A New Analytical Material-Enhanced Laser Desorption Ionization

A New Analytical Material-Enhanced Laser Desorption Ionization (MELDI) Based Approach for the Determination of Low-Mass Serum Constituents Using Fullerene Derivatives for Selective Enrichment Rainer M. Vallant,† Zoltan Szabo,† Lukas Trojer,† Muhammad Najam-ul-Haq, Matthias Rainer, Christian W. Huck, Rania Bakry, and Gu1 nther K. Bonn* Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria Received July 13, 2006

[60]Fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as materialenhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel highspeed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new “top-down” strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation. Keywords: derivatized fullerenes • protein profiling • MELDI • low-mass serum constituents • monolithic MSt/ BVPE capillaries • µ-LC • MALDI-TOF MS/MS

Introduction Clinical proteomics is generally regarded as a promising and progressive discipline for establishing relationships between protein expression profiles and specific disease phenotypes by tracing relevant biomarkers.1 However, from an analytical point of view, proteomics makes great demands on fractionation, separation, and detection techniques and essentially depends on the development of sophisticated workflows, since biological samples such as blood serum and plasma, lymph, urine, or exudates represent mixtures of an extreme level of complexity. Main ingredients are proteins, as well as their degradation products. The presence of organic as well as inorganic salts preclude direct mass spectrometric (MS) analysis, making effective sample fractionation and purification steps indispensable. Routinely employed techniques for proteome analyses can be divided into “top-down” (way of analysis from the protein to the peptide level) and “bottom-up” (from the peptide to the * Address correspondence to: Univ. Prof. Dr. Gu ¨ nther Bonn, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University of Innsbruck, Innrain 52a, 6020 Innsbruck, Austria. Tel., +43(0)512/507-5170; fax, +43(0)512/507-2794; e-mail, [email protected]. † All authors contributed equally to this work.

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Journal of Proteome Research 2007, 6, 44-53

Published on Web 11/23/2006

protein level) approaches.2 Two-dimensional gel electrophoresis (2-DE) is, in the majority of cases, used as a first protein resolution step. Thereupon, proteins of interest can be removed from the gel, digested, and subjected to matrix-assisted laser desorption/ionization (MALDI)-MS or liquid chromatographyelectrospray ionization (LC-ESI)-MS analysis for identification.3 The application of surface-enhanced laser desorption/ ionization (SELDI)4 in the field of proteomics presents another promising top-down approach. Proteins get adsorbed to derivatized mass spectrometric thin layer surfaces and are, after purification and washing steps, directly subjected to on-target analysis by MALDI-MS.5 Multidimensional protein identification (MudPIT), also referred as “shotgun proteomics”, has been shown to be a powerful bottom-up approach.6 This method involves tryptic digestion of protein mixtures followed by twodimensional liquid chromatography (2D-LC) coupled with mass spectrometry, predominately ESI-MS. Recently, material-enhanced laser desorption/ionization (MELDI) has been introduced as refinement of the SELDI approach.7,8 MELDI does not exclusively focus on the impact of surface derivatization on the resulting mass pattern but comprises the properties of the employed support material (support porosity, support hydrophobicity, etc.). Thus, MELDI 10.1021/pr060347m CCC: $37.00

 2007 American Chemical Society

New Approach for Determination of Low-Mass Serum Constituents

includes particle morphology beyond surface chemistry. In a number of publications, it has been shown that different carrier materials such as silica,7 cellulose,8 diamond,9 nanotubes,10 or polymer particles11 result in specific peak patterns, which multiplies the amount of data for serum screening and biomarker discovery. Carbon particles such as underivatized fullerenes, graphite, and carbon black are useful matrices for protein analysis.12-14 The functionalization of fullerenes has received considerable attention because of the successful application of those derivatives in material science.15-17 Although [60]fullerene exhibits outstanding reactivity and is thus an ideal starting material for a vast number of chemically modified compounds, no literature can be found demonstrating the use of fullerene derivatives as a tool in the field of protein profiling for different biological samples. Methano[60]fullerene is frequently studied concerning derivatization reactions because of facile synthetic availability, while maintaining the characteristic properties of [60]fullerenes.18-20 Even if SELDI as well as MELDI can successfully be employed as rapid screening methods for a great amount of biological samples, identification of bound analytes is not possible.3 For the combination of the benefits of MELDI with the identification of sample constituents, new strategies have to be found. In this contribution, we report on the application of novel fullerene derivatives as MELDI supports for selective enrichment of small proteins and peptides from human serum samples. A strategy for biomarker identification of bound analytes in the low molecular mass range (m/z 1000-4000) by elution, high-resolution separation, target-spotting, and MALDIMS/MS evaluation is presented.

