Microwave Photochemical Reactor for the Online Oxidative

Article pubs.acs.org/ac

Microwave Photochemical Reactor for the Online Oxidative Decomposition of p‑Hydroxymercurybenzoate (pHMB)-Tagged Proteins and Their Determination by Cold Vapor Generation-Atomic Fluorescence Detection Beatrice Campanella,† Jose González Rivera,‡ Carlo Ferrari,§ Simona Biagi,† Massimo Onor,† Alessandro D’Ulivo,† and Emilia Bramanti*,† †

National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici − ICCOM − UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy ‡ Chemical Engineering Department, University of Guanajuato, Noria Alta s/n 36050 Guanajuato, Gto. , Mexico § National Research Council of Italy, C.N.R., Istituto Nazionale di Ottica, INO − UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy ABSTRACT: A novel method is presented for the characterization and determination of thiolic proteins. After the labeling with p-hydroxymercurybenzoate, the pHMB-labeled proteins underwent on-line oxidation with a novel microwave (MW)/ UV photochemical reactor, followed by cold vapor generationatomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the conversion of pHMB to Hg(II) with a yield of 89.0 ± 0.5% without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. Hg(II) was reduced to Hg0 in a knotted reaction coil with NaBH4 solution, stripped from the solution by an argon flow and detected. The chromatographic method for labeled thiolic peptides was linear in the 0.2−100 μmol L−1 range, with a LOD as mercury of 57 nmol L−1. This system has proven to be a useful interface for liquid chromatography coupled with CVG-AFS in the determination and characterization of thiolic proteins. This method has been applied to the determination of thiolic peptides after tryptic digestion of serum albumins from different species (human, bovine, rat, horse, and sheep). fluorescence detection. This type of labeling strategies has been recently reviewed by Toyo’oka.16 Among metal ions, organic mercurial compounds are known to be very specific and sensitive for sulfhydryl groups because of the strong affinity of monovalent mercury for −SH in a wide pH range (1−13), both for low molecular weight thiols (such as glutathione and cysteine) and for macromolecules (proteins and humic matter).17−22 In particular, p-hydroxymercurybenzoic acid (pHMB), a monofunctional organic mercurial probe, is widely used for thiol/protein tagging coupled to a mass spectrometric technique (MALDI-MS, ESI-MS, and ICPMS).23−29 Derivatization with pHMB was also used for the identification of selenols in the sulfur pathway in seleniumenriched yeast by liquid chromatography coupled to orbitrap mass spectrometry.30 Over the last 10 years, we extensively studied the interaction between pHMB and −SH groups for analytical and diagnostic purposes for proteins,31−35 nitrosothiols,36−38 mercaptans,39 and low molecular weight thiols39

C

hemical labeling represents a common and powerful approach for protein identification and quantification in biochemical, clinical applications, and quantitative proteomics. Labeling strategies include isotopic-coded affinity tags (ICAT), stable isotope labeling by amino acids in cell culture (SILAC), and isotope tags for relative and absolute quantification (iTRAQ). The isotopic labeling is mainly based on the use of 2 H, 13C, 15N, and 18O coupled with soft ionization sources like electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI).1,2 Other approaches require the labeling of peptides and/or proteins with an element coupled to elemental mass spectrometry, particularly inductively coupled plasma-mass spectrometry (ICP-MS), in order to improve detection limits and sensitivity.3,4 Two different labeling strategies have been explored: (i) elemental labeling of antibodies with lanthanides and/or gold nanoparticles and5−13 (ii) direct labeling of the protein via derivatization reactions with metals of reactive groups such as sulfhydryl groups or primary amines.14,15 Other quantification methods that do not use MS detection utilize derivatization with a suitable labeling reagent (a chromophore or fluorophore containing reagent) followed by chromatographic or electrophoretic separation and UV or © 2013 American Chemical Society

Received: October 18, 2013 Accepted: November 25, 2013 Published: November 25, 2013 12152

