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Originally published In Press as doi:10.1074/mcp.M700152-MCP200 on July 11, 2007.
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Molecular & Cellular Proteomics 6:1907-1916, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Metal-coded Affinity Tag Approach to Quantitative Proteomics*

Robert Ahrends{ddagger},§,, Stefan Pieper{ddagger},, Andreas Kühn{ddagger}, Hardy Weisshoff{ddagger}, Meike Hamester||, Torsten Lindemann||, Christian Scheler§, Karola Lehmann§, Kerstin Taubner** and Michael W. Linscheid{ddagger},{ddagger}{ddagger}

From the {ddagger} Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor Str. 2, 12489 Berlin, Germany, || Thermo Fisher Scientific, Hanna-Kunath-Str. 11, 28199 Bremen, Germany, ** Institute for Analytical Sciences (ISAS), Albert-Einstein-Str. 9, 12489, Berlin, Germany, and § Proteome Factory AG, Dorotheenstr. 94, 10117 Berlin, Germany


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.

Proteomics as a field of research is based on the characterization of an entire proteome of a biological system. A variety of approaches have been developed during the last decades to characterize such mixtures of proteins and peptides, and necessarily, all of them use separation techniques. At the protein level, separation has been achieved using 2-D1 gel electrophoresis (1) and densitometry of stained proteins or fluorescence detection (2). After digestion of the proteins, peptides were identified using liquid chromatography, mass spectrometry, or both (3, 4). However, this information was only qualitative. It became rapidly evident that quantitative data were definitively required, e.g. for the characterization of dynamic biological systems or the search for biomarkers in clinical proteomics. Subsequently methods have been developed for the quantitative determination of proteins and peptides mainly based on chemical or metabolic isotopic labeling combined with LC/MSn detection (5, 6). Label-free LC/MS quantitative strategies are under development as well (7).

Using such techniques, the investigation of changes of the proteome in biological systems has become possible. However, only relative changes can be monitored, and currently, with a few exceptions such as the absolute quantification (AQUA) method (8) or QCAT (9), no labeling techniques are available to determine accurate amounts particularly of whole proteins.

Here we describe a novel strategy for the relative and absolute quantification of peptides and proteins. The method is based on chemical labeling, but instead of isotopes, the different lanthanide ions in macrocyclic complexes are used (10). The molecular structures of the metal labels used are shown in Fig. 1. If required, the structures of peptides can be determined using ESI or MALDI MS/MS data, but the quantitative information comes from inductively coupled plasma (ICP) MS measurements.


Figure 1
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  		FIG. 1. Structures of the two MeCAT reagents used in this study for thiol group labeling. MeCAT exists in different metal variants depending on the incorporated trivalent lanthanide ion (M). A shows MeCATBnz consisting of the DOTA macrocycle for metal coding (I); a spacer (II), which connects the macrocycle; and the maleimido group (III) for thiol-specific labeling. B shows MeCATBio that additionally carries a biotin group (IV) for further affinity purification.

 
ICP MS has been used already for the accurate and sensitive quantification of elements in many biological samples (11–13). Thus, it should be well suited for quantification of metal-labeled, low abundance proteins particularly in combination with enrichment and preconcentration procedures. The technique provides extraordinary detection capability, and it is unaffected by the nature of chemical structures (12), which allows the use of simple internal standards containing appropriate metals. Detection limits of 80 pg/g (parts per trillion) for 157Gd and 2 fg/g (parts per quadrillion (fg/ml)) for 175Lu have been reported by Zhang et al. (15).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The Reagents and Workflow
The synthetic access to DOTA metal complexes has been shown already (16). We used two different labeling reagents named MeCATBnz and MeCATBio (Fig. 1). They contain maleimide as the thiol-specific group and DOTA macrocycle, which has been used previously as an immunoglobulin recognition site for affinity purification (16). The two reagents differ in the spacer region, which is short for MeCATBnz, whereas MeCATBio possesses a biotin-bearing spacer to allow affinity purification (17, 18) The reaction of the DOTA macrocycle with lanthanide metal ions (M3+) results in stable metal chelate complexes (see Table I) (19, 20). The different masses of the complexes (21) allow detection of differently labeled species using ESI MS and ICP MS.


