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In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have mu...
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Anal. Chem. 2008, 80, 5251–5259

Fluorescein as a Versatile Tag for Enhanced Selectivity in Analyzing Cysteine-Containing Proteins/Peptides Using Mass Spectrometry Shu-Hui Chen,* Jue-Liang Hsu, and Fong-Sian Lin Department of Chemistry, National Cheng Kung University, Tainan, Taiwan Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics. Sample complexity and wide dynamic range are big challenges in proteomic analysis. Many techniques including multidimensional protein identification technology (MudPIT) and affinity * To whom correspondence should be addressed.E-mail: shchen@ mail.ncku.edu.tw. 10.1021/ac800436j CCC: $40.75  2008 American Chemical Society Published on Web 05/31/2008

selection of specific targets or residues such as immobilized metal ion affinity chromatography (IMAC) for phosphoproteome,1–3 lectin affinity for glycoproteins,4,5 covalent chromatography for cysteine-containing peptides,6,7 isotope-coded affinity tag (ICAT) for selecting and comparatively quantifying cysteine-containing peptides,8–10 and fluorous affinity tags for peptide subsets11 are developed to reduce the sample complexity or to extract target proteins for comprehensive proteomic characterizations. In addition to selecting subsets of proteins, a new type of protein tagging reagent called “visible” isotope-coded affinity tag (VICAT) could be used not only to isolate cysteinyl proteins but also track and quantify their absolute amount during chromatographic processes by incorporating radioactive 14C atoms.12 VICAT is also a versatile tag that contains a biotin moiety for isolating target proteins by affinity capturing and photocleavable linkers for removing the tag moiety before analysis to prevent a complicated fragment pattern of MS/MS spectra caused by tag molecules. However, radioisotope reagents are hazardous and they require film exposures to visualize. Alternatively, several fluorescence-based tagging methods using fluorogenic thiol derivatization such as ammonium-7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, 4-(dimethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole, 6-chloro-2,1,3benzoadiazole-4-sulfonylaminoethytrimethylammonium chloride, and 5-({2-[(iodoacetyl)aminoethyl]amino}naphthalene-1-sulfonic acid (1,5-I-AEDANS) were developed for tracking and quantifying proteins/peptides by in situ fluorescence measurement. (1) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883–2892. (2) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301–305. (3) Cao, P.; Stults, J. T. Rapid Commun. Mass Spectrom. 2000, 14, 1600–1606. (4) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667–672. (5) Geng, M.; Zhang, X.; Bina, M.; Regnier, F. J. Chromatogr., B: Biomed. Sci. Appl. 2001, 752, 293–306. (6) Wang, S.; Zhang, X.; Regnier, F. E. J. Chromatogr., A 2002, 949, 153–162. (7) Ren, D.; Julka, S.; Inerowicz, H. D.; Regnier, F. E. Anal. Chem. 2004, 76, 4522–4530. (8) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994–999. (9) Baliga, N. S.; Pan, M.; Goo, Y. A.; Yi, E. C.; Goodlett, D. R.; Dimitrov, K.; Shannon, P.; Aegersold, R. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14913– 14918. (10) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946–951. (11) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nat. Biotechnol. 2005, 23, 463–468. (12) Lu, Y.; Bottari, P.; Turecek, F.; Aebersold, R.; Gelb, M. H. Anal. Chem. 2004, 76, 4104–4111.

