Top-Down FTICR MS for the Identification of Fluorescent Labeling

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Top-Down FTICR MS for the Identification of Fluorescent Labeling Efficiency and Specificity of the Cu-Protein Azurin Simone Nicolardi,† Alessio Andreoni,‡ Leandro C. Tabares,‡,§ Yuri E.M. van der Burgt,† Gerard W. Canters,‡ André M. Deelder,† and Paul J. Hensbergen*,† †

Biomolecular Mass Spectrometry Unit, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands Leiden Institute of Physics, Huygens Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands § CEA, Institut de Biologie et de Technologies de Saclay, 91191 Gif-sur-Yvette, France ‡

S Supporting Information *

ABSTRACT: Fluorescent protein labeling has been an indispensable tool in many applications of biochemical, biophysical, and cell biological research. Although detailed information about the labeling stoichiometry and exact location of the label is often not necessary, for other purposes, this information is crucial. We have studied the potential of top-down electrospray ionization (ESI)-15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to study the degree and positioning of fluorescent labeling. For this purpose, we have labeled the Cu-protein azurin with the fluorescent label ATTO 655-N-hydroxysuccinimide(NHS)-ester and fractionated the sample using anion exchange chromatography. Subsequently, individual fractions were analyzed by ESI-15T FTICR to determine the labeling stoichiometry, followed by top-down MS fragmentation, to locate the position of the label. Results showed that, upon labeling with ATTO 655-NHS, multiple different species of either singly or doubly labeled azurin were formed. Top-down fragmentation of different species, either with or without the copper, resulted in a sequence coverage of approximately 50%. Different primary amine groups were found to be (potential) labeling sites, and Lys-122 was identified as the major labeling attachment site. In conclusion, we have demonstrated that anion exchange chromatography in combination with ultrahigh resolution 15T ESI-FTICR top-down mass spectrometry is a valuable tool for measuring fluorescent labeling efficiency and specificity.

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stoichiometry can straightforwardly be obtained from total mass measurements. To specify the position of a label, the most common approach makes use of a proteolytic enzyme (e.g., trypsin) to digest the protein into smaller peptides. Within the context of fluorescent labeling, there is the advantage that it can be combined with chromatographic separation of peptides followed by fluorescence detection.7 The comparison of the mass spectra from an unlabeled and labeled protein digest is often sufficient to determine the (approximate) location of the label. Both the absence of specific unlabeled peptides in the labeled sample as well as the appearance of peptides with a mass increment owing to the fluorescent label has been used for this purpose,8,9 but preferably, additional tandem MS experiments are used to unambiguously assign the exact attachment site(s).7 To determine the number and location of labeled amino acids using the approach described above, a protein has to be measured preferably both before and after proteolytic digestion, and tandem MS experiments of putatively labeled peptides

he analysis of protein expression and (re)localization is of great importance in many areas of biochemical and cell biological research. Often, the visualization of these processes relies on fluorescent detection, for example by fusion of the target protein with protein or peptide tags.1−3 Alternatively, direct chemical protein labeling with a fluorophore, like in the field of antibody staining or visualization of protein uptake, is common practice, and many chemical functionalities have been used for this purpose. In general, the labeling efficiency and site of attachment are determined by the chemical functionality of the fluorescent compound, the side chain properties of the target amino acids, the accessibility of potential labeling sites, and the labeling conditions (e.g., pH). Apart from incorporation of specific targetable amino acids (e.g., cysteine) through site-directed mutagenesis, the labeling procedure is mostly considered as a “black box” and information about the labeling efficiency, stoichiometry, and positioning is often lacking, although these parameters can greatly influence the conclusions drawn from experiments that use these conjugates.4−6 Therefore, the development of robust and accurate methods to determine the above-mentioned labeling parameters are of great importance, and mass spectrometry (MS) based technologies are inherently suitable for this. In such experiments, the labeling efficiency and © 2012 American Chemical Society

Received: December 19, 2011 Accepted: February 7, 2012 Published: February 9, 2012 2512