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  		TABLE I Exact masses and stability constants of the DOTA complexes of the lanthanides used (20, 21)

 
For the quantitative analysis of a protein mixture, MeCAT was combined with high resolution 2-D electrophoresis using dissolvable gels (DPAGE, Proteome Factory AG). Samples were denatured and reduced before reaction at the cysteine residues with M-MeCAT reagents (M = lanthanides). Then the samples were mixed, and the mixtures were divided into two portions, which were separated using analytical and micropreparative 2-D electrophoresis, respectively. After staining, the spots of interest were picked, dissolved for quantification by FIA/ICP MS, or digested for identification using HPLC/MSn. The current workflow is displayed in Fig. 2.


Figure 2
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  		FIG. 2. Current scheme of the MeCAT workflow. Cysteine thiol residues of proteins from different samples were differently labeled with metal-coded M-MeCAT reagents. After combination of the samples, the protein mixture can be analyzed by any separation method such as SDS-PAGE or 2-D electrophoresis. For absolute quantification, the slices/spots were dissolved and analyzed for lanthanide content using FIA/ICP MS. Because the proteins from the different samples are modified with a specific M-MeCAT label, the products differ in mass. This allows for the determination of the intact proteins using MALDI and ESI-MSn as well as for the metal detection with ICP MS. After proteolysis, any necessary identification may follow at the peptide level using LC/ESI MSn. The tag is indicated by a star.

 
Labeling of Peptides
The commercially available peptides (Bachem, Weil am Rhein, Germany) were reduced with 2 mM TCEP at 37 °C for 30 min before a 10-fold excess of M-MeCATBio reagent (M = Lu(III), Ho(III), Tb(III), Tm(III)) was added. The reaction was carried out in 10 mM HEPES (pH 6.5) for 2 h at 37 °C. Finally the reaction was stopped using an excess of DTT.

Labeling of Standard Proteins
The proteins used in this study were bovine serum albumin (Bos taurus) and {alpha}-lactalbumin (B. taurus) from Sigma-Aldrich. Proteins (1.25 nmol) were reduced in 2 mM TCEP for 30 min at 37 °C and labeled using different M-MeCATBnz reagents (M = Lu(III), Ho(III), Tm(III)) in a 20-fold excess to each cysteine residue. The reaction was carried out in a solution containing 5 mM EDTA, 50 mM sodium acetate (NaOAc), and 5% acetone for {alpha}-lactalbumin and 5 mM EDTA, 25 mM NaOAc, and 10% acetone for BSA. Acetone and EDTA were used to achieve complete denaturation of proteins. The reaction mixture was incubated for 12 h at 37 °C, and the reaction was stopped using DTT.

Labeling of Eye Lens Proteins for 2-D Electrophoresis
Fresh eyes lenses from a 1-year-old pig (Sus scrofa) were freshly prepared on ice as described previously (22). The proteins were dissolved in 9 M urea, 70 mM TCEP, and 1% CHAPS. The amount of protein was determined by the Bradford assay (23). Two hundred and fifty micrograms of lens protein (samples A and B) were reduced with 10 mM TCEP for 30 min at room temperature. The protein samples A and B were labeled with 800 µg of Lu(III)- or Tm(III)-MeCATBnz, respectively. The reaction was carried out in a solution containing 5 mM EDTA, 50 mM NaOAc, and 10% acetone at 37 °C, and the reaction was stopped after 12 h using an excess of DTT.