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157

Analytical Chemistry

Article

muscle, ovalbumin (OVA, chicken egg, grade VI), and betalactoglobulin (β-LG, EC 232.928.9) were purchased from Fluka Chemie, Buchs, Switzerland Biochemika. Trypsin (EC 232-6508), human serum albumin (HSA, A-1887), serum albumin from sheep (sheep SA, A6289), rat (rat SA, A 6272), horse (horse SA, A-9888), bovine (BSA, A8531), and L-cysteine (Cys, 30089, Fluka) were purchased from Sigma-Aldrich (Milan, Italy). The concentration of the protein stock solution was determined spectrophotometrically. The protein stock solutions were prepared in 0.1 mol L−1 PBS pH 7.4 and diluted before injection in the chromatographic eluent phase. Solutions of 0.2% sodium dodecyl sulfate (SDS, SigmaAldrich-Fluka, Milan, Italy) were prepared from the corresponding salt in 0.1 mol L−1 PBS, pH 7.4, or 0.1 mol L−1 PBS, pH 8. The solutions was kept at room temperature to avoid salt precipitation. Trifluoroacetic acid (TFA), methanol (MeOH), and acetonitrile (ACN) for RPC were purchased from Carlo Erba (Rodano, Milan, Italy). Tetrahydroborate (THB, NaBH4) solutions (2.5 mol L−1) were prepared by dissolving the solid reagent (Merck & Company, Inc., N.J., pellets, reagent for AAS, minimum assay >96%) into 0.3% m/v NaOH solution. The solutions were microfiltered through a 0.45 μm membrane and stored in a refrigerator. Diluted solutions of NaBH4 (0.05 mol L−1) were prepared daily by appropriate dilution of the stock solutions in 0.3% m/v NaOH, in order to keep the same concentration of NaOH that guarantees the stability of NaBH4 throughout the working day. HCl diluted solution was prepared from 37 wt % HCl (Carlo Erba, Rodano, Milan, Italy). Ultrapure water prepared with an Elga Purelab-UV system (Veolia Environnement, Paris, France) was used throughout. Safety Considerations. pHMB is toxic. Inhalation and contact with skin and eyes should be avoided. All work should be performed in a well-ventilated fume hood. Instrumentation and Chromatographic Conditions. An HPLC-MW/UV combined reactor with CVG-AFS detection system was used for all the measurements, and it has been described previously in detail.61 An HPLC gradient pump (P4000, ThermoQuest) equipped with a Rheodyne 7125 injector (Rheodyne, Cotati, CA) and a 50 μL injection loop was used. Separations by reversed-phase chromatography (RPC) were carried out using a C4 Vydac Grace 214TP5414 reversed-phase column (150 mm ×4.6 mm i.d., silica particle size 5 μm) at a pump flow rate of 1.0 mL min−1. Samples were eluted with a linear gradient specified in the Experimental Section. A detailed description of the MW apparatus and photoreactor for the on line digestion of pHMB, the operating conditions, and the NDAF detector has been previously reported.34,35,61 More details of the CVG-AFS system can be found in a previous paper.45 The output data from the lock-in amplifier were collected with a personal computer equipped with a data acquisition card (DAC, National Instruments, Austin, TX) and its acquisition software (LabVIEW version 6, National Instruments). Labeling Procedure. All the working solutions containing pHMB and Cys were prepared freshly by appropriate dilution with a solution containing 0.01 mol L−1 PBS, pH 7.4. Solutions containing pHMB and Cys (1:1 molar) lead to the formation of mercury−Cys complexes (pH 7.4). Since the reaction of mercury with sulfhydryl groups is instantaneous at room

by means of liquid chromatography coupled with CVG-AFS, a high sensitivity, selectivity, and relatively inexpensive technique for mercury determination. CVG coupled with atomic spectrometry represents, indeed, the most popular method for ultratrace determination and speciation of mercury and mercury compounds. In particular, AFS is the election technique for mercury determination, reaching LOD ≤ 0.1 ng L−1.40 Recently, also Tang et al. published a study on the determination of low molecular thiols by means of pHMB as the tag and detection by means of CVG-AFS.41 In CVG-AFS, the use of online oxidation systems for the conversion of organomercury compounds to Hg (II) is mandatory to obtain higher sensitivity and reproducible results, using atomic spectrometric detector. Common chemical oxidants include KBr/KBrO3,32−39,42−47 K2S2O8,48,49 and K2Cr2O7.50,51 However, KBrO3 is a toxic and carcinogenic reagent classified as R 45-9-E25 and, currently, greener techniques that combine chemical oxidation with UV irradiation52−55 or MW digestion have been reported.56−60 Recently, we have proposed a new photochemical on-line oxidation method that combines MW and UV irradiation in a unique photochemical reactor followed by CVG-AFS detection for the online digestion of pHMB and its complexes with low molecular weight thiols, including cysteine (Cys), glutathione (GSH), homocysteine (HCys), and cysteinyl-glycine (CysGly).61 The MW/UV system effectively replaced the use of chemicals, and this represents a significant contribution toward the implementation of “green” interfaces between the separative apparatus and the detection system. After the conversion of the organomercury compounds into Hg(II), this is reduced to Hg0 in a knotted reaction coil with the NaBH4 solution and detected. In this paper, the new MW/UV-assisted on-line digestion followed by CVG-AFS detection has been applied to the analysis of thiolic proteins. First, MW/UV oxidation of pHMB and pHMB-protein complexes were studied in the flow injection mode (FI) with the aim to evaluate the efficiency of the on-line digestion/step. Second, we explored the hyphenation of reversed-phase (RP) chromatography with MW/UVCVG-AFS system in the separation and determination of thiolic proteins.