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should be performed. Recent advances in the analysis of intact proteins using ultrahigh-resolution MS systems allow intact proteins to be selected as precursors for tandem MS-analysis instead of their smaller peptides: i.e., top-down proteomics.10−13 Consequently, the analysis of intact proteins allows for resolving the number of labels and assignment of the labeling sites in a single MS-experiment. The most common approach for top-down proteomics is electrospray ionization (ESI) tandem MS with precursor isolation in combination with various fragmentation techniques (collision-induced dissociation (CID), electron capture detector (ECD), or electron transfer dissociation (ETD)),14−16 although there is a growing interest using in source decay (ISD) during MALDI ionization, where no precursor ion selection takes place.17−19 Proteins of a few hundred kDa have been successfully analyzed using ESI20,21 even in an intact protein assembly.22 The technique of topdown MS is not limited to proteomics but has been used to study other molecules, for example, RNA, as well.23,24 Key to the interpretation of the resulting complex MS/MS spectra, even from relatively small proteins (10−20 kDa), is the necessity of a high resolving power in combination with accurate mass measurements, and this is typically achieved using Fourier transform ion cyclotron resonance (FTICR) or Orbitrap mass analyzers.25,26 Besides studying the possibilities to use top-down MS for the identification of proteins,27 it has, among others, been used for the analysis of post-translational modifications,28−33 to study an ATP binding site34 and to localize reactive cysteines.35 The aim of the current study was to develop a pipeline to determine the efficiency and specificity of fluorescent protein labeling using ultrahigh resolution top-down ESI-FTICR-MS. As a model system, we have used the 14 kDa Cu-protein azurin from Pseudomonas aeruginosa and the fluorophore ATTO 655 [N-hydroxysuccinimide(NHS)-ester]. Azurin is a member of the ubiquitous group of cupredoxins involved in the shuttling of electrons between proteins.36,37 The usage of fluorescently labeled oxido-reductases has led to applications ranging from biosensing of analytes in biological matrixes38−40 to biochemical studies at the single-molecule level.8,41−45 It has been shown that upon labeling of azurin with a fluorescent molecule whose emission spectrum overlaps with the absorption spectrum of the oxidized copper site it is possible to follow its redox state via fluorescence intensity. Because the reduced copper site has no absorption, the label fluorescence is (partly) quenched only when the site is oxidized as a result of Förster resonant energy transfer (FRET).8,45 Interestingly, azurin can induce apoptosis of cancer cells, most probably by stabilizing p53.46 In relation to this, fluorescent labeling of azurin has been used to study the uptake of azurin and several truncated constructs by mammalian cells.47 The crystal structure of azurin shows an eight-stranded ßhelical structure with a highly hydrophobic core.37,48−50 The protein contains a type I Cu center, responsible for the blue color, showing a slightly distorted trigonal bipyramidal geometry where the 5 ligands are His46, His117, Cys112, Met121, and Gly45.48−52 The presence of the Cu ion together with a disulfide bridge between Cys3 and Cy26 has been proven to stabilize the overall structure of the protein scaffold.53 For the purpose of this work, it is important to remark that, in addition to the N-terminus, azurin contains eleven primary amines (lysine residues) that may be targeted by the NHS-moiety.

