Organometallic derivatizing agents in bioanalysis

REVIEW

Organometallic derivatizing agents in bioanalysis

Susanne Bomke & Michael Sperling & Uwe Karst

Received: 2 January 2010 /Revised: 22 February 2010 /Accepted: 22 February 2010 /Published online: 21 March 2010# Springer-Verlag 2010

Abstract Over the last few decades, the development ofseveral innovative hyphenated analytical techniques andtheir routine use in laboratories has led to new possibilitiesfor the quantitative analysis of biomolecules. Today, theidentification and quantification of biomolecules such aspeptides and proteins are essential to answer importantmedical, pharmaceutical, and biological questions. To allowefficient detection and structure elucidation of biomolecules,several approaches including derivatization strategies wereinvestigated and applied during recent years. This articlesummarizes the current approaches for labeling and presentsthe different types of organometallic derivatizing agents usedas labels. Furthermore, their analytical potential with respectto quantification and structure elucidation for different ap-plications in the field of bioanalysis is discussed.

Keywords Mass spectrometry . ICP-MS . Organometals .

Bioanalytical methods

Introduction

The systematic acquisition of information relevant to ge-nomes, gene transcripts, and proteins including the identifi-cation of structure and function is a fundamental aspect in

current bioanalytical chemistry. Most of the recent achieve-ments in this field benefit from the developments of massspectrometric detection techniques such as electrospray (ESI)[1], matrix-assisted laser desorption and ionization (MALDI)[2, 3], and inductively coupled plasma–mass spectrometry(ICP–MS) [4]. Moreover, the routine use of hyphenatedtechniques combining a separation module such as liquidchromatography (LC) coupled with mass spectrometricmethods such as ESI–MS and ICP–MS offers new possibil-ities for the identification and quantification of biomolecules.The potential of these techniques for protein analysis hasbeen discussed in several reviews focusing on MS [5, 6] andICP–MS [7–9].

It rapidly became evident that apart from identificationand relative quantification, absolute quantitative data arerequired for further characterization of biological samplesand that the whole field of proteomics “had to turn quan-titative” [10]. Thus, dynamic biological systems should bequantitatively examined and the search for biomarkers inclinical proteomics must be expanded with new technolo-gies aimed at absolute quantification. In particular, theability of ICP–MS for simultaneous isotope abundancemeasurements leads the way to innovative metabolic studiesand the quantification of biomolecules [11]. Since the ICP isa plasma open to the surrounding atmosphere, the detectioncapabilities for C, N, and O (i.e., the main constituents ofatmospheric air) are rather limited, and therefore quantifica-tion is frequently based on other elements. Fortunately, somepeptides and proteins contain natural heteroelements apartfrom H, C, N, and O. The role of such natural element tagshas been reviewed recently by Prange and Pröfrock [12]. Theanalysis of naturally covalently incorporated heteroelementssuch as 32S [13–15], 80Se [16–19], 127I [20, 21], or 31P [22–25] by ICP–MS is enjoying increased interest in recent years.However, isobaric interferences, the high first ionization

S. Bomke :M. Sperling :U. Karst (*)Institut für Anorganische und Analytische Chemie,Westfälische Wilhelms-Universität Münster,Corrensstr. 30,48149 Münster, Germanye-mail: [email protected]

M. SperlingEuropean Virtual Institute for Speciation Analysis,Mendelstr. 11,48149 Münster, Germany

Anal Bioanal Chem (2010) 397:3483–3494DOI 10.1007/s00216-010-3611-1

energies of these heteroelements, and the resulting highlimits of detection often lead to unsatisfactory results. Betteranalytical results can be expected by introducing elementaltags via an additional derivatization step. Additionally, sincestandards for most biomolecules of natural origin areunavailable, such tagging using different derivatizationapproaches is a valuable alternative for quantification [26].

In analytical chemistry, derivatization reactions are oftencarried out due to missing chromophoric, fluorophoric, orelectroactive groups in the analyte of interest. A suitablederivatization reaction must convert the analyte into a stableproduct, and the conversion should be quantitative andenable or improve the use of more sensitive detection tech-niques. In order to be practicable, the derivatization, whichalways is an extra reaction step during sampling prepara-tion, should be simple and straightforward.

To date, several derivatization approaches have beendeveloped mainly based on chemical, metabolic, and enzy-matic labeling. The structure and properties of the differentderivatizing agents are intended to match the analyticaldemands with respect to target analyte, detection technique,and application field. With respect to their structures, thederivatization reagents can be divided into different com-pound classes.

This review focuses on current labeling approaches, andthe main focus is directed to the presentation of the variouscompound classes of labeling reagents (Table 1). Moreover,the analytical possibilities resulting from formed deriva-tives, and the advantages and disadvantages of thederivatizing agents will be further discussed.