Affinity Purification
Prior to affinity purification, 35 µl of water and 10 µl of 10-fold binding buffer (500 mM Tris-HCl, pH 7.4, and 10 mM DTT) were added to 55 µl of solution containing 20 pmol of M-MeCATBio (M = Ho(III), Lu(III), Tm(III), Tb(III))-labeled peptide (HIV integrase inhibitor) and 10 µg of tryptically digested Escherichia coli cell lysate. For affinity purification of MeCATBio-labeled peptides, a modified protocol of Girault et al. (18) was used using streptavidin-coated magnetic beads (M-280, Invitrogen). After incubation and immobilization of 10 µl of streptavidin-coated magnetic beads with a magnetic concentrator, the supernatant was removed, and the beads were washed twice with 100 µl of cleaning buffer (500 mM Tris-HCl, pH 7.4, 10 mM DTT, and 1 mg/ml BSA) followed by 100 µl of binding buffer (50 mM Tris-HCl, pH 7.4, and 1 mM DTT). Next the beads were mixed with 100 µl of peptide mixture, prepared as described above, and incubated for 60 min at 25 °C with gently shaking. The supernatant was removed, and the beads were washed four times with 100 µl of washing buffer (50 mM Tris-HCl, pH 7.4, and 0.01% (w/v) SDS), four times with 100 µl of 1 mM DTT, and finally three times with 100 µl of water. After removal of the supernatant, peptides were eluted with 50 µl of 0.1% (v/v) TFA and 40% (v/v) ethanol by incubation for 5–10 min at 60 °C, and the supernatant containing the eluted labeled peptides was dried in a SpeedVac and stored at –20 °C until analysis.

SDS-PAGE to Monitor Efficiency of Metal-coded Labeling
To demonstrate labeling efficiency, following the labeling reaction MeCAT-coded proteins were subjected to SDS-PAGE as described by Laemmli (24) using 4% (w/v) stacking gel and 15% (w/v) separating gels. To visualize the mass shift of metal-coded proteins, gels were stained with silver (25) or a colloidal Coomassie G-250 solution (26).

2-D Electrophoresis
Without further purification or fractionation, the M-MeCAT-labeled samples (M = Tm(III), Lu(III)) were subjected to 2-D electrophoresis analysis. Soluble proteins were separated by the high resolution 2-D electrophoresis technique according to Klose and Kobalz (26). Large (30 x 23-cm) soluble 2-D gels (DPAGE, Proteome Factory AG) were prepared from a ready-made gel solution. Analytical and preparative gels were 1 mm thick. For analytical runs 100 µg and for preparative runs up to 500 µg of the combined lens samples were applied at the anodic side of the gel. In the first dimension vertical IEF was performed using carrier ampholytes in the range of pH 2–11. The second dimension was a vertical SDS-PAGE as described by Laemmli (24) using 15% (w/v) DPAGE gels. After electrophoresis, the analytical gels were stained with silver nitrate, and preparative gels were stained with Coomassie Brilliant Blue G-250. For the subsequent analyses protein spots were digested either in gel or solubilized according to the DPAGE protocol for FIA/ICP MS analysis.

Digestion
For in-gel digestion pieces were cut out, transferred to vials, and destained (10% acetic acid, 30% ethanol, and 60% water (v/v/v)). The pieces were then rinsed with 100 µl of 50 mM ammonium hydrogen carbonate buffer, dehydrated with 100 µl of 100% acetonitrile four times, and dried in a SpeedVac for 15 min. Fifty microliters of 50 mM ammonium hydrogen carbonate buffer and 2 ng of trypsin were added to each vial; the digestion was carried out for 12 h at 37 °C. After centrifugation, the supernatant was carefully removed, and the pellets were reconstituted in 50 µl of 50 mM ammonium hydrogen carbonate buffer for 30 min, centrifuged, and freed from the supernatant. Finally all pellets were extracted with 50 µl of acetonitrile. The supernatants were pooled and analyzed.

Peptide Analysis by Nano-LC/ESI MS/MS
Digested samples were dissolved in 0.1% (v/v) formic acid to a final concentration of 500 fmol/µl. For LC/MS/MS, an 1100 nano-LC system (Agilent, Santa Clara, CA) was used. Zorbax 300SB-C18 3.5-µm, 150-mm x 75-µm and Zorbax 300SB-C18 3.5-µm, 0.3 x 5-mm (Agilent) enrichment columns were used. Separation was carried out using a binary mobile phase water/acetonitrile gradient with a maximum flow rate of 0.29 µl/min. The eluents were: A (isocratic pump): 98.5% deionized water, 1% acetonitrile, and 0.5% (v/v) formic acid; B (gradient pump): 94.9% deionized water, 5% acetonitrile, and 0.1% (v/v) formic acid; C (gradient pump): 99.9% acetonitrile and 0.1% (v/v) formic acid. For peptide separation, the sample was injected via autosampler onto the enrichment column using a 20-µl flow (A).