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EXPERIMENTAL SECTION

Chemicals. Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, USA). Azurin Expression and Purification. Wild type azurin (Pseudomonas aeruginosa) was expressed in E. coli and purified as previously described.54 Cells from E. coli JM109 were transformed using a pUC-derived plasmid containing the azurin gene followed by a signal peptide for periplasmic translocation. After culturing, cells were harvested and resuspended in a solution of 20% (w/v) sucrose in 30 mM TRIS/HCl, pH 8.0, containing 1 mM EDTA for 20 min at room temperature. Subsequently, the solution was centrifuged at 8000 rpm for 15 min, and the supernatant was collected (sucrose fraction). The cells were resuspended in Milli-Q water at 4 °C, stirred for 20 min, and centrifuged at 8000 rpm for 15 min. The supernatant was collected and added to the above obtained sucrose fraction while the pellet was discarded. In E. coli, azurin is normally expressed in its apo-form. Therefore, after cells were lysed, copper sulfate was slowly added to the medium, to a final concentration of 600 μM in order to incorporate Cu in the polypeptide matrix. Potassium ferricyanide was added to the solution to a final concentration of 100 μM to produce an oxidizing environment. A stepwise precipitation step was included by lowering the pH of the solution to pH 4 by adding concentrated acetic acid. The precipitated proteins were removed by centrifugation (8000 rpm, 20 min). The resulting clarified solution containing azurin was loaded on a homepacked CM Sepharose Fast Flow (Amersham Biosciences) column, and elution was performed using a pH gradient from pH 4 to pH 6.9 (50 mM ammonium acetate). Fractions containing azurin were collected and, after buffer exchange and reduction with sodium dithionite, loaded onto a home-packed DEAE Sepharose Fast Flow (Amersham Biosciences) column and eluted using a salt gradient from 0 to 50 mM of NaCl in 5 mM TRIS/HCl at pH 8.5. After a buffer exchange and oxidation using 1 mM potassium ferricyanide, azurin containing fractions were loaded on a 5 mL HiTrap SP column (GE Healthcare) and eluted using a pH gradient of pH 4 to pH 6.9 (50 mM ammonium acetate). All the chromatographic steps were performed on an Ä kta Purifier system (GE Healthcare). The purification process was monitored by checking the purity of the protein after each chromatographic step on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by means of UV/vis spectroscopy (Cary 50 spectrophotometer, Varian Inc., Agilent Technologies, USA). The final product, after the last cation exchange column, appeared on an SDS-PAGE gel as a single band with apparent mass of ∼14 kDa and showed an UV/vis spectrum with a Abs628 nm/Abs280 nm ratio of ∼0.57 which indicates full loading of the Cu-site.54 Fluorescent Labeling of Azurin and Chromatography of Labeled Species. Protein labeling was performed using a modified version of a previously described protocol.8 Azurin was incubated in a molar ratio of 2:1 with the NHS-ester of the fluorescent label ATTO 655 (ATTO-TEC, GmbH, Germany) in 20 mM HEPES buffer, pH 8.3, for 30 min. The unreacted label was then removed using a 5 mL HiTrap Desalting column (GE Healthcare). During the desalting step, a buffer exchange to 5 mM TRIS/HCl, pH 8.5, was also performed. The (labeled) azurin was subsequently oxidized using K3[Fe(CN)6] (potassium ferricyanide) as described above. The potassium 2513