Experimental Procedures Chemicals and Reagents. All chemicals were used without further purification unless otherwise noted. [C60]Fullerene g99.5% was purchased from MER Corporation (Tucson, AZ). For fullerene derivatization, 1-octadecanol 99%, iminodiacetic acid (IDA) 98%, t-butyl bromoacetate 99%, dimethyl sulfide 99%, p-toluenesulfonic acid 97%, thionyl chloride g99%, and malonyl dichloride 97% were obtained from Aldrich (Milwaukee, WI). Lysozyme (from chicken egg white) (HEWL) for the characterization of fullerene derivatives was purchased from Sigma (St. Louis, MO). For monolith fabrication, magnesium, p-vinylbenzyl chloride, p-methylstyrene (MSt), 1-decanol, and toluene were purchased from Aldrich, and R,R′-azoisobutyronitrile (AIBN) was purchased from Fluka (Buchs, Switzerland). Before use, toluene was distilled over sodium, and p-methylstyrene was extracted with 10% NaHCO3 and water, dried over Na2SO4, and finally distilled under vacuum. The oligodeoxynucleotide mixture [d(pT)12-18] and the peptide standard used for quality control of the fabricated MSt/BVPE monoliths were purchased from Sigma. Copper(II)-sulfate p.a. 99%, trifluoroacetic acid (TFA) g99.5%, and HPLC-grade acteonitrile (ACN) were obtained from Merck (Darmstadt, Germany). R-Cyano4-hydroxycinnamic acid (HCCA, g99.0%) and 4-hydroxy-3,5dimethoxy-cinnamic acid (sinapinic acid, SA, g99.0%) used as MALDI matrices were purchased from Fluka. All water used in this study was purified by a NANOpure Infinity unit (Barnstead, Boston, MA). Instrumentation. Elemental analysis of the derivatized fullerenes was carried out on a Carlo Erba EA 1110 CHNS instrument (Carlo Erba Reagents, Rodano, Italy). The infrared spectra were recorded on a Nicolet 5700 Attenuated Totally Reflection Fourier Transform Infrared (ATR-FTIR) instrument