The separation column was directly coupled to a Bruker Esquire 3000 plus (Bruker Daltonics, Bremen, Germany) via a nano-ESI source using a 10-µm-inner diameter PicoTip emitter (New Objectives, Woburn, MA). For MS and MS/MS experiments, the following mode and tuning parameters were used: scan range, 100–3000 m/z; polarity, positive; capillary voltage, 1550 V.

High resolution MS and MS/MS experiments were carried out using a Finnigan LTQ FT mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) interfaced to a nano-HPLC system (Agilent 1100) using the conditions described above. MS and MS/MS data were recorded using the following parameters: scan range, 350–2000 m/z; polarity, positive; capillary voltage, 1600 V; tube lens voltage, 10 V.

Peptide Analysis by MALDI-TOF MS
Peptides were analyzed on a Bruker Daltonics MALDI-TOF mass spectrometer (Reflex II) with {alpha}-cyano-4-hydroxycinnamic acid as matrix. The samples were prepared by the dried droplet method. A mixture of angiotensin I, angiotensin II, and substance P was used for external mass calibration. The accuracy of peptide mass measurement was about 10 ppm.

FIA/ICP MS
LC/ICP Quadrupole MS—
For analysis of lens proteins in samples extracted from polyacrylamide gels, a combination of a Famos Ultimate II instrument (LC Packings, Sunnyvale, CA) and Elan 6000 ICP mass spectrometer (PerkinElmer Life Sciences) (plasma power, 1100 watts; nebulizer gas flow, 1.5 liters/min) was used. As an interface between the LC and ICP MS instruments the microconcentric nebulizer MCN-6000 with a membrane desolvation system was used (Cetac, Omaha, NE). Standard solutions for external calibration (18 fmol, 36 fmol, 72 fmol, 144 fmol, 288 fmol, and 2.88 pmol) were prepared using a multi-element standard mixture (Merck) containing holmium and lutetium.

FIA/ICP HR Sector Field MS—
The analysis of labeled standard proteins was carried out on an Element XR (Thermo Fisher Scientific) (plasma power, 1450 watts; nebulizer gas flow, 0.87 liters/min; nebulizer, concentric PFA in combination with a Surveyor HPLC system (Thermo Fisher Scientific)) with a flow rate of 200 µl/min. Standard solutions for external calibration (2.5, 5, 10, 100, 1000, 5000, and 10,000 fmol) were prepared using a multi-element standard mixture containing holmium and lutetium.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
To demonstrate MeCAT as a viable alternative to other labeling techniques, to show its use in relative determinations, and to prove that it is a unique option for absolute quantitative determination of proteins, it is necessary to show that the labeling of peptides and proteins is complete, reproducible, and robust. To this end, the method described above was applied to peptides (HIV integrase inhibitor peptide and tryptic BSA digest), to single proteins (bovine serum albumin and {alpha}-lactalbumin), and to the proteome of the porcine lens (S. scrofa).

Initially it was necessary to prove that the differently tagged peptides had the same retention time in HPLC separations. Two tryptic digests of BSA were labeled with M-MeCATBnz, one with holmium and the second with lutetium as metal core. Both digests were mixed in a ratio of 2:1, and the mixtures were analyzed using LC/MS and LC/MS/MS. The chromatogram in Fig. 3 shows two coeluting tagged peptides. The two peptides in each peak were present at the expected ratio of 2:1 (Ho(III):Lu(III); ESI MS).