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ferricyanide was removed from the solution with repeated cycles of concentration and dilution using Amicon ultra-0.5 centrifugal filters (Millipore, USA) with a cutoff of 3 kDa. Ion exchange chromatography (IEC) of the labeled protein species was performed on a 1 mL MonoQ column (GE Healthcare) using an Ä kta Purifier (GE Healthcare) system. The labeled azurin fraction was loaded on the column (equilibrated with 5 mM TRIS, pH 8.5), and subsequently, protein species were eluted with a gradient from 0 to 100 mM NaCl in 5 mM TRIS, pH 8.5, in 30 column volumes at a flow rate of 1 mL/min. The elution process was followed by monitoring absorbance at 280 nm (protein) and 660 nm (characteristic absorption of ATTO 655). The fractions corresponding to each peak were then collected and checked by means of UV/vis spectroscopy to confirm the presence of protein. ESI-FTICR-MS. Anion-exchange azurin fractions were desalted using Oasis HLB cartridges (Waters). Cartridges were first washed with elution solution (MeOH/water, 50:50 containing 0.1% formic acid) and subsequently equilibrated with 0.1% formic acid. After application of the samples, cartridges were washed with 3 mL of 0.1% formic acid, and azurin species were then eluted with 150 μL of elution solution. These samples were analyzed by direct infusion on a solariX ESI-FTICR mass spectrometer (Bruker Daltonics) equipped with a 15 T magnet. To this end, a 100 μL syringe (Hamilton) was connected by a peek tube to the ESI source, and samples were infused at 120 μL/h using a syringe infusion pump. For removal of the copper from the holo-azurin, formic acid was added (final concentration 5%) and samples were stored at room temperature for 2 h prior to MS analysis. Typically, full scan ESI-FTICR spectra were acquired in the m/z range of 600−3500 using the broadband detection mode with 1 M data points. Eight scans were summed for each final spectrum. In general, ions were first accumulated for 100 ms in the hexapole and then transferred into the ICR cell for the detection. A flight time to the acquisition cell of 1.1 ms was set with trapping potentials of 1 V at both the front and back plate of the ICR cell. For MS/MS analysis, precursor ions were isolated using the quadrupole with an isolation window of 10 m/z units and then fragmented with collision induced dissociation (CID). The acquisition settings used to carry out these experiments were dependent on both the m/z-value and the intensity of the selected precursor ion. That is, the accumulation time in the collision cell, the applied collision energy, the flight time to the ICR cell, and the number of summed scans were optimized for each precursor ion. Typically, the CID-ESI-FTICR spectra were acquired in the m/z range of 600−3500 using the broadband detection mode with 1 M data points and trapping potentials of the ICR cell of 1 V. DataAnalysis Software 4.0 SP 3 (Bruker Daltonics) was used for the visualization and the calibration of the spectra. Prior to the ESI-FTICR measurements, the system was externally calibrated using a commercially available tune mix (Agilent). All the measured CID spectra were internally calibrated using a list of theoretical fragment ions. Such a list included labeled fragment ions when the CID spectra were obtained from labeled azurin. The theoretical CID fragments of azurin were obtained using the MS-Product tool (http://prospector.ucsf. edu/prospector/mshome.htm).

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RESULTS AND DISCUSSION

Efficiency and Stoichiometry of Azurin Labeling with ATTO 655. The aim of the experiments was to study the efficiency, stoichiometry, and positioning of fluorescent labeling using a top-down approach. For this purpose, we selected the Cu-protein azurin and the fluorescent label ATTO 655 (PubChem CID 16218785) harboring an amine-reactive (NHS-ester) moiety. After labeling, the sample was fractionated using high resolution anion exchange chromatography (Figure 1) recording both overall protein absorbance (280 nm) as well as the specific absorbance of ATTO 655 (660 nm).

Figure 1. Chromatographic separation of azurin after labeling with ATTO 655. Azurin was labeled with ATTO 655, and the resulting species were separated with anion exchange chromatography, recording both overall protein absorbance at 280 nm and the specific absorbance of the ATTO 655 label at 660 nm.

UV−vis spectra of each of the eluting species are shown in Figure S1 (see Supporting Information). Fraction I shows the same spectrum (Figure S1a, Supporting Information) as (oxidized) wt azurin8 and is ascribed to unlabeled protein whereas peaks II−IV display features of the protein as well as the label (Figure S1b, Supporting Information) and are ascribed to singly labeled protein fractions. Fraction V also displays combined features but the absorption band of the label is modified in a manner characteristic of noncovalent dimerization of the fluorescent probe (Figure S1c, Supporting Information).55 This fraction is therefore ascribed to a doubly labeled species. The observation of multiple peaks agrees with the occurrence of multiple labeling sites (lysines) on the azurin surface. Labeling with the NHS-ester converts an amine into an amide leading to loss of one positive charge. Since in ion exchange chromatography not only the total charge but also the surface charge distribution affects the separation, it is reasonable to assume that the various fractions correspond with different surface locations of the label.9,56 The integrated peak areas (280 nm) of the individual peaks, after correction for the absorbance of the ATTO 655, were used to calculate the overall degree of labeling (DoL%) (see Supporting Information, Methods). This showed that, in total, approximately 40% of the azurin was labeled. In the case of azurin, the redox state of the copper also has a strong influence on the chromatographic behavior of azurin (data not shown). To simplify the purification 2514

