisher) and adjust the protein concentration to

1.5 mg/ml using 50 mM Tris, 0.1 percent SDS, pH 8.0. Separately process a test sample

and a control sample made up of different cell populations.

2. Reduce disulﬁ des in the two protein samples by the addition of 2 l of 50 mM TCEP

(Thermo Fisher) to each 100 l aliquot of protein solution. Cover and boil the samples

for 10 minutes in a water bath to completely denature and reduce the proteins. Avoid the

use of thiol-containing reductants, such as DTT, as these will react with the iodoacetyl

group on the ICAT compound.

1. ICAT Reagents 655

656 16. Mass Tags and Isotope Tags

3. Dissolve one vial of heavy isotope, cleavable ICAT reagent (Applied Biosystems) in 20  l

acetonitrile (use a fume hood for handling solvents). Dissolve a second vial contain-

ing the normal isotope ICAT compound in 20 l acetonitrile. Vortex mix each vial to

dissolve.

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine pep-

tides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured

peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-

cleavable site to release isolated peptides from a (strept)avidin afﬁ nity resin. This compound adds a ﬂ uorescent

group to better detect labeled peptides as they are being isolated from a sample.

2. ECAT Reagents 657

4. Add 100 g of the control protein solution to one vial of dissolved normal isotope ICAT

reagent. Mix to dissolve. Add 100 g of the test protein solution to one vial of dissolved

heavy isotope ICAT reagent. Mix to dissolve.

5. React both solutions for 2 hours at 37°C.

6. Combine the test sample with the control sample in a single vial. Mix well.

7. Prepare a solution of TCPK-trypsin in 50 mM ammonium bicarbonate, pH 8.0, at a con-

centration of 100 ng/ l. Add 10 l of the trypsin solution to every 10 g of combined,

labeled protein solution from Step 6. Incubate at 37°C for 12–16 hours or overnight

with mixing.

8. Centrifuge the digested peptide mixture to remove any insoluble material. The bioti-

nylated peptides then are puriﬁ ed on 20 l immobilized monomeric avidin column. The

column ﬁ rst is primed with elution buffer (0.4 percent TFA in 30 percent acetonitrile)

and then washed with binding buffer (100 mM ammonium bicarbonate, pH 8.0). The

sample is applied and washed through with binding buffer until all not-bound peptides

are completely removed. The acetonitrile/TFA elution is subsequently used to cleave off

the biotin group from the eluted peptides. Note: Additional fractionation may be done to

reduce further the complexity of the sample, such as the use of ion-exchange chromatog-

raphy. The eluted, labeled peptides ﬁ nally are analyzed by LC–MS/MS.

2. ECAT Reagents

Element-coded afﬁ nity tags represent a new type of isotope tag for mass spec analysis

(Corneillie et al., 2003, 2004; Whetstone et al., 2003; Meares et al., 2007). This system uses

a bifunctional metal chelate group that securely coordinates a lanthanide metal ion. A reactive

group also is present for the modiﬁ cation of certain amino acids in proteins, which typically

consists of a bromoacetyl group. This group reacts similarly to an iodoacetyl group and forms

thioether linkages with cysteine thiols. The ECAT design includes a DOTA chelating group

(Chapter 10) containing four nitrogen atoms and four carboxylates to complex with any of

the lanthanide series metal ions via eight coordination bonds. Simply by using different lantha-

nide elements within the complex the result will be a unique set of isotope tags having differ-

ent mass signatures by MS. At least in theory, up to 15 different ECAT mass tag compounds

could be created by using all of the different lanthanide metals representing elements 57–71. In

addition, the lanthanides are naturally mono-isotopic in that they occur mainly in nature with

only a single isotope. Only cerium contains a high percentage of another isotope in nature (88

percent

140

Ce and 11 percent

142

Ce), all the other lanthanides are 97 percent a single isotope.