Figure 3
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  		FIG. 3. Extracted ion chromatograms (EIC) of two selected BSA peptides (RPC*FSALTPDEYVPK and LC*VLHEK where C* represents the tagged cysteine) from a 2:1 mixture of two digests, each labeled separately with a M-MeCATBnz reagent coded with M = Ho(III) or Lu(III). The two differently tagged peptides coelute, and the two spectra reflect the expected ratio of 2:1. Integration of the chromatograms also results in a Ho(III):Lu(III) ratio of 2:1. This indicates that the metal ratio is unchanged during analysis; this is the prerequisite for the reliable relative quantification. Intens, intensity.

 
We also used this experiment to demonstrate that the labeled peptides could be identified by a common database search (Mascot data are not shown). As one example, the spectrum of the peptide LCVLHEK is shown in Fig. 4. In general, the CID MS/MS fragmentation pattern shows the typical y and b series.


Figure 4
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  		FIG. 4. A CID-ESI MS/MS spectrum of a Ho(III)-MeCATBnz-labeled BSA peptide obtained by separation of 800 fmol of tryptically digested BSA. The tag is indicated by a star. The spectrum shows the y and b series of the doubly charged precursor ion. In addition, the immonium ion of the labeled cysteine, the thiol-Ho(III)-MeCATBnz (HS-MeCATBnzHo) as base signal, and the precursor with loss of water are visible. Due to the relatively large mass shift of the tag, all the b ions carrying the tag are completely separated from the y ions. Intens, intensity.

 
In the next step we had to prove the complete reaction of all the available cysteines. At the peptide level, four samples of the HIV integrase inhibitor (sequence, HCKFWW) were labeled with four different metal-coded M-MeCATBio reagents (M = Tb(III), Ho(III), Tm(III), Lu(III)). Then the resulting reaction mixtures were combined in a ratio of 2:1:2:1 (Tb(III):Ho(III):Tm(III):Lu(III)). A survey mass spectrum (MALDI-reflectron-TOF MS) of the labeled peptide mixture is given in Fig. 5A. The expected ratios were found, whereas no unlabeled peptides were detected. Generally unlabeled peptides are detectable at least with the same sensitivity as labeled peptides. Thus, the results indicate that the labeling is complete.


Figure 5
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  		FIG. 5. A, MALDI-TOF MS spectrum showing the completely labeled HIV integrase inhibitor peptide (HIVII; HCKFWW; molecular weight, 905.4) with four different M-MeCATBio reagents (M = Tb(III), Ho(III), Tm(III), Lu(III)) and traces of the reagent and DTT adducts. The relative Tb(III):Ho(III):Tm(III):Lu(III) ratios of 2:1:2:1 in the M-MeCATBio are clearly visible. B, labeled HIV integrase inhibitor peptide spiked with a digested E. coli cell lysate. C, MALDI MS of the labeled peptide after affinity purification using streptavidin magnetic beads from the spiked E. coli cell lysate. The peptides were recovered, and the metal ratio remains unchanged. Intens, intensity.

 
To prove the quality of the recovery with affinity purification, the mixture of tagged peptides was spiked with a tryptically digested E. coli cell lysate. Fig. 5B shows the survey spectrum of the peptide mixture. Only peptides from the E. coli cell lysate are visible. After affinity purification, only the M-MeCAT-labeled HIV integrase inhibitor and traces of M-MeCAT reagents were detected. The ratio of metals remained unchanged (Fig. 5C).

For proteins, the reaction behavior of M-MeCATBnz was tested using BSA and {alpha}-lactalbumin. We monitored the labeling reaction of different ratios of proteins and reagents. For comparison, the products were separated by 15% SDS-PAGE. Because the MeCAT label changes the mass and the pI of the proteins considerably, the reaction progress could be monitored. Thus, the slowest migrating band contains the highest number of tags, whereas the unlabeled protein should be found in the first band. In Fig. 6, the results of the separation of differently tagged {alpha}-lactalbumin are shown. Depending on the excess of MeCATBnz reagent (M = Ho(III)) and the amount of acetone added to improve denaturation, all eight cysteines in the protein reacted. The nine {alpha}-lactalbumin species were observed, ranging from unlabeled to fully labeled species. At a 20-fold excess of M-MeCAT/cysteine, no further shift of the protein band was apparent.