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AEDANS-labeled peptides were further shown to be fractionated by Ga/IMAC or o-nitrobenzyl-based photocleavable resins to reduce the sample complexity.13 Most of these dyes are water soluble and exhibit favorable ion signals. Compared to other tagging derivatives that commonly form complicated fragment patterns, fluorescence-based derivatives generally exhibit predictable fragmentation, and thus, peptide sequencing can be performed without removing tag molecules. These fluorescencebased tagging methods were primarily designed as an alternative spectroscopic technique for protein quantification, and mass spectrometry was used solely for protein identification. However, most of the nongel separation techniques do not have sufficient power to resolve individual proteins for accurate quantification from samples with high complexity. Another type of fluorescence tagging method is specifically designed with matched protein charge and size, which are compatible with two-dimensional gel electrophoresis. These labeling methods also have a low level of labeling degree such that the spectra pattern derived from the gel digests will not be interfered with by the labeling method since most detected peptides were not labeled. It is, however, possible that the proteins identified by unlabeled peptides using mass spectrometry do not match the protein quantified by labeled peptides using the gel image. It will be better for fluorescence-based tagging method to bear not only favorable chemistries for reactions but also favorable characteristics for selecting, identifying, and quantifying target proteins by both fluorescence and MS. As an alternative, we investigated the potential of fluorescein derivatives for proteomics studies. Fluorescein derivatives are one of the most popular classes of fluorescence labeling dyes for qualitative and quantitative analysis. Because of their wellrecognized optical properties for quantification by fluorescence, we would like to explore fluorescein derivatives as fluorescencebased tagging reagents for MS-based proteome. These dyes are water soluble and commercially available with reactive groups for different amino acid residues. We chose to target cysteine residues by using a sulfhydryl-reactive 5-iodoacetamidofluorescein (IAF) derivative. From in silico studies of Escherichia coli, yeast, and human proteome, over 80% of proteins contain at least one cysteine reside but these 80% of proteins can be represented by less than 20% of their tryptic peptides.6 Furthermore, we showed that many metal-chelating beads made of iminodiacetic acid or tricarboxymethylethylene diamine-modified porous materials could be used to enrich fluorescein-labeled peptides either with or without metal ions attached. By combining IAF derivatization, stable isotope dimethyl labeling, and affinity enrichment, cysteinyl proteins can be tracked, identified, and quantified. We also examined the fragmentation pattern of fluorescein-labeled cysteinyl peptides to explore unique spectroscopic characteristics such as specific reporter ions which could be very useful for selective proteome analysis by MS. EXPERIMENTAL SECTION Materials. Acetonitrile, urea, and formaldehyde (37% solution in H2O) were purchased from J. T. Baker (Phillipsburg, NJ). Trifluoroacetic acid (TFA), D,L-dithiothreitol (DTT), tris(2(13) Clements, A.; Johnston, M. V.; Larsen, B. S.; McEwen, C. N. Anal. Chem. 2005, 77, 4495–4502.