■

EXPERIMENTAL SECTION Reagents. Analytical reagent-grade chemicals were used without further purification. pHMB [4-hydroxymercuric be n z o i c a c i d , s o d i u m s a l t ( C A S n o . 1 3 8 -8 5 - 2 , HOHgC6H4CO2Na)] was purchased from Sigma (SigmaAldrich, Milan, Italy). A 1 × 10−2 mol L−1 stock solution of pHMB was prepared by dissolving the sodium salt in 0.01 mol L−1 NaOH in order to improve its solubility, stored at 4 °C and diluted freshly, just before use. The precise concentrations of pHMB solutions were determined from the absorbance at 232 nm (ε232 = 1.69 × 104 cm−1 M−1). Stock solution of 1000 ± 5 μg mL−1 of inorganic HgII in the form of Hg(NO3)2 was purchased from Merck Laboratory Supplies (Poole, Dorset, U.K.). The phosphate buffer solutions (PBS) were prepared from monobasic monohydrate sodium phosphate and dibasic anhydrous potassium phosphate (BDH Laboratory Supplies, Poole, England). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, EC 1.2.1.12), aldolase (ALS, EC 4.1.2.13) from rabbit skeletal 12153

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157

Analytical Chemistry

Article

temperature, no incubation time was required before measurement. Freshly prepared aqueous solutions of proteins (OVA, ALS, GAPDH, β-LG, HSA, BSA, sheep SA, horse SA, and rat SA) were diluted to the appropriate concentration range and denatured in 0.1 mol L−1 PBS, pH 7.4, 0.2% SDS. The employment of denaturing agents as SDS is important to improve the accessibility of −SH groups to the mercurial probe. pHMB−protein complexes were obtained by incubating the proteins with a 5-fold excess of pHMB at room temperature (21 ± 1 °C) and analyzed after 180 min if not differently specified. Solutions were stable during the working day (time tested 8 h). The applied molar excess of pHMB was calculated on the basis of the number of free cysteine residues in proteins (4 −SH groups for OVA, 16 for GAPDH, 32 for ALS, 1 for βLG, HSA, BSA, sheep, horse, and rat serum albumin).31 All the solutions were filtered before injection by a 0.45 μm cellulose acetate filter (Millipore). The excess of pHMB was chromatographically separated and did not interfere with the analysis (see Results), without the need of removing the unreacted reagent. In the FI experiments, the pHMB concentration was kept constant (2.5 μmol L−1) and the protein was in molar excess (5 μmol L−1) in order to guarantee that all pHMB was complexed with proteins. Trypsin ProTeolysis and Labeling of Albumins. The tryptic digestion of proteins was adjusted in order to get a moderate number of peptides. One milliliter of 5 μmol L−1 of each albumin was incubated with trypsin (protein/trypsin ratio = 500), and a five molar excess of pHMB in 0.1 mol L−1 PBS, pH 8, at 37 °C for 1 h. After this, a second aliquot of trypsin has been added to reach a protein/trypsin ratio = 250 and incubated overnight at 37 °C. After 24 h, the tryptic mixture was diluted 1:1 in 0.1% TFA and injected.