Figure 6
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  		FIG. 6. Silver-stained SDS-PAGE of pure {alpha}-lactalbumin and six labeling reaction mixtures with different reaction conditions. The amount of acetone and the excess of M-MeCATBnz reagent (M = Ho(III)) were varied. The stained bands represent the different labeling stages up to the completely, 8-fold-labeled protein.

 
To study the migration behavior of the tagged proteins, a reduced sample of BSA was divided into two aliquots and labeled using M-MeCATBnz (M = Tm(III), Lu(III)). Then different amounts of labeled protein were separated by SDS-PAGE and Coomassie-stained. Following this analysis, only one band was visible (Fig. 7).


Figure 7
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  		FIG. 7. A, migration experiments on SDS-PAGE of M-MeCATBnz-labeled BSA (M = Lu(III), Tm(III)) mixed in equal amounts. The marked band (1 µg) was cut out, dissolved, and diluted for FIA/ICP MS analysis (B). Integrated peak areas resulted in a Lu(III):Tm(III) ratio of 1:1.

 
In Fig. 7 the analysis of a 1:1 (Tm(III):Lu(III)) mixture of M-MeCATBnz-tagged BSA is shown. To confirm that both metals appear at the expected ratio in the bands of the one-dimensional gel separation (Fig. 7A), the FIA/ICP MS data of one band are shown (Fig. 7B). The 1-µg BSA band was cut out, solubilized, and subjected to FIA/ICP MS for quantification (Fig. 7B). The two signals for thulium and lutetium are identical.

In Fig. 8, the data for the calculation of the detection limit of ICP MS for protein quantification are shown. To this end, different amounts of labeled BSA reaction mixture (16, 31, 63, 125, 250, 500, and 1000 ng) were separated using SDS-PAGE. After silver staining, the protein bands were cut out, solubilized, and diluted for FIA/ICP MS analysis. From these data an external calibration curve has been constructed (Fig. 9) that is linear over the complete range covered here (4 orders of magnitude). Detection limits of 2.31 fmol for lutetium and 2.29 fmol for holmium were calculated using standard procedures.


Figure 8
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  		FIG. 8. A, photograph of a silver-stained SDS-PAGE gel loaded with a dilution series of Ho(III)-MeCATBnz-labeled BSA. B shows the detection of the lowest amount of labeled protein by FIA/ICP MS not visible in gel.

 

Figure 9
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  		FIG. 9. A, FIA/ICP MS of holmium and lutetium standard solutions made up using the same conditions as those used for proteins samples (dissolved gel matrix and metal salt). B, shown are the calibration curves calculated from the data of the measured metal standards. c, concentration.

 
In an attempt to estimate the detection capabilities of the approach, BSA and lactalbumin were tagged and analyzed. With ICP MS, 3.95 fmol of holmium from Ho(III)-MeCATBnz-labeled BSA were detected. Assuming that all 35 cysteines in BSA were labeled, a BSA amount of 110 amol was calculated. For Ho(III)-MeCATBnz-labeled {alpha}-lactalbumin, 5.37 fmol of holmium was determined, corresponding to 670 amol of 8-fold labeled protein.

In the next step, the application of the technique to an analysis of protein mixtures was tested. Proteins from the porcine eye lens (S. scrofa) were tagged and analyzed. The proteins were diluted under denaturing conditions and reduced, and two samples were labeled using M-MeCATBnz (M = Lu(III), Tm(III)). Then the samples were combined, and the proteins were separated using micropreparative 2-D electrophoresis. The Coomassie-stained spots were cut out and digested with trypsin (27) for LC/ESI MS/MS analysis and identification (Table II).


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  		TABLE II Results of sequence analysis and quantitative analysis of S. scrofa eye lens proteins obtained from 2-D electrophoresis

Ss, S. scrofa; NCBInr, National Center for Biotechnology Information non-redundant; EST, expressed sequence tags (cDNA).

 
To quantify the proteins using ICP MS, the spots from a silver-stained analytical gel were cut out and dissolved (Fig. 10). Because the concentration of the metals was too high, the samples were diluted 25-fold and then analyzed using FIA/ICP MS.