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carboxyethyl)phosphine hydrochloride (TCEP), sodium cyanoborohydride, bovine serum albumin (BSA), and ovalbumin (from chicken egg) were provided by Sigma (St. Louis, MO). Iodoacetamide (IAM) was purchased from Fluka (Buchs, Switzerland). R-Cyano-4-hydroxycinnamic acid and formaldehyded2 (20% solution in D2O) were purchased from Sigma-Genosys (Woodlands, TX). Formic acid (98∼100%) and sodium acetate were purchased from Riedel-de Hae¨n (Seelze). IAFwas purchased from Molecular Probes (Eugene, OR). Ammonium bicarbonate was purchased from Wako (Osaka, Japan). Ammonium hydroxide (ammonia solution, 29.8%) was purchased from TEDIA (Fairfield, OH). Sequence grade-modified trypsin was purchased from Promega (Madison, WI). Microcon YM10 centrifugal filter devices and ZipTips C18 desalting tips were purchased from Millipore (Bedford, MA). Poros MC 20 beads were purchased from PerSeptive Biosystems (Framingham, MA). Chelating Sepharose beads were purchased from Amersham Phamacia (Uppsala, Sweden). Deionized water used was from an E-pure water purification system (Barnstead Thermolyne Co., Dubuque, IA). IAF Labeling and Trypsin Digestion. Ovalbumin or BSA (50 µg) was reduced with 10 mM TCEP (or DTT) in 100 mM ammonium bicarbonate (pH 8.8) containing 8 M urea at 37 °C for 1 h. The reduced proteins were added with 30 mM 5-IAF, and the reaction mixture was incubated at room temperature in the dark for 4 h. Excess reagents were removed with Microcone YM10 by centrifugation following procedures described in the manufacturer’s protocol. Additional washings with 100 mM ammonium bicarbonate (100 µL × 3) were necessary to remove the excess dyes. The fluorescein-labeled proteins were then redissolved in 100 µL of 100 mM ammonium bicarbonate (pH 8.8), and the labeled protein solutions were then digested by adding 1 µg of trypsin and reacted at 37 °C for 16 h. Enrichment of Fluorescein-Labeled Peptides by Poros MC20 Beads. A volume of 10 µL of the fluorescein-labeled digest (∼5 µg) was acidified to pH 2-3 by TFA, and then the acidified digest was loaded onto 0.1% TFA pre-equilibrated Poros MC20 beads packed in a 20-µL cartridge. Most of the nonfluoresceinlabeled peptides were washed away with 0.1% TFA, and the remaining fluorescein-labeled peptides were eluted with 100 mM ammonium bicarbonate (pH 8.8). The resulting fluorescein-labeled peptides were acidified, desalted by ZipTips, and examined by MALDI-TOF. Dimethyl Labeling of Fluorescein-Labeled Peptides. For comparative quantification, two sets of ovalbumin (50 µg for each) were labeled and digested following the same protocol described in IAF Labeling and Trypsin Digestion. For each set, 5 µg of the fluorescein-labeled ovalbumin digest dissolved in 10 µL of 100 mM sodium hydrogen carbonate buffer was diluted with 90 µL of sodium acetate buffer (100 mM, pH 5-7), mixed with 5 µL of H2- and D2-formaldehyde (4% in water), respectively. After vortexing, each set was then added immediately with 4 µL of freshly prepared sodium cyanoborohydride (600 mM). The final mixtures were vortexed again and then allowed to react for 10 min. After reaction, 4 µL of ammonium hydroxide (4% in water) was added to each mixture to consume the excess aldehyde. Two sets of samples were combined, acidified, and then enriched by Poros MC20 beads following the same

Figure 1. MALDI-MS spectra for the tryptic digest of (A) IAM-modified ovalbumin and (B) IAF-modified ovalbumin.

procedure described in Enrichment of Fluorescein-Labeled Peptides by Poros MC20 Beads. Mass Spectrometry. The MS data were obtained using a MALDI-TOF spectrometer equipped with a 337-nm N2 laser (M@LDI, Micromass, Manchester, UK). The sample was acidified with 0.5 M HCl to minimize the salt effect,14 and then it was mixed with the matrix at a matrix/HCl/sample ratio of 2:1:1 (v/v/v). The resulting mixture was deposited onto the target plate and dried before detection. The MS/MS data of fluorescein-labeled tryptic peptides were acquired from a Q-TOF micro spectrometer (Micromass, Manchester, UK) equipped with a nanoflow HPLC system (LC Packings, Amsterdam, The Netherlands). Briefly, a tryptic digest solution (1 µL, ∼300 pg of protein) was injected onto a column (NAN75-15-03-C18 p.m.; 75 µm × 15 cm) packed with C18 beads (3-µm, 100-Å pore size, PepMap). Mobile-phase buffer A consisted of 0.1% formic acid in water; mobile-phase B consisted of 95% acetonitrile in 0.1% formic acid. The peptides were separated using a linear gradient of 0-70% solvent B over 40 min at a flow rate of 200 nL/min. Typically, 20-60 spectra were combined because the average peak duration for a peptide was ∼20-60 s, and each individual spectrum was acquired within 1 s at an interscan time of 0.1 s. For sequencing, the MS/MS spectra were obtained through a survey scan and the automated datadependent MS analysis was carried out using the dynamic exclusion feature built into the MS acquisition software. Peptide sequence assignment was performed using a peptide sequencing program (MassLynx, Micromass) either manually or processed through a database MS/MS ion search (Mascot MatrixScience) with variable of modifications of fluorescein (C) indicating fluorescein-labeled cystein residue, dimethylation (N) and dimethylation (K) for dimethylation of N-terminus and Lys with light atoms, and RA (N) and RA (K) for dimethylation of N-terminus (14) Huang, S. H.; Hsu, J. L.; Morrice, N. A.; Wu, C. J.; Chen, S. H. Proteomics 2004, 4, 1935–1938.