Current labeling strategies

Isotope labeling

Stable isotope labeling has emerged as a powerful tool toidentify and quantify peptides and proteins in complex mix-tures. This labeling strategy can be applied for chemical,metabolic, and enzymatic labeling. The first of these involveschemical reactions for the labeling of biomolecules prior totheir analysis. For example, functional groups of proteins canbe selectively labeled with stable-isotope-containing affinityreagents, allowing fast analysis of complex protein mixtures.The prototype of these chemical labeling approaches wasintroduced by Gygi et al. as isotope-coded affinity tags(ICAT) in 1999 [27]. This method is based on the differentialisotope labeling of the rare cysteine residues with tagscontaining a thiol-selective reactive group, an affinity tag(biotin), and an isotopically marked linker group. Generally,ICAT is used to determine the relative expression of peptidesin diseased versus healthy states (Fig. 1). Because ICAT isrestricted to only cysteine-containing proteins, attempts to

broaden the applicability of this approach were made. Forexample, isotope-coded protein labels (ICPL) were devel-oped, which selectively label all free amino groups inproteins [28]. However, the lack of robustness, differentialelution of identical peptides labeled with the hydrogen/deuterium isotope pairs on reversed phases, and complicatedinterpretation of the resulting tandem mass spectra [29]severely restricted the application of ICAT despite manyrefinements [30–32]. A ‘bottom-up’ approach involving thedigestion of proteins into peptide fragments that can bedetected and sequenced with liquid chromatography coupledwith tandem mass spectrometry (LC/MS/MS) uses isobarictags of the proteolytic peptides for relative and absolutequantification (iTRAQ). This method is based on the in-corporation of up to four mass tags that label the N-terminusand lysine residues of peptides resulting from a tryptic digestof the proteins [33, 34]. Furthermore, the introduction ofstable isotopes can be performed in vivo by using metaboliclabeling such as SILAC (stable isotope labeling of aminoacids in cell culture) [35]. Metabolic labeling has beenwidely used for the relative quantification of protein ex-pression in differently treated cell cultures. The enzymaticlabeling approach makes use of the incorporation of isotopesby using H2

18O during a tryptic digest [36, 37]. A seriousdrawback of this approach is the pH-mediated back ex-change of 16O and 18O [36]. Also, the variation resultingfrom the exchange of one or two carboxyl oxygen atomsmakes the interpretation of data very complex. Furthermore,missing separations at the protein level seriously restrict theanalytical value of this in vivo labeling strategy [38].

Chelating compounds

Historically, the most common tool for the analysis ofproteins makes use of chelating compounds in combinationwith radioactive tracers. As a result of developments in massspectrometry in recent years, chelating compounds withdifferent incorporated metals came into use for the detectionof biomolecules. The term chelate implies the involvementof a complex, in which a metal ion is bound to two or moreatoms of the chelating agent, whereas the bonds may be anycombination of coordination or ionic bond [39]. Accordingto the strict definition of the term “organometallic com-pounds”, metal chelates are not included, as the presence of ametal–carbon bond is essential for this classification.Bifunctional chelating agents have various medical applica-tions, e.g., they are frequently used in combination withradiopharmaceuticals and contrast agents [40] as well as inbioanalytical assays [41]. Macrocyclic metal chelates such as1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) loaded with different lanthanide (Me3+) ions formvery stable metal complexes with high stability constants(Fig. 2) that can be applied to the metal-coded affinity tag

3484 S. Bomke et al.

(MeCAT©) technique that allows absolute quantitativedetermination of peptides and proteins [42–44] by ICP–MSwith the help of an external standard.

Bifunctional chelating agents like diethylenetriamine-pentaacetate (DTPA) have been used to label conjugatedfunctional groups on peptides and proteins [45] and che-lated with 111In or other radioactive isotopes to produceradiopharmaceuticals. Other DTPA derivatives containingmaleimide, bromoacetamide, and pyridyldithio linkers were

used as luminescent probes in combination with Eu3+ andTb3+ as chromophores [46]. Recently, DTPA was subse-quently labeled with naturally and isotopically enrichedEu3+ and was used as a labeling tag for relative quantifi-cation [47]. Liu et al. demonstrated the labeling of peptides byusing yttrium and terbium–DTPA complexes [48]. Furtherlanthanide ions such as Eu, Tb, and Ho were implemented ina 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidsuccinimide ester (SCN–DOTA) complex to label bovine

Table 1 Overview of the most important classes of labeling reagents

S

NHNH

O

O

NH

OD

DD

DO

O NH

D

DD

DO

I

Chemical structure types Reference cited

[27]