Figure 10
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  		FIG. 10. Silver-stained 2-D electrophoresis of S. scrofa eye lens proteins. A shows the separation of unmodified proteins. In B the mixture of M-MeCATBnz-labeled proteins (M = Lu(III), Tm(III)) was separated. Spots 1–9 mark analyzed eye lens proteins. For the technical details, see "Experimental Procedures." Note that only the proteins with low numbers of tags are visible. The highly modified proteins do not migrate when the indicated pH range of the buffer system is used.

 
In Table II, qualitative data with names of proteins, searched database, accession number, Mascot score, and sequence coverage obtained from micropreparative 2-D gel are compiled. Spots 1–4 were identified as {alpha}-crystallin A species, spots 5/6 were identified as ß-crystallin B2, and spots 7–9 were identified as ß-crystallin B3 isoforms. Furthermore Table II shows quantitative data of the crystallins. The amounts of proteins range from 37 to 970 ng for the Lu(III)-MeCATBnz-labeled sample and from 44 to 760 ng for Tm(III)-MeCAT-labeled sample.

To explore the detection capabilities further, one major spot on this gel was thoroughly analyzed with a spatial resolution of 1 mm. The measured metal abundance was used to construct a three-dimensional profile of the entire spot (Fig. 11). The data provide evidence for the excellent sensitivity of ICP MS that allows detection of low protein concentrations even at the border of the spots.


Figure 11
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  		FIG. 11. Analysis of one single spot from a 2-D gel separation of two mixed S. scrofa eye lens protein samples tagged with Tm(III)/Lu(III)-MeCATBnz. A, the grid is shown indicating the dissection lines. The spot contains {alpha}-crystallin A, analogous to spot 1 of Fig. 10B. This spot was dissected in 45 gel pieces (1 x 1 mm), and each piece was screened for Lu(III) and Tm(III) using FIA/ICP MS. B and C show a three-dimensional surface map based on the metal amount and distribution of Tm(III)-MeCATBnz- (B) and Lu(III)-MeCATBnz-labeled (C) protein. The data are color-coded, ranging from 6 pmol (dark blue) to 22 fmol (green) of protein.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
The data given here present evidence that MeCAT is not only an alternative to other labeling techniques but opens a new dimension for protein quantification. We showed that with optimized reaction conditions the complete labeling of proteins and peptides was achieved. In addition, we demonstrated coelution of tagged peptides in HPLC and the option for identification of labeled peptides using LC/MSn data and a Mascot search as required. Differently labeled BSA and other proteins appear in one spot on SDS-PAGE separation, and we are presently investigating the generality of this observation for other proteins. The affinity purification of differentially labeled peptides turned out to be very efficient, and we never observed loss or exchange of metals from the tag. The thermodynamic (see Table I) and especially the kinetic stability of the complexes is such that loss or exchange even with possibly competing highly abundant endogenous metal ions is insignificant (28). Thus, the ratio of labeled peptides always remained stable during the analyses. This clearly demonstrates that the MeCAT approach is suitable for quantitative proteomics.

ICP MS for the quantitative detection of the metals was introduced to enhance the efficiency, the sensitivity, and the precision of MeCAT quantification. Because the metal tag increases the molecular mass considerably, for ESI MS there can be limitations. However, in our experience MALDI MS for control of the labeling reaction was always possible, and ESI MS was successful in most cases, generally for the more polar proteins. In fact, the number of charges for smaller proteins or larger peptides is increased. Another issue of concern is that, as with other chemical labeling techniques, this tagging technique changes the pI values of peptides and proteins. In this case, the pI values became more acidic. Therefore, the 2-D gel separations of tagged proteins may need adjusted pH ranges to optimize resolution. When large numbers of cysteine residues in proteins were tagged, the proteins would migrate to more acidic regions of the gel. This adds to the complexity of the analysis; however, the information about the larger number of tags also becomes immediately evident.