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Figure 2. High-resolution 15T ESI-FTICR mass spectrometry of unlabeled and ATTO 655 labeled azurin. (a) ESI-FTICR MS spectra of azurin after labeling with ATTO 655 and strong anion exchange chromatography (Figure 1). Fraction I (top), fraction II (middle, fraction III and IV resulted in a similar spectrum), and fraction V (bottom). In all fractions, azurin was found both in the folded holo-form (containing Cu) and the unfolded apoform (after loss of the Cu). (b) The comparison of the ion species with the same charge state was used to reveal the number of ATTO 655 units bound to azurin in the different chromatographic fractions.

unlabeled azurin was observed in fraction I, singly labeled azurin in fractions II−IV, and doubly labeled azurin in fraction V. Top-Down Mass Spectrometry Analysis of Unlabeled and ATTO 655-Labeled Azurin. Next, we performed ESIFTICR-MS/MS of unlabeled copper containing azurin (fraction I, Figure 1). For this purpose, the [M + 8H]9+ ion was selected and fragmented using CID (Figure 3a). Due to the presence of the disulfide bridge between Cys-3 and Cys-26, no fragments related to this region were observed within the spectrum. Still, the top-down fragmentation data allowed assignment of multiple cleavages, overall covering 45% of the sequence. Removal of the copper using 5% formic acid did not have a strong influence on the overall fragmentation spectrum when the protein was still folded (Figure 3b) although some differences were apparent (see inset Figure 3a,b) and the overall sequence coverage was slightly higher (50%). However, upon unfolding and removal of the copper, the fragmentation spectrum changed considerably (Figure 3c). Interestingly, this revealed that the total fragmentation efficiency was improved, evident from the increase in the relative intensity of the fragment ions compared to the parent ion, but this did not result in an overall better sequence coverage (41%). As a next step, we analyzed the ATTO 655-labeled azurin to identify the location of the label. Specifically, we systematically looked for diagnostic fragment ions that could discriminate between the different labeling positions. We started by analyzing the most prominent ATTO 655 labeled azurin peak (peak III, Figure 1) that accounts for ∼16% of the area of all peaks in Figure 1 and 50% of all peaks corresponding to labeled species (calculated using the area under the 280 nm curve). The fragmentation spectrum of the labeled folded apo-azurin (Figure 4a) revealed a clear y14-ion containing one label (m/z (982.0126), [M + 2H]2+), and several y-ions at lower m/z

procedure, we therefore fully oxidized the labeled sample prior to fractionation. We then measured the fractions using ESI-FTICR-MS to determine the labeling efficiency and stoichiometry of the individual chromatographic peaks (Figure 2). Copper containing azurin (holo-azurin, C607H955,N164O194S9Cu, designated as “M” throughout the manuscript when referring to the whole protein) is a highly folded protein which slowly unfolds after the loss of copper (apo-azurin, C607H956,N164O194S9, designated as “M−Cu” throughout the manuscript) under acidic conditions.57 Under our ESI conditions (0.1% formic acid in water/methanol (50:50)), this resulted in the appearance of two separate charge state distributions (Figure 2a). The highly charged ion species (from 11+ to 17+) correspond to the unfolded apo-azurin, while the lower charge states (from 7+ to 10+) match with the folded holo-azurin. Removal of the copper prior to MS analysis, using 5% formic acid, revealed that under these conditions the apo-azurin slowly unfolds over time (data not shown). As expected from the relative absorbance at 280 and 660 nm observed in the chromatograms, we found only unlabeled azurin (Figure 2a, upper part) within chromatographic peak I (Figure 1). Of note, the oxidized copper forms a covalent bond with cysteine-112,58 and therefore, the major 9+ ion at m/z 1557.084 (most abundant isotope peak) is represented by [M + 8H]9+ (C607H963,N164O194S9Cu) and not by [M + 9H]9+ (C607H964,N164O194S9Cu). In the later eluting fractions, we observed either singly labeled (Figure 2a, middle part) or doubly labeled (Figure 2a, lower part) azurin. Taking the highest ion in the individual spectra ([M + 8H]9+), the measured mass difference between the differentially labeled azurin forms was 509.199 Da, which corresponds well with the mass of ATTO 655 (C27H33N3O6S, 527.208 Da) taking into account the water loss as a result of the labeling reaction. On the whole, and in accordance with the UV−vis spectra, 2515