Reagent

Isotope labeling

ICAT

iTRAQ [33, 34]

Mercury tags

Chelatingcompounds

e.g. MeCAT

CO2R

NN

RhCl1 R = CH32 R = C6F5

L(CO)4W COR1

R2Carbenecomplexes

[76, 77]

[78-81]

CO

O N

O

OMLn

OC Me

1 M = Mo, L = CO, n = 22 M = Fe, L = CO, n = 13 M = W, L = CO, n = 3

Carbonylcomplexes

N N

N N

O

OO O

O

OHO

HN

O

N

O

OLa3+

O

CH3HgCl

CH3CH2HgCl

[127-129][135, 136]

[130-134]

[42-44]

[59-71]

HOOC Hg OH

Metallocene-based

reagents

Fe

RR = (CO)H, (CO)Cl, CH2COOH, B(OH)2

R = N

O

O

N

O

O

O O

C

O

O N

O

OCo+

[96-117]

[120-126]

ON

O

O

ON

N

1 L = CO, R1 = R2 = Me2 L = CO, R1 = Et, R2 = Ph3 L = PBu3, R = Et, R2 = PH4 L = CO, R2 = Li, R2 = Ph

1

S

NHNH

O

O

NH

OD

DD

DO

O NH

D

DD

DO

I

Chemical structure types Reference cited

[27]

Reagent

Isotope labeling

ICAT

iTRAQ [33, 34]

Mercury tags

Chelatingcompounds

e.g. MeCAT

CO2R

NN


L(CO)4W COR1

R2Carbenecomplexes

[76, 77]

[78-81]

CO

O N

O

OMLn

OC Me


Carbonylcomplexes

N N

N N

O

OO O

O

OHO

HN

O

N

O

OLa3+

O

CH3HgCl

CH3CH2HgCl

[127-129][135, 136]

[130-134]

[42-44]

[59-71]

HOOC Hg OH

Metallocene-based

reagents

Fe

RR = (CO)H, (CO)Cl, CH2COOH, B(OH)2

R = N

O

O

N

O

O

O O

C

O

O N

O

OCo+

[96-117]

[120-126]

ON

O

O

ON

N


1

Organometallic derivatizing agents in bioanalysis 3485

serum albumin and hen egg white lysozyme [48]. Theresulting derivatives were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE).Until recently, only a few groups have made use of chelatingcompounds in combination with atomic spectrometry analysistechniques such as ICP–MS. For example, peptide quan-tification was performed by using In–DOTA complexes incombination with complementary hyphenation of LC/ESI–TOF–MS and LC/ICP–MS [49]. Commercially availablefluorescent probes (DELFIA™) containing the lanthanidesEu, Tb, and Sm were employed for DOTA labeling ofantibodies and detection by ICP–MS [50] even in combina-tion with multiplex analysis [51]. Additionally, the firstICP–MS-based multiplex profiling of glycoproteins waspublished recently, in which lectins conjugated to lanthanide-chelating compounds were used [52].

Despite the excellent detectability of lanthanide ionsby ICP–MS and the high stability of those reagentsavoiding metal loss or metal exchange, the highly polarcomplexes and their derivatives are often not suitablefor separations on reversed-phase (RP) columns. Unfortu-nately, without a satisfactory separation of the derivatized

biomolecules, an absolute quantification cannot be ex-pected. Baseline separation of the derivatives is necessaryto avoid suppression effects when using LC/ESI–MS.However, the use of chelating compounds as derivatizingagents is mainly combined with LC/ICP–MS as the

N N

N N

HOOC

COOH

HOOC

N

O

O

La3+

COOH

R

chelate complex (e.g. DOTA)

lanthanide ion

R = biotin

cysteine selectivemaleimide group

N N

N N

HOOC

COOH

HOOC

N

O

O

La3+

COOH

R

chelate complex (e.g. DOTA)

lanthanide ion

R = biotin

cysteine selectivemaleimide group

Fig. 2 Structure of the DOTA-based metal-coded affinity tag(MeCAT©) reagent. The substituted maleimide reacts selectively withcysteine residues. The biotin group provides the possibility of avidin-biotin chromatography for purification. According to the low limits ofdetection in ICP–MS, the lanthanide ion allows absolute quantificationof the analytes