Figure 1. Yield of MW/UV-assisted on-line digestion of pHMB (2.5 μmol L−1 concentration injected) as a function of increasing percentages of MeOH (sparse pattern) and ACN (dense pattern) in 0.1% TFA.

area of AF signal with respect to uncomplexed Hg(II) analyzed under the same conditions, which gave the same results in the range of 0−100% MeOH and ACN. Analogous experiments were performed on protein−pHMB complexes. Figure 2 shows the results obtained for the MW/

■

RESULTS Efficiency of MW-UV-Assisted On-Line Oxidation. Preliminary Experiments by FIA-CVG-AFS. The conversion degree of uncomplexed pHMB and pHMB complexed to proteins into Hg(II) by the MW/UV apparatus was evaluated. This would allow us to evaluate the digestion yield and to get quantitative information from HPLC analysis. MW/UV online oxidation was studied in FI mode by injecting uncomplexed pHMB, ALS-pHMB, OVA-pHMB, and GAPDH-pHMB complexes, as model proteins. For the MW-UV-assisted online digestion, we adopted the operating conditions previously optimized for the on-line digestion of pHMB−thiol complexes.61 The oxidation efficiency for each complex was calculated as the ratio S1/S0 × 100, where S1 and S0 are the integrated signals obtained by MW/UV FIA-CVG-AFS for equimolar concentration of pHMB complexed with protein and uncomplexed, respectively, and processed under the same operating conditions. pHMB signal was taken as a reference based on its 89.0% ± 0.5% oxidation efficiency with respect to an equimolar concentration of Hg(II) analyzed under the same conditions. Analysis was performed in 0.1% TFA as the eluent solution with increasing percentages of organic solvent (ACN and MeOH), in order to investigate eventual changes in the oxidative decomposition yield of pHMB. This is fundamental in view of using these eluents in RPC. Figure 1 shows the oxidative decomposition yield of pHMB as a function of the percentages of MeOH and ACN calculated from the FI peak

Figure 2. Yield of MW/UV-assisted on-line digestion of OVA-pHMB (dense pattern), GAPDH-pHMB (sparse pattern), and ALS-pHMB complexes (no pattern), as a function of increasing percentages of ACN in 0.1% TFA.

UV-assisted on-line digestion of ALS-pHMB, OVA-pHMB, and GAPDH-pHMB complexes, as representative protein samples using ACN as the organic phase. Also, in this case we found that MeOH does not affect the recovery of pHMB-protein complexes and ACN affects the oxidation efficiency of the pHMB-ALS complex by 30%, only for percentages higher than 90%. The S/N ratio was not worse in the presence of ACN nor MeOH. This is an improvement with respect to the employment of bromine, generated in situ by KBr/KBrO3 and HCl for the on line oxidation. ACN was, indeed, proven to be unsuitable because of S/N ratios ten times smaller than those obtained with methanol as the eluent.32 On the other hand, MeOH concentration itself had to be kept below 50% because for higher percentages, the signal decreased up to about 55% when the methanol concentration was 100%.32 12154

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157

Analytical Chemistry

Article

with pHMB. On the basis of the pHMB calibration curve and the area of chromatographic AF peak, the average number of −SH groups titrated (expressed as mole −SH/mol of protein) was 0.72 ± 0.03 for BSA, 0.98 ± 0.01 for β-LG, and 2.71 ± 0.01 for OVA, in agreement with the literature data.28,62,63 pHMB Tagging of Tryptic Peptides. pHMB tagging coupled to RPC-MW/UV-CVG-AFS analysis was applied to characterize the peptides from the tryptic digestion of serum albumin from different species (bovine, human, from sheep, rat, and horse). Serum albumin is the most abundant soluble protein in the body of all vertebrates and is the most prominent protein in plasma. The human isoform of serum albumin is a protein formed with 585 amino acids, of which 35 are cysteine (Cys) and cystines (Cys-Cys), with a molecular weight of 67 kD. Only the first Cys in the sequence remains as a free thiol, while the rest participate in the formation of 17 disulfide bonds. These characteristics of the cysteine residues are conserved in the serum albumin of all vertebrates. The aim of this part of the work is to show how pHMB labeling coupled to RPC-MW/UV-CVG-AFS system can be applied to identify Cys-containing peptides in a peptide mixture, and as it can be employed to identify species-specific protein peptides from the chromatographic elution pattern. Trypsin is a serine protease that cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. The cleavage may occur in some or all possible cleavage points. Thus, in the case of albumins, many peptides containing the Cys35 may be obtained, depending on the cleavage sites. The digestion procedures are in general employed with different aims: (i) to reconstruct the protein sequence from the overlapping of the digestion fragments obtained with different proteolytic enzymes and (ii) to determine the molecular weight and, thus, to identify a protein in a mixture. In our case, we aimed to get a reproducible digestion pattern of the native albumins in order to distinguish them. Thus, we are focused on the achievement of different, reproducible, characteristic digestion patterns, more than the complete digestion of the proteins. Figure 4 shows a representative comparison of the mercuryspecific chromatograms of the tryptic digests of SDS denatured pHMB-horse SA and SDS denatured pHMB-BSA complexes. The data shows that depending on the species, the elution pattern of albumin tryptic digests was different. Figures 5 and 6 show the mercury-specific chromatograms of the tryptic digests of HSA and sheep SA (N = 3 replicates of the whole procedure), demonstrating the repeatability of the digestion procedure. Table 1 summarizes the retention times from AF chromatogram of the characteristic fragments derived from the tryptic digestion of the albumins from the five different species. The retention times corresponding to the pattern of the different species-specific fragments is reported in bold. The results show that the elution pattern of the pHMBcomplexed fragments is very different in the five species considered, except for the peptide eluting around 46 min that corresponds to a fraction of undigested protein. This was confirmed by the analysis of undigested proteins in the same chromatographic conditions (tr: HSA = 47.2 min, BSA = 47.7 min, sheep SA = 46.9 min, horse SA = 46.1 min, and rat SA = 46.4 min). Different elution patterns could allow, in principle, the identification of the species-specific protein.