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(Thermo Electron Corporation, MA) in the range of 500-4500 cm-1. The copper content of IDA-[60]fullerene was determined by an atomic absorption spectrometer (AAS) (PU9100X, Philips) at 324.6 nm. The protein adsorption capacity studies of the fullerene derivatives were done on a double-beam spectrophotometer (U-2000, Hitachi High-Technologies, Japan) at 280 nm. All MALDI and MELDI analyses were performed on a MALDI/TOF-MS (Ultraflex MALDI TOF/TOF, Bruker Daltonics, Bremen, Germany) employing stainless steel targets (MTP 384 target ground steel TF, Bruker Daltonics) for protein profiling and anchor chip targets (MTP anchor chip 600, Bruker Daltonics) for serum spotting and MS/MS analysis. Spectra were recorded in reflector mode for characterization of fullerene derivatives and characterization of serum peptides (m/z 10004000) and in linear mode for serum protein profiling (m/z 2000-20 000). All chromatrographic analyses, as well as target spotting experiments, were done on a Dionex Ultimate 2DµHPLC system, consisting of a Famos autoinjector, a Schwichos loading pump, Ulimate Gradient pump, a 3 nL cell detector and a spotter. Synthesis of Fullerene Derivatives. Dioctadecyl methano[60]fullerene was prepared by cyclopropanation of [60]fullerene with dioctadecyl propanedionate,21 which was synthesized by esterification of malonic acid with 1-octadecanol as described earlier22 (Anal. Calcd for C99H74O4: C, 89.6; H, 5.6. Found: C, 88.3; H, 8.9). [60]Fullerenoacetic acid was synthesized from [60]fullerene according to Ito et al.23 by nucleophilic attack of a carbonylstabilized sulfonium ylide (t-butyl(dimethylsulfuranylidene)acetate).24 The resulting t-butyl [60]fullerenoacetate was converted into [60]fullereneoacetic acid by hydrolysis with p-toluenesulfonic acid (Anal. Calcd for C62H2O2: C, 95.6; H, 0.26. Found: C, 93.2%; H, not significant). [60]Fullerenoacetic acid (200 mg, 0.26 mmol) was reacted with thionyl chloride (10 mL, 140 mmol) for 8 h under nitrogen. The excess of thionyl chloride was evaporated under vacuum, and the resulting [60]fullerenoacetyl chloride (175 mg, 0.21 mmol) was treated with IDA (57 mg, 4.3 mmol) in the presence of triethylamine (60 µL, 8.2 mmol) for 8 h at 65 °C using dry tetrahydrofuran as solvent. Finally, the solvent was evaporated, and the IDA-[60]fullerene was washed thoroughly with hot water to remove unreacted IDA and dried (176 mg, 0.2 mmol) (Anal. Calcd for C66H7NO5: C, 88.7; H, 0.78%. Found: C, 87.4; H, 1.0%). Fabrication of Poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) (MSt/BVPE) Capillary Columns. Monolithic MSt/ BVPE capillary columns were produced by thermally initiated free radical polymerization of methylstyrene (MSt) and 1,2-bisp(vinylphenyl)ethane (BVPE), which was synthesized by Grignard dimerization of p-vinylbenzyl choride as described elsewhere.25 Totals of 78.33 mg of BVPE (17.5 vol %), 5 mg of AIBN (1 wt %), 87.5 µL of MSt (17.5 vol %), 70 µL of toluene (14 vol %), and 255 µL of 1-decanol (51 vol %) were mixed, sonicated at 40-50 °C, and filled into preheated, silanized capillaries. The polymerization was allowed to proceed for 24 h at 65 °C in a water bath under gentle agitation. Before use, the capillary columns were extensively washed with ACN and checked regarding their performance and efficiency by the separation of a 9-peptide and an oligothymidylic acid [p(dT)12-18] standard, employing reversed-phase (RP) and ion-pair reversed-phase (IP-RP) conditions, respectively. Characterization of the Fullerene Derivatives. For the determination of Cu(II) capacity of IDA-[60]fullerene, 12 mg Journal of Proteome Research • Vol. 6, No. 1, 2007 45

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Figure 1. Fullerene derivatization reactions based on buckminster fullerene (Educts, fullerene derivatives and reaction conditions: see Table 1).

of IDA-[60]fullerene was loaded with Cu(II) by suspending in a 50 mM CuSO4 solution. After a washing step, Cu(II)-IDA[60]fullerene was suspended in 700 µL of 50 mM Na2EDTA for 20 min to release the complexed Cu(II) ions. The Cu(II) concentration was then determined by AAS after calibration was performed by standard solutions ranging from 4-20 ppm. The protein binding capacity of all fullerene derivatives was evaluated using HEWL according to a method described in detail by Finette et al.26 Protein solutions (2 mL each) of different concentrations were incubated on the equilibrated materials for 15 h at 20 °C. The protein was dissolved in 10% ACN/0.1% TFA for dioctadecyl methano [60]fullerene and in PBS buffer (20 mM and 0.5 M NaCl, pH 6) for [60]fullerenoacetic acid and Cu(II)-IDA-[60]fullerene. Suspensions were centrifuged, and the protein content of the supernatant was measured with a UV/vis spectrophotometer at 280 nm. Incubation/Elution of Human Serum on/from Fullerene Derivatives. Incubation of human serum (10 µL serum/1 mg fullerene) on the fullerene derivatives was performed as described earlier.27 For MELDI application, 1 µL of the suspended, loaded fullerene carrier was directly spotted on a target and mixed with 1 µL of saturated SA solution (50% ACN/0.1% TFA). The elution of the serum constituents adsorbed on the particles was achieved by suspension in 50% ACN/0.1% TFA in the case of [60]fullerenoacetic acid and ACN/0.1% TFA in the case of dioctadecyl methano[60]fullerene, followed by washing the particles with 0.1% TFA (two times). The eluate and washing solutions were combined and concentrated (lyophilized) to 1.7 µL/mg fullerene. µ-HPLC Separation and Target Spotting of Serum Eluate. Eluates were separated using RP conditions (solvent A, 0.1% TFA in water; solvent B, 0.1% TFA in ACN), employing a monolithic MSt/BVPE capillary column (80 × 0.2 mm). The 46

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column eluates were spotted on anchor chip targets (500 nL/ spot). Prior to spotting, the targets were subjected to a thin layer matrix (HCCA) preparation according to a protocol given by Gobom et al.28 After spotting, the fractions were desalted by 0.1% TFA, and the spots were recrystallized by addition of 0.1% TFA in 70% ACN (500 nL each spot). All targets were measured automatically in the m/z range of 1000-4000 using the Flex control autoexecute add-on (Bruker Daltonics).