„Normal“ mouse „Giant“ mouse

a b

S

NHNH

O

O

NH

OH

HH

HO

O NH

H

HH

HO

I

S

NHNH

O

O

NH

OD

DD

DO

O NH

D

DD

DO

I

Extraction of proteins from blood

Addition of

heavy (D) label

Mixture of both & enzymatic digest

light (1H) label

„Normal“ mouse „Giant“ mouse

S

NHNH

O

O

NH

OH

HH

HO

O NH

H

HH

HO

I

S

NHNH

O

O

NH

OD

DD

DO

O NH

D

DD

DO

I

Extraction of proteins from blood

Addition of

heavy (D) label

Mixture of both & enzymatic digest

light (1H) label

Fig. 1 Labeling procedureusing isotope-coded affinity tags(ICATs) for the determination ofindividual proteins originatingfrom two different samples(here: “normal” and “giant”mice). The ICAT reagentconsists of on isotope-codedlinker group (containing eight1H or eight D atoms) and athiol-reactive iodoacetamidegroup for the selectivealkylation of cysteine residues.Additionally, a biotin moietyallows the enrichment of thecysteine-containing labeledproteins via biotin-avidinaffinity chromatography


hyphenated technique. Through its compound- and matrix-independent ionization, large dynamic range of over6 decades, and excellent limit of detection in the nanogramper liter range the ICP–MS technique has become oneof the most versatile and sensitive tools in bioanalyticalchemistry today.

Metallocarbonyl complexes

During recent years, several new metallocarbonyl com-plexes have been designed which allow side-chain-selectivereactions in the formation of protein adducts. Metallocar-bonyl complexes endow the resulting bioconjugates withunusual redox properties [53]. Their IR absorption [54] andluminescence features [55, 56] enable their sensitive detec-tion in biological samples. The complexes and their conju-gates display sharp and intense absorption bands resultingfrom the stretching vibrations of metal-coordinated COligands in the 1,850–2,150 cm−1 region. Since this wave-length region is free of any absorption band caused by bio-molecules and biologically matrices, undisturbed detectiondown to the picomole level is possible [57]. Furthermore, theintroduction of these species facilitates the crystallization ofthe corresponding adducts and, hence, its identification byX-ray analysis [58], which provides structural informationat the atomic level. Resolution of the three-dimensionalstructure of biological molecules—in particular proteins—isone of the key steps in the study of biochemical mechanismsat the molecular level.

So far, metallocarbonyl complexes have been used forthe labeling of drugs, proteins, and oligonucleotides.Several studies on the synthesis of η5-Cp-metallocarbonyllabeling reagents with M = Mo [59], W [59, 60], Re [61,62], Co [63], and Fe [64–67] and their reactions withbiomolecules have been reported (Fig. 3). Furthermore,Metzler-Nolte et al. investigated the use of molybdenumallyl dicarbonyl compounds for the coupling of amino acidsand C-terminal functionality by standard peptide chemistrymethods [68]. Other metallocarbonyl complexes containingCo [69], Cr [70] Ru, Os, and Ir [71] as markers forbiomolecules are also presented in the literature. Atomicspectroscopy devices such as ICP–MS have not been used

for the detection of metallocarbonyl complex-labeled com-pounds in any of the studies presented so far.

One further application integrating metallocarbonylcomplexes is the development of the carbonylmetalloimmunoassay (CMIA), which was introduced by Jaouenet al. [72–74]. This assay is based on IR detection, makinguse of the stretching vibrations of organometallic carbonylgroups used in labeled antigens. The obtained sensitivity iscomparable to that of a radioactive tracer. For routineanalysis, for example, in hospitals, this approach must becompared with the established, nonradioactive assays basedon fluorescence detection or enzymatic methods, whichguarantee high selectivity and sensitivity in addition tosimplicity. However, these traditional methods are limited tothe analysis of only one analyte [75].

Carbene complexes

The presence of an electrophilic carbenic carbon atom makescarbene complexes suitable for nucleophilic substitution byprimary and secondary amines, yielding very stable metal-loaminocarbenes. Proteins possess several potentially nucle-ophilic residues which can be modified by using carbenecomplexes (Fig. 4).

Fischer-type carbene complexes

The electrophilic property of tungsten carbene complexesof the Fischer-type was first used to label amino acidderivatives and the model protein bovine serum albumin(BSA) [76]. Side-chain-selective labeling was observed,involving the α-amino group borne by some of the lysineresidues. The derivatives were characterized and confirmedby IR and UV–vis spectroscopy. Other tungsten-containingFischer carbene complexes were employed to derivatizelysozyme to form stable protein–aminocarbene conjugates,which were analyzed using RP-HPLC, combined withMALDI time-of-flight (ToF) mass spectrometric analysis[77]. Today, one of the main aims in this field is to provideprotein crystallographers with new heavy metal reagents tobe used in the course of the determination of proteinstructures by X-ray diffraction.