These results suggest that both organic modifiers can be used for RPC separations using MW/UV-CVGAFS detection. Application of MW/UV-CVGAFS detection in RPC separations. The MW/UV-assisted on-line mercury oxidation method was applied to the determination of the pHMB− protein complexes as an useful interface between RPC and CVG-AFS detection, and it was also tested as a diagnostic tool in the formation of different thiolic peptides after a mild tryptic digestion of serum albumins from different species. pHMB elutes from the C4 Vydac RPC column in a 5 min broad peak, only with 100% MeOH or ACN, not interfering in the “analytical window” of the chromatogram. Calibration was evaluated by analyzing three replicates of standard Cys-pHMB solutions at 0.25, 0.5, 1, 5, 10, 25, 50, and 100 μmol L−1 concentration levels. This complex elutes in a sharp peak in the dead volume of the column, allowing us to better quantify the mercury peak area. The eight-point calibration curve determined by a least-squares regression algorithm was linear over the range of 0.2−100 μmol L−1. The fitting equation was y = 234.23x − 4.25, with a mean correlation coefficient R2 = 0.999. The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated as 3sb/slope and 10sb/slope, when sb is the standard deviation of 10 blank measurements and were 57 nmol L−1 and 192 nmol L−1, respectively. It must be underlined that by using a mercury-specific detector, both the detection limit and sensitivity for each protein depends on the number of −SH groups reacting with pHMB. With consideration that the recovery of uncomplexed and protein-complexed pHMB did not differ significantly, by knowing the protein concentration, this method can also be employed to estimate the number of −SH groups reactive with pHMB. pHMB Derivatization Coupled to RPC-MW/UV-CVG-AFS Analysis: A Tool for Protein Tagging. Figure 3 shows the UV absorbance chromatogram at (a) 254 nm and the (b) mercuryspecific trace of a mixture of SDS-denatured BSA (tR = 27.2 min), β-LG- (tR = 30.1 min), and OVA (tR = 34.7 min) labeled

Figure 3. RP absorbance chromatogram at (a) 254 nm and the (b) mercury-specific chromatograms of pHMB−SDS-denatured BSA (tR = 27.2 min), pHMB−SDS-denatured β-LG (tR = 30.1 min), and pHMB−SDS denatured OVA (tR = 34.7 min) complexes in Vydac C4 column. Chromatographic conditions: 1 min isocratic elution in 60% of 0.1% TFA aqueous solution-40% MeOH followed by a 43 min gradient up to 90% MeOH, 1 min gradient up to 100% MeOH, 10 min isocratic elution in 100% MeOH, and column re-equilibration. Flow rate = 1.0 mL min−1, with a 50 μL injection. 12155

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157

Analytical Chemistry

Article

Figure 6. Overlapping triple mercury-specific chromatograms of pHMB−SDS denatured sheep SA tryptic digest in the Vydac C4 column. Chromatographic conditions: see Figure 4. N = 3 replicates.