and Lys with heavy atoms. All results were further verified using manual interpretation. For precursor ion scans, two collision energies were used simultaneously (low collision energy, 10 V; high collision energy, 60 V) and the resulting collision-induced dissociation (CID) spectra of selected precursors were acquired using two MS/MS channels. The molecular mass tolerance of the product ion was 10 mDa, and the threshold of intensity was 30 counts. RESULTS AND DISCUSSION IAF Derivatization and Trypsin Digestion. Proteins were first reduced by either TCEP or DTT and then were alkylated by 5-IAF. TCEP is a nonthiol-containing reducing agent and was used to minimize the dye consumption by excess DTT. Moreover, excess dyes were removed prior to trypsin digestion to avoid interferences. Fluorescein-labeled ovalbumin was digested by trypsin and then was detected by MALDI-TOF for mass mapping and ESI-Q-TOF micro for peptide sequencing. As shown in Figure 1, it was found that IAF derivatization did not affect trypsin digestion and the spectra acquired were similar to those without labeling or with IAM derivatization. From the peak list depicted in Table 1, a majority of tryptic ions of fluorescein-labeled ovalbumin were detected with expected masses. Ovalbumin contains five cysteine residues, which were contained in five cysteinyl tryptic peptides. All five cysteinyl peptides were detected with complete IAF derivatization by ESI-Q-TOF micro; while two of them, LPGFGDSIEAQCGTSVNVHSSLR (m/z 2841) and VHHANENIFYCPIAIMSALAMVYLGAK (m/z 3364), were not detected by MALDI-TOF, and it was likely due to the large m/z values (2841 and 3364, respectively), which were beyond the detection window of the reflectron MALDI instrument. Nevertheless, the rest of the three tryptic peptides, GSIGAASMEFCFDVFK (m/z 2137.8), YPILPEYLQCVK (m/z 1852.7), and ADHPFLFCIK (m/z 1577.6), were detected by MALDI-TOF with appreciable Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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intensity. Compared to the common derivatization method using IAM, the ion signal of fluorescein-labeled peptides was much stronger and it is likely due to a more abundant electron density associated with fluorescein molecules. The IAF labeling on another model protein, BSA, was also examined. BSA contains 35 cysteinyl residues, among which 32 sites were detected with complete IAF derivatization by Q-TOF micro as listed in Supporting Information, Table S1. It is particularly notable that some adjacent cysteines on the same tryptic peptide, such as ECCDKPLLEK, were both detected with complete labeling, indicating no significant steric hindrance effect was present for such substitutional reaction. There were another three cysteinyl peptides undetected, and we believe these sites were labeled and the failure in detecting them by nanoLC-ESI-Q-TOF was due to their coelution with other peptides with stronger ion signals. 3-2 Fragmentation and Reporter Ion Characterization for Fluorescein-Labeled Cysteinyl Peptides. The fragmentation of fluorescein-tagged peptides under CID conditions was investigated by a Q-TOF micro instrument. Figure 2 displays a typical MS/ MS spectrum of a fluorescein-labeled peptide, ADHPFLFCIK (m/z 1577.6), derived from ovalbumin. Similar to those of unlabeled or IAM-derivatized ions, the fragmentation was dominated by the cleavage of the amide backbone with intact label. Thus, the resulting spectra were readily interpretable for automatic peptide sequencing with high confidence. The peptide scores obtained from MASCOT search were compatible for two labeling methods. More interestingly, a strong reporter ion (m/z 422) was readily detected in all fluorescein-containing peptides including those derived from ovalbumin (Figure 2), BSA (Figure 3A), and HSP27 (Figure 3B) and complement component 1 Q subcomponentbinding protein (Figure 3C) from the lysates of MCF-7 cells. This reporter ion was also detected by the MALDI-Q-TOF instrument (Supporting Information, Figure S1). In addition, an immonium ion (m/z 463, f C) corresponding to the fluorescein-labeled cysteine residue was detected from almost all the MS/MS spectra of fluorescein-labeled peptides. Since the rest of the fragment ions remain intact, a possible pathway for producing the reporter ion was derived from the immonium ion: The positive charge on the immonium ion initiated the loss of the fluorescein moiety, and the resulting fluorescein moiety was protonated to give the m/z 5254