CO

O N

O

OMLn

OC Me


+ R NH2

CO

HN

MLn

OC Me

R

HO N

O

O

-

CO

O N

O

OMLn

OC Me


+ R NH2

CO

HN

MLn

OC Me

R

HO N

O

O

-

Fig. 3 Reaction of ametallocarbonyl complex withan amine. The derivativescan easily be detected byinfrared spectroscopy


N-heterocyclic carbene complexes

In the past, N-heterocyclic carbene (NHC) complexes havebeen widely investigated for their potential as catalysts fororganic or industrial synthesis [78]. Recently, the use ofruthenium- and rhodium-containing complexes with func-tionalized NHC ligands was presented for the labeling ofthe neuropeptide enkephalin via solid-phase peptide syn-thesis (SPPS) [79]. The modified peptide was furthercharacterized by HPLC, 1H- and 13C-NMR as well as byESI–MS. Other groups have used NHC–metal complexesas labeling reagents for sugars [80] and peptides [81]. Theinvestigation of the biological properties of analytes, forinstance their cytotoxicity, is one of the main goals for thefuture.

NHC–metal complexes provide excellent properties forthe modification of biomolecules with respect to the stability,solubility, and analytical properties of the resulting con-jugates. It will be interesting to investigate the synthesis ofNHC–metal complexes for biolabeling without the use ofSPPS. Additionally, the incorporated metals Ru and Rhprovide the best detectability of the formed bioconjugates byusing atomic spectroscopymethods like ICP–MS and show ahigh potential for quantitative analysis of proteins. With suchpromising properties, the synthesis of new NHC complexescontaining other trace metals is conceivable in future work.

Metallocene-based derivatizing agents

Commonly, metallocenes with the general formula M(C5H5)2 contain two cyclopentadienyl anions bound toa metal center (M), usually appearing in the oxidationstate of +2. Since the discovery of the most importantand best-known representative—ferrocene—in 1951, manyapplications and synthesis of other metallocenes have beenpublished.

To date, metallocene derivatives have been coupled withamino acids, peptides, and proteins, to mention a few.Moreover, metallocene reagents were used for the labelingof drugs [82], in biological assays [83], and for the labelingof the DNA [84, 85]. Because of the high stability ofmetallocene moieties towards 9-fluorenylmethoxycarbonyl(Fmoc), amino acid side-chain-deprotecting reagents, andresin cleavage, a lot of metallocene–peptide conjugateswere prepared by SPPS [86] (Fig. 5). It is known that theincorporated metal influences the stability of the formedconjugates in biological media; metal-specific modes ofaction such as lipophilicity and redox potential of the metalcomplex were implicated. Moreover, metallocene derivativeshave become significant in drug design, whereby theintroduction of organometallic ferrocenyl groups enhancesthe activity of the compound and changes the pharmacody-namic profile [87]. Whereas titanocene dichloride (Cp2TiCl2)[88] and, to a more limited extent, the structurally relatedmetallocenes of the type Cp2MCl2 with M = Mo, V, Nb, Re[89–91] have entered clinical trials, only ferrocene andcobaltocinium-based reagents have been directly used for theclassical labeling of biomolecules. Although cyclopenta-dienyl compounds can be formed with any transition metal,most of them are not suited for labeling of biomolecules. Therequired properties for their use as labeling agents includehigh stability, inexpensive and efficient synthesis, and lowtoxicity. Since such properties, at least in part, can beprovided by the two main representatives—ferrocene and itsisostructural cobaltocinium—their potential for labeling shallbe further discussed.

Ferrocene bioconjugates

Ferrocene is the most important metallocene and the firstone investigated. Ferrocene-based derivatization has raisedconsiderable interest in many fields of analytical chemistry.

CO2R

NN


L(CO)4W COR1

R2

a)

b)

R3R4NH

-R1OHL(CO)4W C

NR3R4

R2


1

CO2R

NN


L(CO)4W COR1

R2

a)

b)

R3R4NH

-R1OHL(CO)4W C

NR3R4

R2


1


1

Fig. 4 a Structure and reactivityof a Fischer carbene complex.The complexes undergo facileaminolysis with primary andsecondary amines, leading tostable aminocarbenes [76].b Structure of a rhodiumN-heterocyclic carbene (NHC)complex [79]


Two reviews nicely summarize the formation of ferroceneconjugates with amino acids, peptides, and proteins and theiranalytical and biological applications [92, 93]. Ferrocene canundergo electrophilic aromatic substitution reactions on thecyclopentadienyl ring(s). Other metallocenes frequentlydecompose under such reaction conditions [94]. Over thepast few decades, a large number of ferrocene-based reagentswere developed for several functional groups, whereby thelargest number of reagents is dedicated to the analysis ofamino functionalities. This is due to the fact that the aminoacid lysine with its aliphatic ε-amine group is present tosome extent in peptides and proteins and is often quiteabundant in biomolecules [95]. Apart from substitutedamine-reactive groups, reagents which react selectivelytowards alcohols [96–98], aldehydes [99], carboxyl groups[100, 101], dienes [102, 103], imino groups [104], isocya-nates [105, 106], and thiols [107] are now well known andestablished.