Table 1. Retention Times of the pHMB-Complexed Tryptic Fragments from the Albumins from Different Species

Figure 4. The mercury-specific chromatograms of pHMB−SDS denatured horse SA tryptic digest (black line) and pHMB−SDS denatured BSA tryptic digest (red line). Chromatographic conditions: 1 min gradient from 5% of 0.1% TFA aqueous solution-95% to 40% MeOH, followed by a 40 min gradient up to 47% MeOH, 1 min gradient up to 65% MeOH, 10 min gradient up to 75% MeOH, 1 min gradient up to 100% MeOH, 10 min isocratic elution in 100% MeOH, and column re-equilibration. Flow rate = 1.0 mL min−1, with 50 μL injection.

albumin HSA BSA sheep SA horse SA rat SA

retention time (min) 2.9 − − − −

5.8 − 5.5 − −

− − 6.6 − 7.3

− − 8.3 − −

8.9 9.6 − 9.3 9.4

10.4 10.1 − 10.2 10.1

47.2 47.9 46.9 46.1 45.4

− 50.2 47.8 48.9 46.6

reactor that allowed us to obtain the digestion of pHMB and pHMB−protein complexes to Hg(II) at room temperature with a yield ranging between 80% and 95%. MW/UV oxidative decomposition of pHMB and pHMB− protein complexes were studied in the flow injection mode (FIMW/UV-CVGAFS) for increasing concentrations of ACN and MeOH (0−90%), in order to evaluate the possible application of this on-line digestion method to RP chromatographic separations that require organic solvents, finding that the oxidative decomposition efficiency of pHMB was approximately the same from 0 to 100% MeOH and 0−90% ACN. RPC-MW/UV-CVGAFS allowed us to identify Cys-containing peptides in the tryptic digests of albumin from different species and to distinguish the species-specific albumins from the chromatographic elution pattern of the tryptic digest.

■

Figure 5. Overlapping triple mercury-specific chromatograms of pHMB−SDS denatured HSA tryptic digest in the Vydac C4 column. Chromatographic conditions: see Figure 4. N = 3 replicates.

AUTHOR INFORMATION

Corresponding Author

■

*E-mail: [email protected]. Tel: +39 050 3152293. Fax: +39 050 315 2555.

CONCLUSION Biological thiols have no specific physical-chemical properties, which are required to detect proteins with high sensitivity. A direct determination method like ESI-MS is not specific for thiolic proteins. The direct determination of proteins using the sulfur signal in ICP-MS techniques is affected by many interferences and does not have enough sensitivity for protein analysis. Thus, derivatization techniques are required in order to get specificity and sensitivity. pHMB represents a good labeling reagent for thiolic proteins because it is a monofunctional probe containing mercury, which has a high affinity for −SH groups and it forms pHMB− protein complexes that are soluble and stable. The combination between MW and UV radiation gave a unique photochemical

Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Anal. Bioanal. Chem. 2007, 389, 1017−1031. (2) Ong, S. E.; Mann, M. Nat. Chem. Biol. 2005, 1, 252−262. (3) Bettmer, J.; Jakubowski, N.; Prange, A. Anal. Bioanal. Chem. 2006, 386, 7−11. (4) Sanz-Medel, A.; Montes-Bayón, M.; del Rosario Fernández de la Campa, M.; Encinar, J.; Bettmer, J. Anal. Bioanal. Chem. 2008, 390, 3− 16. (5) Tanner, S. D.; Ornatsky, O.; Bandura, D. R.; Baranov, V. I. Spectrochim. Acta, Part B 2007, 62, 188−195. 12156