Results and Discussion Product Identification. [60]Fullerene was reacted to dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA[60]fullerene (Figure 1). Table 1 provides details on the educts and the prepared derivatives, as well as the reaction conditions. Success of the derivatizations was confirmed by various techniques such as MALDI/TOF-MS, IR, and elemental analysis. Figure 2A shows the MALDI/TOF-MS spectra for dioctadecyl methano[60]fullerene. The distinctive peak at m/z 1327.6 describes the desired product. The prominent peak at m/z 1936.92 may be attributed to an adduct (hydrophobic interactions) of the reversed-phase linker and the dioctadecyl [60]fullerene. Figure 2B demonstrates the successful synthesis of [60]fullerenoacetic acid. The dominant peak at m/z 777.81 represents the product obtained after the hydrolysis of t-butyl [60]fullerenoacetate. Figure 2C depicts a mass peak at m/z 892.97 ascribed to the successful synthesis of IDA-[60]fullerene, since the obtained mass is consistent with the theoretical mass (Table 1). The mass signal observed at m/z 833.07 may be attributed to a neutral loss (acetic acid). The peak at m/z 778, belonging to [60]fullerenoacetic acid, is the product of hydrolysis of unreacted [60]fullerenoacetyl chloride. The different functionalizations on fullerenes are also ana-

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Table 1. Summary of the Educts (a-e and 1), Synthesized Fullerene Derivatives (2-6), and Reaction Conditions Employed for Fullerene Derivatization name

empirical formula

a b c d e

dioctadecyl propanedionate t-butyl(dimethylsulfuranylidene)acetate p-toluenesulfonic acid monohydrate thionyl chloride iminodiactic acid

C39H76O4 C8H16OS C7H8S*H2O SOCl2 C4H7NO4

1 2 3 4 5 6

[60]fullerene dioctadecyl methano[60]fullerene t-butyl[60]fullerenoacetate [60]fullerenoacetic acid [60]fullerenoacetyl chloride IDA-[60]fullerene

C60 C99H74O4 C66H19O2 C62H2O2 C62HO2Cl C66H7NO5

theoretical mass [g/mol]

CAS

609.04 160.28 190.22 118.97 133.10

16832-80-7 58719-71-4 6192-52-5 7719-09-7 142-73-4

720.64 1327.66 834.07 778.00 811.96 893.03

99685-96-8 193271-96-4 311336-89-7 155116-19-1 ---

reaction conditions

solvent

RT, 12h RT, 8h reflux, 8h reflux, 8h reflux, 8h

toluene toluene toluene -THF

Figure 2. Identification and purity check of synthesized dioctadecyl methano[60]fullerene (A), [60]fullerenoacetic acid (B), and IDA[60]fullerene (C).

lyzed by comparing IR spectra of dioctadecyl methano[60]fullerene, [60]fullernoacetic acid, and IDA-[60]fullerene in the range of 4500-450 cm-1 with the characteristic absorptions. A distinctive peak in the range of 1143-1190 cm-1 for C-O group confirms the presence of mentioned functionalities. There is a strong and sharp band in the region of 1704-1727 cm-1 due to CdO asymmetric stretching. The stretching peaks at 3020 and 3427 cm-1 justify the presence of O-H in [60]fullerenoacetic acid and IDA-[60]fullerene, respectively. Three alkane peaks are present in the IR spectra for dioctadecyl methano[60]fullerene around 719, 1462, and 2115 cm-1 for C-18 chain, whereas these peaks are not present in other derivatives. Protein Binding Properties. The Cu(II) content of IDA-[60]fullerene was found to be 12, 14, and 12 µg/mg for three independently prepared and loaded fullerenes, indicating the synthesis was highly reproducible. For investigation of the effect of fullerene derivatizations on the binding properties toward proteins, adsorption isotherms of a model protein were recorded at 20 °C (Figure 3). HEWL was chosen, since it is frequently used for this purpose8,26 and, thus, allows comparison of the binding properties of newly introduced materials with literature data. Data points fitted the theoretical adsorption model of Langmuir,26 which can be taken from the excellent linear behavior (R2 ) 0.995 for dioctadecyl methano[60]fullerene; R2 ) 0.992 for [60]fullerenoacetic acid;

Figure 3. Adsorption isotherms of HEWEL at 20 °C determined for dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and Cu(II)-IDA-[60]fullerene.