The non-polar ferrocene-based derivatizing agents turnhighly polar analytes into less polar reaction products andthus make them suitable for separation on reversed-phase

columns. Ferrocenes (Fe(II)) can be easily oxidized to thecorresponding ferrocinium cation (Fe(III)). As a result of thisone-electron redox behavior, ferrocenes enable the detectionof originally non-electroactive analytes by electrochemicalmethods like cyclic voltammetry [108]. For the detection ofthe derivatized biomolecules, mass spectrometric approacheslike ICP–MS [109, 110], ESI–MS [111, 112], and MALDI–MS [104] were applied (Fig. 6). Additionally, applicationsusing atomic spectroscopic detection such as atomic absorp-tion spectroscopy (AAS) [113, 114], atomic emissiondetection (AES) [115, 116], and electrochemiluminescence[117] have been published.

Cobaltocinium bioconjugates

The cobaltocinium ion is isoelectronic with ferrocene,thus providing even higher stability against strong oxi-dation reagents such as fuming nitric acid, potassiumpermanganate, and ozone [118, 119]. The presence of thepositive charge of the cobaltocinium unit may increase thehydrophilic properties of the analytes [120], making

a)

b)

Fmoc1) Fmoc deprotection

H2) Coupling to amino acid

Phe Fmoc

3) Repeating steps 1 and 2

Phe Arg Lys Fmoc

4) Fmoc-Deprotection & reaction with Metallocene-COOH

Phe Arg Lys

O

NH Fe

5) Cleavage & side chain deprotection

Phe Arg Lys

O

NH FeH2N

O

O N

O

OCo+ + R-NH2

O

NHCo+

N

O

O

O

O

NH

O

+ R-NH2Fe Fe

R

PF6

R

N

O

O + R-SHFe

O

NH

N

O

OFe

O

NH

SR

PF6

[107]

[109]

[125]

a)

b)

Fmoc1) Fmoc deprotection

H2) Coupling to amino acid

Phe Fmoc

3) Repeating steps 1 and 2

Phe Arg Lys Fmoc

4) Fmoc-Deprotection & reaction with Metallocene-COOH

Phe Arg Lys

O

NH Fe

5) Cleavage & side chain deprotection

Phe Arg Lys

O

NH FeH2N

O

O N

O

OCo+ + R-NH2

O

NHCo+

N

O

O

O

O

NH

O

+ R-NH2Fe Fe

R

PF6

R

N

O

O + R-SHFe

O

NH

N

O

OFe

O

NH

SR

PF6

[107]

[109]

[125]

Fig. 5 a Different ferrocene-and cobaltocinium-basedderivatizing agents for thioland amino functionalities.b Derivatization of anamino group by a ferrocenederivative using solid-phasepeptide synthesis (with =polymer and Fmoc =fluorenylmethoxycarbonyl)


cobaltocinium salts very attractive as haptens and tracersin biological systems [121, 122]. Metzler-Nolte et al.described the successful synthesis of a conjugate of acobaltocinium ion with an antigen nuclear localizationsignal (NLS) by SPPS, which specifically delivers theorganometallic species into the nucleus of a cell [123].The much higher redox potential and chemical stability ofthe cobaltocinium ion over the ferrocene moiety were usedfor enhancing cellular uptakes of bioconjugates. Moredetailed information on the synthesis of cobaltocinium–peptide bioconjugates prepared by solid-phase peptidesynthesis is published elsewhere [86, 124]. The potentialof these bioconjugates for biological assays is discussed,and metallohaptens of the activated cobaltocinium esterwith psychostimulant drugs such as amphetamine anddesipramine [125] and antiepileptic drugs such as pheno-barbital and phenytoin [126] were synthesized.

To date, the stable cobaltocinium conjugates werecharacterized by using 1H- and 13C-NMR, IR spectroscopy,X-ray crystallography, and ESI–MS, but no attempt hasbeen made so far to use separation techniques such asHPLC and capillary electrophoresis in combination withatomic spectroscopy detection possibilities like ICP–MS.

Unfortunately, as cobalt is a monoisotopic element, theuse of isotope dilution analysis for the quantification ofmodified biomolecules is not possible; however, evenexternal calibration and detection by ICP–MS offer limitsof detection in the lower parts per billion range, making thisapproach an attractive alternative.