The virtually absent background due to the low natural lanthanide concentration and the detection capability of ICP MS provide the method with sufficient detection power for the absolute quantification of intact proteins at attomole levels (e.g. BSA, 110 amol). In the examples shown here, we have used the monoisotopic lanthanides to reduce the complexity in the ESI or MALDI spectra. In principle, the multi-isotopic metals may also be used at the expense of simplicity of molecular clusters when isotopic overlap (e.g. 158Gd and 158Dy) is excluded. Normally at least one isotope may be found for each metal that shows no overlap with others.

Optimized affinity preconcentration and purification schemes ultimately should lead to the quantification and subsequent identification of low abundance proteins. However, the unique metal tag extends the application for quality control and validation. We can always rely on simple, structurally unrelated internal standards because the only requirement is a lanthanide metal ion. Thus, each step in the analysis may be monitored. This means that all parameters can be optimized for reproducibility and increased yield. A further advantage of this technique is that a direct comparison of biologically unrelated proteins in complex mixtures is feasible, providing access to the analysis of protein complexes and protein species ("isoforms") with unique properties.

Another challenge for the quantitative protein analysis is the dynamic range of concentration in biological systems, spanning at least 10 orders of magnitude (29). As yet, only ICP MS offers a matching dynamic range. We have demonstrated in the course of this work a dynamic range of more than 3 orders of magnitude down to the attomole range. In principle, for ICP MS coverage up to 12 orders of magnitude for lanthanide samples has been shown (30), meaning that the dynamic range is not limited by the analytical technique. Furthermore analysis of one spot using FIA/ICP MS is rapid (60 s per spot), and automation can be implemented. It is evident that MeCAT cannot only be used for absolute quantification but also for relative quantification of four or more samples simultaneously. Because absolute determination is uncomplicated and accurate, even quite different samples and preparations can be compared; this is another unique aspect of this strategy. The prospect of covering such a dynamic range and the superior sensitivity encourages us to develop the technical parameters necessary to make MeCAT a viable alternative to other techniques available or that are under development.


   CONCLUSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In the course of the research described here, we could demonstrated that MeCAT can be used to quantify proteins in an absolute and a relative fashion. The method was established on several model peptides and proteins and by analysis of an eye lens proteome. The approach will be used in the future to quantify intact protein and protein species in complex mixtures using ICP MS and ESI MSn. For the identification of proteins, top down sequencing strategies and, after digest, peptide sequencing and database verification can be applied. Such studies are in progress. In addition, 2-D LC/MS and capillary electrophoresis techniques are under development to avoid the specific problems associated with the 2-D gel separation. For the analyses shown here, the change in pI values of proteins has been addressed. This change in pI makes adjustments of the pH profiles mandatory. The capillary electrophoresis or LC techniques may be able to circumvent these additional steps. The possibility to monitor each step in the analysis based on internal standards should provide reliable data that ultimately can be validated due to the traceability of ICP MS measurements.

Furthermore an adaptation of MeCAT to specific biological questions can be envisaged by variation of functional groups. The multiplexing screening ability of the MeCAT approach provides answers to specific quantitative questions and can be particularly useful for e.g. patient profiling and monitoring in medicine. With the reliable absolute quantitation possibilities, a technique is available for the first time that allows comparison of protein distributions in distant species. This opens up a way to compare different but related species based on proteomes and protein complex stoichiometry across species boundaries (14).


  FOOTNOTES
 
Received, April 3, 2007, and in revised form, July 2, 2007.

Published, MCP Papers in Press, July 11, 2007, DOI 10.1074/mcp.M700152-MCP200

1 The abbreviations used are: 2-D, two-dimensional; DPAGE, dissolvable PAGE; FIA, flow injection analysis; ICP, inductively coupled plasma; MeCAT, metal-coded affinity tag; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; M, lanthanide metal; HIV, human immunodeficiency virus. Back

* This work was supported by Deutsche Forschungsgemeinschaft Grant Li 309/25-3), Stiftung Industrieforschung Grant T3/2005, and Thermo Fisher Scientific. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Both authors contributed equally to this work. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Laboratory of Analytical and Environmental Chemistry, Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany. Tel.: 493020937575; E-mail: m.linscheid{at}chemie.hu-berlin.de


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