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Figure 3. Top-down 15T FTICR mass spectrometry of holo- and apo-azurin. ESI-FTICR MS/MS spectra obtained from unlabeled azurin in fraction I (Figure 1). (a) Collision-induced dissociation (CID) spectrum of the folded holo-azurin ([M + 8H]9+). (b) CID spectrum of the folded apo-azurin ([M−Cu + 9H]9+) (c) CID spectrum of the unfolded apo-azurin ([M−Cu + 14H]14+). A higher sequence coverage was obtained from the fragmentation of the holo-forms (45% and 50% for the form with and without copper, respectively) compared to the fragmentation of the apo-form (41%). However, the fragmentation efficiency was higher for the unfolded form. The sequence coverage was calculated considering only b and y fragment ions.

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Figure 4. Top-down 15T FTICR mass spectrometric identification of the ATTO 655 labeling sites within azurin. (a) Top-down ESI-FTICR MS/ MS from singly labeled apo-azurin from fraction III (Figure 1). A clear y14 + ATTO 655 (m/z 982.0126) fragment in combination with unlabeled yions at lower m/z values demonstrates labeling at Lys-122. The identification of only unlabeled b121 and labeled b122 (see inlet) additionally shows that this is the major, if not the only, labeling site in this fraction. (b) Enlargement of the ESI-FTICR MS/MS spectrum obtained from holo-azurin [M + 8H]9+ labeled on Lys-122 with ATTO 655. Both copper free and copper containing y14 fragments were detected as doubly charged ions with a resolution of 252 000 at m/z 1012.5. In the central box, the resolved isotopic distribution of the [y14 − 2H + ATTO 655 + Cu]2+ and the [y14 + ATTO 655 + Cu]2+ fragment ions is depicted. The first and the second peak at m/z 1013.47 (as well as and the second and the third peak at m/z 1014.47) correspond to the difference in the Δ mass between 65Cu vs 63Cu and two 12C vs 13C isotopes, respectively. The resolution of such doublet was used as an indicator of copper-containing fragments. (c) Top-down ESI-FTICR MS/MS from a mixture of singly labeled apo-azurin from 2517

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Figure 4. continued fraction IV (Figure 1). The presence of an ATTO 655-labeled y1 fragment in combination with multiple unlabeled y-ions shows that part of the azurin in this fraction is labeled at the C-terminus (Lys-128). On the other hand, the identification of the b35 + ATTO 655 fragment (see inlet) but the absence of a b1 + ATTO 655 fragment indicates that another part of the azurin is labeled on either Lys-24 or Lys-27.

Figure 5. Overview of the azurin species and their diagnostic top-down 15T FTICR CID fragments, after labeling with ATTO 655.

the third peak at m/z 1014.47) corresponds to the difference in the Δ mass between 65Cu vs 63Cu and two 13C vs 12C isotopes, respectively. The isotopic fine structure61 of such a doublet can be used as an indicator of copper-containing fragments. Overall, the above results show that Lys-122 is the most reactive lysine toward the ATTO 655-NHS-ester label. Recently, N-terminal labeling of azurin with Cy5-NHS was identified using MALDI-ToF MS of a tryptic digest.8 We observed N-terminal labeling in fraction II, which resulted, among others, in a clear diagnostic b1 + ATTO 655 fragment (Figure 5). The chromatographic profile of peak IV indicates that it contains multiple unresolved singly labeled species. In line with this observation, top-down analysis of this fraction (selecting the ion at m/z 1613.6609 [M + ATTO 655 + 8H]9+ as precursor ion) revealed that it contains a mixture of at least two different species. The ATTO 655-labeled y1 ion at m/z 656.3114 (Figure 4c) demonstrates labeling at the C-terminal lysine (Lys-128) but the y-ions were also observed unlabeled. Indeed, part of the ATTO 655-labeled azurin in this fraction was labeled at the N-terminal region of the proteins, which was most apparent from the doublet of labeled and unlabeled b35ions. The absence of the diagnostic b1 + ATTO 655 fragment, as observed in fraction II, indicated that the label was located at Lys-24 or -27, although the exact location could not be identified. Top-down analysis of the double labeled azurin (Fraction V) resulted in multiple ATTO 655-labeled fragments. The presence of a double labeled b35-ion (Figure 5) demonstrates that at least part of the species contained two labels at the Nterminal region, with one of these at the N-terminus. We also