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422 ion with a chemical formula of C22H14O6NS. The intensity ratio of isotopic peaks for the compound (C22H14O6NS) was calculated to be 100.0:25.2:6.0 based on the natural abundance of the elements. As displayed in the inset of Figure 2, the abundance ratio of isotopic peaks for m/z 422:423:424 was measured to be 100:25.8:6.7, which matched the expected values for the deduced reporter ion. Reporter ions are unique and very useful in developing MS-based methodology. They can be used to enhance signals by isolating the scan window for the desired ion. Thereby, precursor ion scan was used to select fluorescein-derivatized peptides that produce the reporter ion (422.07 ± 0.01 Da). Figure 4A represents the MS/MS precursor scan chromatogram acquired from fluorescein-labeled ovalbumin digest loaded on column using high collision energy (60 V), and it can be seen that the instrument has switched to this mode of operation five times during the experiment. A total of six fluorescein-labeled peptides were sequenced by MS/MS. This resulted in a 100% yield of cysteinyl peptides for all identified sequences from MS/MS scans. As a comparison, Figure 4B depicts the base peak intensity (BPI) chromatograms acquired under the survey scan during which the instrument had switched to MS/MS mode many times and identified five cysteinyl peptides. It is worth noticing that the cysteinyl peptide, DVLVCNK, was only detected under precursor scan but not detected under the survey scan and this peptide was identified to be derived from ovomucoid as an impurity present in ovalbumin. Using precursor ion scan for the digest of BSA, we had also identified one cysteinyl peptide, CCTESLVNR, which was not identified by the survey scan (Supporting Information, Table S1). 3-3 Coupling Techniques for Enriching and Quantifying Fluorescein-Labeled Cysteinyl Peptides. It is desirable to develop coupling techniques for enriching and for quantifying fluorescein-labeled cysteinyl peptides. In the work of McEwen et al.,13 immobilized Ga metal chelating material was demonstrated to adsorb dye molecules, which bear negative sulfate groups through electrostatic interactions with the metal ions. In this study, we discovered that IMAC solid support (iminodiacetic acidmodified porous materials) such as Poros MC 20 beads could selectively bind fluorescein-labeled peptides either with or without the presence of bound metal ions. The metal ion was found not

Figure 2. MS/MS spectra of the tryptic peptide, ADHPFLFCIK, derived from ovalbumin with (A) IAF modification and (B) IAM modification on cysteine residue. The inset is an enlarged spectrum of the reporter ion that showed an isotopic ratio of (100:26.5:8.5 for z/z + 1/z + 2) “f C” denotes the immonium ion of IAF-modified cysteine (m/z 463).

to be necessarily involved in the capture of fluorescein-derivatized peptides. Moreover, the fluorescein-labeled peptides adsorbed to