This is important for mass spec separations, as the resultant peaks won ’t contain extraneous

mass signatures due to multiple isotopes ( Figure 16.7 ).

The ECAT reagent bromoacetamidobenzyl-DOTA (BAD) can be used like an ICAT tag to

label only those peptides containing cysteine residues ( Figure 16.8 ), thus reducing the total

number of peptides having to be analyzed in a mass spec separation. Unlike the ICAT reagent

design, the ECAT compound does not contain a biotin handle for afﬁ nity separation. Instead,

a monoclonal antibody has been developed with speciﬁ city for the DOTA chelate containing

a bound lanthanide metal. The antibody will recognize any lanthanide element bound in the

chelate and thus function as an afﬁ nity ligand for separating ECAT-labeled peptides. The afﬁ n-

ity of the monoclonal for ECAT chelate structure allows highly stringent washes to be done

658 16. Mass Tags and Isotope Tags

prior to elution of the labeled peptides. The afﬁ nity column typically is washed with low pH,

high salt, high pH, and 30 percent acetonitrile before elution is done. This removes all traces of

nonspeciﬁ cally bound peptides, so they can ’t interfere with the mass spec analysis. The ECAT

labeled peptides then are eluted with 20 percent acetonitrile containing 0.1 percent TFA.

The ECAT system has an advantage over ICAT reagents in being available with more mass

signatures than is achievable using

C or

H labeled tags, which are difﬁ cult to synthesize. In

addition, it is not hampered by the binding idiosyncrasies of a biotin group interacting with

(strept)avidin or the fragmentation problems a biotin tag gives on MS analysis. The ECAT

chelate does not generate fragmentation products during the mass spec analysis. Also, the mass

defect characteristic of lanthanide metals results in a mass signature upon MS separation that

occurs in a relatively unoccupied region of the m/z spectrum (Schneider and Hall, 2005). Thus,

identiﬁ cation of ECAT labeled peptides potentially is simpler than using ICAT reagents.

Another variant of ECAT reagent technology has been developed to analyze the products of

protein oxidation (Lee et al., 2006). Called “oxidation-dependent, carbonyl-speciﬁ c element

coded afﬁ nity tag ” (O-ECAT), the compound contains the same DOTA lanthanide chelating

group, but instead of a thiol-reactive bromoacetyl group, it has an aldehyde- or ketone-reactive

aminoxy group ( Figure 16.9 ). The aminoxy functional group can covalently link to aldehydes

or ketones to give an oxime bond, which is stable under aqueous conditions ( Figure 16.10 ).

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine

(DNPH; Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modiﬁ cation pro-

duces detectable complexes, but it does not provide information as to what amino acids are

involved. O-ECAT modiﬁ es carbonyl end products of protein oxidation and in addition, it can

provide exact information as to the amino acids that were oxidized. Mass spec analysis of mod-

iﬁ ed proteins performed after proteolysis gives the exact amino acid sequences including the

sites of O-ECAT reagent modiﬁ cation. The same antibody that is speciﬁ c for the metal chelate

portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT

Figure 16.7 The ECAT mass tag consists of a DOTA metal chelate group that can coordinate a lanthanide

metal ion and a bromoacetyl group for coupling to cysteine-containing proteins.

labeled proteins or peptides. Thus, O-ECAT-modiﬁ ed proteins can be detected in Western blots

or the sites of oxidation quantiﬁ ed using ELISA-based assays.

3. Isobaric Tags

The use of mass tagging reagents to analyze proteomic data has greatly improved the ability

to compare samples for protein expression differences. However, a major limitation of the

ICAT procedure (Section 1, this chapter) is that it can only compare two samples simultane-

ously, usually a test and a control. Even with the ECAT design (Section 2) using multiple lan-

thanide metals to make a series of different mass tag signatures, it is difﬁ cult to extend the

3. Isobaric Tags 659

Figure 16.8 Reaction of the ECAT reagent with a cysteine-containing protein results in the formation of a

stable thioether bond.