Other metal-containing derivatizing agents

Mercury tags

Based on the strong mercury–sulfur affinity, mercurialreagents are known to be highly selective for thiols even inthe presence of all other types of reactive groups typicallypresent in proteins. Thus, the dissociation constants for themercaptide formed between the inorganic mercuric ion (Hg2+)and cysteine or the organic methylmercury (CH3Hg

+) andcysteine are 10−15.8 and 10−20.3, respectively [127]. Whereasdivalent mercuric ions (Hg2+) are capable of reacting in away to connect two thiol groups and to undergo interactionswith disulfide bridges [128], monofunctional organomercu-rial compounds of the type RHgX have the advantage ofonly reacting with one thiol group [129].

2 4 6 8 10 120

1.0

2.0

x106

Inte

nsi

ty [

cps]

Time [min]

LC/ESI-MS tR = 7.13

Time [min]

0.5

1.0

1.5

x104

0 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 12

123

4

x103

Time [min]

Inte

nsity

[cps

]

02 4 6 8 10 12

56Fe57Fe

LC/ICP-MStR = 5.37 57Fe

1200 1400 16000

0.5

1.5

x105

+13

+12

+10

+11

1231.0

1599.9

1454.4

1333.4

Inte

nsi

ty [

cps]

m/z15000 16000

0

2.0

4.0

15987.0x105

Deconvolution

16227.0

m/z

B

C

N

O

O

O

O

NH

O

+ R-NH2

pH 9Fe Fe

R

N

O

O

HO-

A

2 4 6 8 10 120

1.0

2.0

x106

Inte

nsi

ty [

cps]

Time [min]

LC/ESI-MS tR = 7.13

2 4 6 8 10 120

1.0

2.0

x106

Inte

nsi

ty [

cps]

Time [min]

LC/ESI-MS tR = 7.13

Time [min]

0.5

1.0

1.5

x104

0 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 12

123

4

x103

Time [min]

Inte

nsity

[cps

]

02 4 6 8 10 12

56Fe57Fe


Time [min]

0.5

1.0

1.5

x104

0 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 12

123

4

x103

Time [min]

Inte

nsity

[cps

]

02 4 6 8 10 12

56Fe57Fe


0.5

1.0

1.5

x104

0 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 120 2 4 6 8 10 12

123

4

x103

Time [min]

Inte

nsity

[cps

]

02 4 6 8 10 12

56Fe57Fe


1200 1400 16000

0.5

1.5

x105

+13

+12

+10

+11

1231.0

1599.9

1454.4

1333.4

Inte

nsi

ty [

cps]

m/z15000 16000

0

2.0

4.0

15987.0x105

Deconvolution

16227.0

m/z1200 1400 16000

0.5

1.5

x105

+13

+12

+10

+11

1231.0

1599.9

1454.4

1333.4

Inte

nsi

ty [

cps]

m/z15000 16000

0

2.0

4.0

15987.0x105

Deconvolution

16227.0

m/z

B

C

N

O

O

O

O

NH

O

+ R-NH2

pH 9Fe Fe

R

N

O

O

HO-

AFig. 6 a Reaction scheme forthe derivatization of an aminewith an amine-selectiveferrocene-based reagent to thecorresponding amide. b LC/ESI–MS and LC/ICP–MSmeasurements of the derivatizedprotein lysozyme. Variations inretention time are due to differ-ent void volumes in the usedHPLC instrumentation. c Massspectrum and deconvolution re-sult of the derivatized proteinlysoyzme; all six lysine residuesand the N-terminus of the pro-tein were fully derivatizedwith the ferrocene tag


For example, p-hydroxymercuribenzoic acid (pHMB)has been used for the derivatization of metallothioneins[130] and glutathione [131] with detection by ICP–MS andatomic fluorescence spectrometry, respectively. Moreover,pHMB has been applied for the derivatization of peptidesand proteins (Fig. 7), using hyphenated techniques based onchromatography [132], reversed-phase chromatographycoupled on-line with cold vapor generation atomic fluores-cence spectrometry (CVG–AFS) [133] as well as ESI–MSand ICP–MS [134]. Furthermore, the selective interactionof monomethylmercury chloride and monoethylmercurychloride with thiol groups was exploited for counting thenumbers of free thiols (–SH) and disulfide (S–S) bonds inproteins [135]. The first strategy of absolute quantificationof proteins labeled by CH3Hg

+ was recently presented byWang et al. [136], using HPLC coupled to ICP–MS withCH3HgCl as external standard.