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157

Analytical Chemistry

Article

(6) Bandura, D. R.; Baranov, V. I.; Tanner, S. D. Anal. Chem. 2002, 74, 1497−1502. (7) Ahrends, R.; Pieper, S.; Kuhn, A.; Weisshoff, H.; Hamester, M.; Lindemann, T.; Scheler, C.; Lehmann, K.; Taubner, K.; Linscheid, M. W. Mol. Cell. Proteomics 2007, 6, 1907−1916. (8) Jakubowski, N.; Waentig, L.; Hayen, H.; Venkatachalam, A.; von Bohlen, A.; Roos, P. H.; Manz, A. J. Anal. At. Spectrom. 2008, 23, 1497−1507. (9) Patel, P.; Jones, P.; Handy, R.; Harrington, C.; Marshall, P.; Evans, E. H. Anal. Bioanal. Chem. 2008, 390, 61−65. (10) Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal. Chem. 2002, 74, 1629−1636. (11) Quinn, Z. A.; Baranov, V. I.; Tanner, S. D.; Wrana, J. L. J. Anal. At. Spectrom. 2002, 17, 892−896. (12) Zhang, C.; Wu, F. B.; Zhang, X. R. J. Anal. At. Spectrom. 2002, 17, 1304−1307. (13) Zhang, C.; Zhang, Z. Y.; Yu, B. B.; Shi, J. J.; Zhang, X. R. Anal. Chem. 2002, 74, 96−99. (14) Whetstone, P. A.; Butlin, N. G.; Corneillie, T. M.; Meares, C. F. Bioconjugate Chem. 2004, 15, 3−6. (15) Liu, H. L.; Zhang, Y. J.; Wang, J. L.; Wang, D.; Zhou, C. X.; Cai, Y.; Qian, X. H. Anal. Chem. 2006, 78, 6614−6621. (16) Toyo’oka, T. J. Chromatogr., B 2009, 877, 3318−3330. (17) Reid, R. S.; Rabenstein, D. L. Can. J. Chem. 1981, 59, 1505− 1514. (18) Robert, J. M.; Rabenstein, D. L. Anal. Chem. 1991, 63, 2674− 2679. (19) Xu, M.; Yang, L. M.; Wang, Q. Q. Chem.−Eur. J. 2012, 18, 13989−13993. (20) Xu, M.; Yan, X. W.; Xie, Q. Q.; Yang, L. M.; Wang, Q. Q. Anal. Chem. 2010, 82, 1616−1620. (21) Guo, Y. F.; Xu, M.; Yang, L. M.; Wang, Q. Q. J. Anal. At. Spectrom. 2009, 24, 1184−1187. (22) Guo, Y. F.; Chen, L. Q.; Yang, L. M.; Wang, Q. Q. J. Am. Soc. Mass Spectrom. 2008, 19, 1108−1113. (23) Kutscher, D. J.; Bettmer, J. Anal. Chem. 2009, 81, 9172−9177. (24) Kutscher, D. J.; Busto, M. E. D.; Zinn, N.; Sanz-Medel, A.; Bettmer, J. J. Anal. At. Spectrom. 2008, 23, 1359−1364. (25) Kutscher, D. J.; Fricker, M. B.; Hattendorf, B.; Bettmer, J.; Gunther, D. Anal. Bioanal. Chem. 2011, 401, 2691−2698. (26) Lu, M. L.; Li, X. F.; Le, X. C.; Weinfeld, M.; Wang, H. L. Rapid Commun. Mass Spectrom. 2010, 24, 1523−1532. (27) Rao, Y. L.; Xiang, B. R.; Bramanti, E.; D’Ulivo, A.; Mester, Z. J. Agric. Food Chem. 2010, 58, 1462−1468. (28) Takatera, K.; Watanabe, T. Anal. Chem. 1993, 65, 3644−3646. (29) Zaluzec, E. J.; Gage, D. A.; Watson, J. T. J. Am. Soc. Mass Spectrom. 1994, 5, 359−366. (30) Rao, Y. L.; McCooeye, M.; Windust, A.; Bramanti, E.; D’Ulivo, A.; Mester, Z. Anal. Chem. 2010, 82, 8121−8130. (31) Bramanti, E.; D’Ulivo, A.; Lampugnani, L.; Zamboni, R.; Raspi, G. Anal. Biochem. 1999, 274, 163−173. (32) Bramanti, E.; Lomonte, C.; Galli, A.; Onor, M.; Zamboni, R.; Raspi, G.; D’Ulivo, A. J. Chromatogr., A 2004, 1054, 285−291. (33) Bramanti, E.; Lomonte, C.; Onor, M.; Zamboni, R.; Raspi, G.; D’Ulivo, A. Anal. Bioanal. Chem. 2004, 380, 310−318. (34) Bramanti, E.; Lucchesini, S.; D’Ulivo, A.; Lampugnani, L.; Zamboni, R.; Spinetti, M. C.; Raspi, G. J. Anal. At. Spectrom. 2001, 16, 166−171. (35) Bramanti, E.; Sortino, C.; Lomonte, C.; Onor, M.; Zamboni, R.; Raspi, G.; D’Ulivo, A. Talanta 2004, 63, 383−389. (36) Bramanti, E.; Angeli, V.; Mester, Z.; D’Ulivo, A. Talanta 2009, 79, 554−555. (37) Bramanti, E.; Angeli, V.; Mester, Z.; Pompella, A.; Paolicchi, A.; D’Ulivo, A. Talanta 2010, 81, 1295−1299. (38) Bramanti, E.; Cavallaro, R.; Onor, M.; Zamboni, R.; D’Ulivo, A. Talanta 2008, 74, 936−943. (39) Bramanti, E.; D’Ulivo, L.; Lomonte, C.; Onor, M.; Zamboni, R.; Raspi, G.; D’Ulivo, A. Anal. Chim. Acta 2006, 579, 38−46.