IMAC resin tightly under acidic conditions and then were released easily by alkaline elution. The specificity and efficiency of such Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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Figure 3. MS/MS spectra of fluorescein-labeled tryptic peptides (A) SLHTLFGDELCK derived from BSA, (B) ELEQVCNPIISGLYQGAGGPGPGGFGAQGPK derived from HSP27 in MCF-7 cells, and (C) ALVLDCHYPEDEVGQEDEAESDJFSIR derived from complement component 1 Q subcomponent-binding protein in MCF-7 cells. 5256

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Figure 4. MS/MS chromatograms of the tryptic digest of fluorescein-labeled ovalbumin obtained from (A) precursor ion scan using the reporter ion at m/z 422 ( 0.01 Da (B) BPI using a low collision energy. In (A), the denoted values are the migration time and the indicated sequences are fluorescein-containing peptides that were picked up during the scan. In (B), m/z values of the BPI corresponding to each peak are indicated below the migration time.

Figure 5. MS spectra the fluorescein-labeled ovalbumin digest (A) before, (B) in the wash fraction, and (C) in the elute fraction of fluorescein affinity extraction. After stable isotope dimethyl labeling, the MS spectrum obtained from the elute fraction of the combined mixtures were shown in (D) in which “1/2” denotes the fluorescein-labeled peptides and the expanded spectrum shows 1:1 ratio of the isotopic pair for *ADHPFLFCI*K peptide and “*” denotes dimethyl labeling.

fluorescein affinity selection was demonstrated by fluoresceinlabeled ovalbumin digest shown in Figure 5. Before affinity capturing, the MS spectrum of fluorescein-labeled ovalbumin

digest was displayed in Figure 5A in which the labeled cysteinyl peptides were barely detected and the nonlabeled peptides dominated the ion signals. The spectrum acquired from the wash Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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Table 1. Expected and Observed Tryptic Peptides (MW > 1 kDa) Derived from Fluorescein-Labeled Ovalbumina peptide sequence

position

ILELPFApSGTMpSMLVLLPDEVSGLEQLESIINFEK YNLTSVLMAMGITDVFSSSA NLSGISSAESLK VHHANENIFYCPIAIMSALAMVYLGAK NVLQPSSVDSQTAMVLVNAIVFK LPGFGDSIEAQCGTSVNVHSSLR VTEQESKPVQMMYQIGLFR DILNQITKPNDVYSFSLASR EVVG(p)SAEAGVDAASVSEEFR

229-263 291-322 20-46 159-181 62-84 200-218 85-104 340-359

ELINSWVESQTNGIIR ISQAVHAAHAEINEAGR AcGSIGAASMEFCFDVFK GGLEPINFQTAADQAR LTEWTSSNVMEER YPILPEYLQCVK HIATNAVLFFGR DEDTQAMPFR ADHPFLFCIK

143-158 323-339 1-16 127-142 264-276 111-122 370-381 190-199 360-369

expected mass

observed mass

observed masses for dimethylated isotopic pairs

4022.9 3293.6 3363.5 2460.3 2841.1 2284.1 2281.1 2008.9 2088.9(p)c 1858.9 1773.9 2137.7 1687.8 1581.7 1852.7 1345.7 1209.5 1577.6

nbb nb 1122(+3)a 2460.2 948(+3)a 2284.1 2281.0 2008.8 2088.7 1859.1 1773.7 2138.5 1687.7 1582.4 1853.7 1346.2 1209.5 1577.8

nb nb nb nb nb nb 2337.1, 2345.0 2036.8, 2040.9 nb 1888.0, 1892.0 1801.9, 1805.8 2165.8, 2169.8 1715.9, 1719.9 1609.8, 1613.8 1908.9, 1912.9 1373.8, 1377.8 1237.5, 1241.5 1633.7, 1641.8

a All values observed were from MALDI measurements except subscript a, which were observed from ESI-Q-TOF. b nb, not observed. c (p) denotes phosphorylation.