As a result of the direct covalent bond between sulfur andmercury, avoiding the necessity of any additional “bridge”towards the sulfhydryl group, the mercury bioconjugateshave unsurpassed stability. Unfortunately, the relatively highionization potential of mercury, the noxious properties of allmercury species, and persistent memory effects seriouslylimit the attractiveness of mercury compounds for routineuse as labeling agents for biological samples.

Iodine tags

Apart from mercury, other possibilities exist to introduceelement tags into biomolecules. Thus, the iodination ofproteins followed by the detection of the stable iodineisotope 127I can be exploited for the analysis of biomole-cules [137]. In this approach, the target protein is treatedwith I+, which can attack the ortho-positions of the aminoacid tyrosine and the 2,5-positions of the imidazole ringof histidine residues. Recently, Navaza et al. published anew approach, which applies a specific iodine-containingcompound only for the selective iodination of tyrosine[138]. To date, iodine-containing tags have been used only

rarely. However, the radiolabeling of proteins with 125I hasbeen used to track proteins in an organism. Recently, stableiodine isotopes were applied for the derivatization ofdifferent antibodies, which target forms of the cytochromeP450 enzyme family [139]. For analysis, gel electrophoresiscombined with laser ablation (LA)–ICP–MS was employed.

Concluding remarks and future perspectives

In bioanalysis, effort is progressively shifting towardshighly parallel, high-throughput, and highly multiplexedapproaches to obtain large amounts of data with increasingefficiency [140]. The strategy of ICP–MS-based immuno-assays is straightforward and numerous studies have beenpublished on this research field today. Whereas an immuno-assay can be described as the detection and quantification ofa specific biomolecule (antigen) with a complementaryantibody and is distinguished by its high specificity, thecombination with ICP–MS offers a new approach to theproteomics challenges. Hence, the first successful class ofreagents for elemental tagging of antibodies was reportedrecently [141]. First promising results obtained with ICP–MSas a detection system [142] certainly warrant furtherexploration of its capabilities by more laboratories. In severalpublications, nanogold and lanthanide-tagged antibodies areemployed [143–148], and the labeled antigens are furtherinvestigated by atomic spectroscopy devices. Recent discov-eries include the use of new polymer-based elemental tags inbioassays [149].

As isotopic labeling is a very promising tool for thequantification of biomolecules, but is mostly restricted toisotopic labeling of carbon, hydrogen, and oxygen, thisstrategy is principally also possible with most organometalliccompounds. Some recent publications [47, 150] concentrateon this new approach, which definitely has high potentialfor the accurate relative and absolute quantification of bio-molecules. Moreover, some publications deal with the use ofpost-column isotope dilution in combination with ICP–MS

SH

H2N

COOH

COOH

HgH

+

COOH

HgS

H2N

COOH

SH

H2N

COOH

SH

H2N

COOH

COOH

HgOH

+

COOH

HgS

H2N

COOH

Fig. 7 Structure of p-hydroxymercuribenzoic acid (pHMB) and the reaction with a cysteine-containing peptide. The derivatization of eachsulfhydryl group causes a mass shift of m/z 321


as a detector for bioanalytical purposes and showed verypromising results recently [15, 151]. Especially in this field,progress can be expected in future work.

Furthermore, new approaches including the utilization ofnanoparticles as derivatization reagents for antibodies [152]and oligonucleotides [153] and their subsequent analysis byICP–MS have been presented. All of these approaches com-bine the use of immunoreactions coupled with ICP–MS-baseddetection. This general strategymay offer new possibilities forbiological assays and clinical diagnoses. Recently, a novelsandwich assay for human α-thrombin was reported whichtakes advantage of gold nanoparticles for signal amplificationin combination with ICP–MS detection [154].

New technological developments and their implementa-tion, for instance LA–ICP–MS, are promising tools in thefield of bioanalysis. The excellent separation of proteins bypolyacrylamide gel electrophoresis is well known, but theinvestigation of heteroatom-tagged proteins with LA–ICP–MS is a new, powerful tool for quantification. Monoclonal[139] and polyclonal [155] antibodies have been labeledwith metal-containing chelate complexes or even goldclusters [156]. After the immunoreaction of the labeledantibodies with the antigen, the element label was detectedby LA–ICP–MS.

Other groups separated the modified biomolecules bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS–PAGE) followed by LA–ICP–MS detection. In thisarea, challenges have to be overcome in the future. Forexample, a drawback of the labeling procedure using chelate-based derivatizing agents is the change of electrophoreticmobility during gel electrophoresis due to the relatively highmolecular weight of the chelate added to the weight of thebiomolecule. However, the large variety of chemical andtechnological innovations clearly indicates the excellentfuture perspectives of organometallic derivatizing agents inbioanalysis.

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