(40) Morita, H.; Tanaka, H.; Shimomura, S. Spectrochim. Acta, Part B 1995, 50, 69−84. (41) Tang, L.; Chen, F.; Yang, L. M.; Wang, Q. Q. J. Chromatogr., B 2009, 877, 3428−3433. (42) Baxter, C.; Frech, W. Anal. Chim. Acta 1990, 236, 377−384. (43) Bloxham, M. J.; Hill, S. J.; Worsfold, P. J. J. Anal. At. Spectrom. 1996, 11, 511−514. (44) Bramanti, E.; Jacovozzi, K.; D’Ulivo, L.; Vecoli, C.; Zamboni, R.; Mester, Z.; D’Ulivo, A. Talanta 2008, 77, 684−694. (45) Bramanti, E.; Lomonte, C.; Onor, M.; Zamboni, R.; D’Ulivo, A.; Raspi, G. Talanta 2005, 66, 762−768. (46) Bramanti, E.; Toncelli, D.; Morelli, E.; Lampugnani, L.; Zamboni, R.; Miller, K. E.; Zemetra, J.; D’Ulivo, A. J. Chromatogr., A 2006, 1133, 195−203. (47) Bramanti, E.; Vecoli, C.; Neglia, D.; Pellegrini, M. P.; Raspi, G.; Barsacchi, R. Clin. Chem. 2005, 51, 1007−1013. (48) Hintelmann, H.; Wilken, R. D. Appl. Organomet. Chem. 1993, 7, 173−180. (49) Munaf, E.; Haraguchi, H.; Ishii, D.; Takeuchi, T.; Goto, M. Anal. Chim. Acta 1990, 235, 399−404. (50) Palmisano, F.; Zambonin, P. G.; Cardellicchio, N. Fresenius’ J. Anal. Chem. 1993, 346, 648−652. (51) Schickling, C.; Broekaert, J. A. C. Appl. Organomet. Chem. 1995, 9, 29−36. (52) Bendicho, C.; Pena, F.; Costas, M.; Gil, S.; Lavilla, I. TrAC, Trends Anal. Chem. 2010, 29, 681−691. (53) Falter, R.; Ilgen, G. Fresenius’ J. Anal. Chem. 1997, 358, 407− 410. (54) Falter, R.; Scholer, H. F. J. Chromatogr., A 1994, 675, 253−256. (55) Falter, R.; Scholer, H. F. Fresenius’ J. Anal. Chem. 1995, 353, 34− 38. (56) Liang, L. N.; Jiang, G. B.; Liu, J. F.; Hu, J. T. Anal. Chim. Acta 2003, 477, 131−137. (57) Morales-Rubio, A.; Mena, M. L.; McLeod, C. W. Anal. Chim. Acta 1995, 308, 364−370. (58) Tsalev, D. L.; Sperling, M.; Welz, B. Analyst 1992, 117, 1735− 1741. (59) Tsalev, D. L.; Sperling, M.; Welz, B. Analyst 1992, 117, 1729− 1733. (60) Welz, B.; Tsalev, D. L.; Sperling, M. Anal. Chim. Acta 1992, 261, 91−103. (61) Angeli, V.; Ferrari, C.; Longo, I.; Onor, M.; D’Ulivo, A.; Bramanti, E. Anal. Chem. 2011, 83, 338−343. (62) Raspi, G.; LoMoro, A.; Spinetti, M.; Tesi, A. Anal. Commun. 1997, 34, 307−309. (63) Brownlow, S.; Cabral, J. H. M.; Cooper, R.; Flower, D. R.; Yewdall, S. J.; Polikarpov, I.; North, A. C. T.; Sawyer, L. Structure 1997, 5, 481−495.

12157

dx.doi.org/10.1021/ac403389z | Anal. Chem. 2013, 85, 12152−12157