fraction was displayed in Figure 5B and that acquired from the elute fraction was displayed in Figure 5C. Clearly, the labeled cysteinyl peptides were isolated and enriched in the elute fraction and the nonlabeled peptides were almost washed away as seen in Figure 5B. The results indicated that fluorescein-labeled peptides were enriched successfully with only a trace amount of nonspecific binding. The adsorption mechanism associated with such fluorescein affinity selection is not understood. The dye moiety adsorbs on the porous iminodiacetic acid-modified materials under acidic conditions (0.1% TFA) and is released under basic conditions (100 mM ammonium bicarbonate). Since carboxylic groups on both the dye molecule and the solid support are protonated without net charge under acidic conditions, we suspect that fluorescein moieties are adsorbed on the solid support via van der Waals or π-π interactions. Under basic conditions, such interactions will diminish due to the presence of negative charges on both the surface and the molecule, leading to the release of adsorbed dyes. Since fluorescein molecules contain three benzene rings, fluorescein-labeled peptides are expected to have stronger van der Waals or π-π interactions than any amino acid residues, and thereby, such adsorptions are specific for peptides with fluorescein labeling. We had also tested other stationary phases such as chelating Sepharose beads, SCX, SAX, or RP, but only the chelating beads were found to have applicable affinity selection. Reversed-phase materials adsorb dye molecules irreversibly, and only very stringent conditions such as overnight washing by organic solvents can remove adsorbed dyes. The loading capacity for fluorescein-labeled peptides was roughly estimated to be ∼0.1 µmol of labeled peptides for 20 µL of Poros MC 20 beads, and the recovery was estimated to be ∼90% for the first use and reduced to be less than 60% when reused. For quantifying the enriched fluorescein-labeled peptides, stable-isotope dimethyl labeling15 was applied to the digested (15) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem. 2003, 75, 6843–6852.

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peptides prior to fluorescein affinity selection. This strategy was demonstrated by using two sets of ovalbumin with an equal amount of total proteins. Two samples were labeled with IAF on cysteine residues, digested by trypsin, and dimethylated with H2and D2-formaldehyde, respectively, on both the N-termini and lysine residues of tryptic peptides. From MALDI-TOF inspections, most of the peptides were detected with the expected m/z values (Table 1). Two labeled samples were then mixed together for fluorescein affinity enrichment and MS analysis. As shown in Figure 5D, dimethyl labeling did not appear to affect fluorescein affinity enrichment. Nonfluorescein-labeled peptides were still washed away and fluorescein-labeled peptide pairs were found predominantly in the elute fraction with expected 1:1 isotopic ratio. As displayed in the enlarged spectrum shown in the inset of Figure 5D, the isotopic pair of the peptide, *ADHPFLFCI*K, was detected at m/z 1633.7 and 1641.8, respectively, with 1:1 intensity ratio. Because of two labeling sites, there was a 8-Da mass difference for the isotopic pair. These results demonstrate a feasible method for enriching and quantifying fluorescein-tagged cysteinyl proteins by coupling fluorescein affinity selection and stable isotope dimethyl labeling. CONCLUSION In this study, we demonstrated that fluorescein tagging owns multiple characteristics to track, enrich, identify, and quantify target proteins by fluorescence as well as by MS. A unique reporter ion (m/z 422) containing the fluorescein moiety was generated by CID during MS analysis, and it was applied to the selection of fluorescein-tagged proteins/peptides using precursor ion scan. Moreover, the fluorescein-tagged proteins/ peptides adsorbed to multicarboxylate-modified stationary phases and were selectively enriched by simple acid/base elution. In this study, these advantages in selectivity were shown to analyze the subset of proteins containing cysteines. This approach, however, can be applied to select other subsets in targeting proteomics by choosing various fluorescein dyes with different reacting functional groups for peptides containing

other amino acids such as lysines, or modified proteins such as phosphorylation or glycosylation. Furthermore, visible dyes can be monitored by the naked eye, and it allows the sample preparation and separation processes to be optimized in situ.

ACKNOWLEDGMENT This work was supported by the National Science Council.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 3, 2008. Accepted April 23, 2008. AC800436J

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