without the label, indicating that Lys-122 was the labeling site (Figure 5). The fact that we observed the b121-ion only without a label but the b122-ion with the label demonstrated that Lys-122 was the major, if not the only, labeled amino acid in this fraction. Resolving the b122-ion at m/z 1728.5775 (most abundant isotope) [M + ATTO 655 + 8H]8+ underscores the absolute requirement of ultrahigh resolution for accurate assignment of the labeling site. We subsequently also analyzed the fragmentation spectrum of the copper-containing ATTO 655labeled azurin from the same chromatographic peak. In folded azurin, the copper is coordinated by five residues (Gly45, His46, Cys112, His117, and Met121) while in the unfolded state the major binding site is formed by Cys112 and His117 and possibly Met121.58 Upon top-down fragmentation of the 9+ ion at m/z 1613.6609 (most abundant isotope) [M + ATTO 655 + 8H]9+, representing holo-azurin in its folded form, most of the fragments lost the copper ion. However, some Cucontaining fragments were observed, corresponding to the copper coordinating C-terminal region,59 showing that topdown mass spectrometry can also be used to identify the copper binding site in azurin, similar to what has been observed recently for a chloroplast protein from C. reinhardtii.60 As an example, we observed the ATTO 655-labeled y14-ion both with and without the copper (Figure 4b). The ultrahigh-resolution measurements of the labeled y14-ion showed that it actually resembles two species with overlap in the isotopic distribution ([y14 − 2H + ATTO 655 + Cu]2+ and the [y14 + ATTO 655 + Cu]2+, see inset Figure 4b). Furthermore, the observed split of the first two peaks at m/z 1013.47 (as well as the second and 2518

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observed the y1 + ATTO 655, showing labeling at the Cterminus (Figure 5). Because we did not observe doubly labeled y-ions in this fraction, it was not possible to unambiguously annotate labeling at Lys-122. However, we observed a singly labeled b35-ion and the b2 + ATTO 655, so we assume that at least part of the doubly labeled azurin is labeled both N-terminally and C-terminally.

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CONCLUSIONS Using top-down 15 T ESI-FTICR MS in combination with high resolution anion exchange chromatography of ATTO 655labeled azurin, we were able to identify multiple attachment sites of the fluorescent probe on the surface of azurin and attribute, in most cases, the precise residue where the coupling occurred. In a “one-go” experiment, without prior proteolytic digestion, Lys-122 was identified as the main reaction site on wt azurin during labeling with NHS-ester ATTO 655 in the conditions used for the reaction in this study (pH 8.3). This agrees with recent findings that indicated Lys-122 as having a lower than average pKa due to the location in a nonpolar region of the polypeptide matrix. This enhances the nucleophilic character of the lysine and explains its high reactivity.62 Further research is ongoing to study the distribution of the labeled species as a function of the pH at which the labeling reaction is performed. We anticipate that the approach presented here could be applied not only to fluorescent dyes but also to spin labels.63 We were able to produce different species of singly labeled protein, without the need of mutagenesis, by a single labeling reaction followed by chromatography. In the near future, it may be easier to label a protein and subsequently determine the location of the label instead of performing site-directed mutagenesis to target the label to a particular position. Due to the possible coelution of different species, as observed in our current study, more elaborate or multidimensional separation techniques may be needed to separate all the different components before MS analysis to properly and fully assign the label positions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +31-71-5265078. Fax: +31-71-5266907. E-mail: p. [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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