R2 ) 0.999 for Cu(II)-IDA-[60]fullenene) when plotting the Cequ against Cequ/mads, where Cequ is the concentration in equilibrium and mads is the mass of HEWL adsorbed at Cequ. The calculated maximal binding capacity was found to be 9.22, 9.67, and 20.53 µg/mg for dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and Cu(II)-IDA-[60]fullerene, respectively. These results prove the strong influence of derivatizations on the behavior Journal of Proteome Research • Vol. 6, No. 1, 2007 47

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Figure 4. Representative reproducibility study of the fullerene derivatization, the serum incubation, and the MELDI process for [60]fullerenoacetic acid. Conditions, Bruker Ultraflex MALDI-TOF/ TOF; each spectrum, addition of 350 shots; matrix, SA; m/z, 2000-10 000. Sample: diluted human serum.

of the carrier materials compared to the blank [60]fullerene. Phosphate-buffered saline (PBS) was employed for HEWL incubation in the case of the carboxylated as well as the IMAC fullerene support, whereas 10% ACN/0.1% TFA was used for protein adsorption on the RP support (denaturing conditions). The tremendous differences in msat between [60]fullerenoacetic acid and Cu(II)-IDA-[60]fullerene might be attributed to the PBS buffer used during HEWL incubation. While a phosphate buffer is generally known to be the most effective for sample loading on IMAC, HEWL adsorption on the [60]fullerenoacetic acid seems to be, to a certain degree, suppressed by the buffer. Derivatized Fullerenes as MELDI Carrier Materials. The fullerene derivatives were investigated regarding their applicability as MELDI carrier materials for screening of biological samples. Sample preparations on particles were accomplished according to the method described in Experimental Procedures and directly subjected to MALDI-MS analysis after matrix addition. One important prerequisite for the successful utilization of fullerene derivatives as functionalized adsorbents for protein profiling is the reproducibility of their synthesis, as well as the reproducibility of the whole MELDI process, including incubation and MS evaluation. Figure 4 presents MELDI-MS spectra of three independently synthesized and incubated charges of [60]fullerenoacetic acid. High qualitative reproducibility in the range of m/z 2000-10000 is given in these spectra. Minor divergences in signal intensity are attributed to MALDI used for sample desorption and ionization.8 Comparable results are also found for dioctadecyl methano[60]fullerene as well as Cu(II)-IDA- [60]fullerene (data not shown). Because fullerene derivatizations were found to alter the adsorption behavior toward proteins, differences in the protein profiling mass patterns are also expected when employing the 48


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fullerene derivatives as MELDI carrier materials. In fact, the spectra in the mass region of m/z 2300-7300 provided distinct differences in terms of the number of mass signals because of different chemical properties and polarity effects of the fullerenes (Figure 5A-C). While in the case of dioctadecyl [60]fullerene adsorption of serum constituents is based upon hydrophobic interaction (van der Waals interactions) due to the presence of the long alkyl chains, the adsorption mechanism of [60]fullerenoacetic acid is to a great extent influenced by the carboxyl group (as a polar, hydrophilic functionality).29 Taking into account the low acidic properties of this functional group, it can be concluded that analyte interaction is probably affected by hydrogen bonding and hydrophilic interactions rather than electrostatic interaction.29 The adsorption of serum constituents in the case of Cu(II)-IDA-[60]fullerene is based on different interaction between the analytes and the immobilized Cu(II) ion. [60]Fullereneoacetic acid provided the highest diversity of signals (Figure 5B), while both dioctadecyl methano[60]fullerene (Figure 5A) and Cu(II)-IDA-[60]fullerene (Figure 5C) were strongly limited in the number of evaluable masses. Further differences were established in the m/z region 10 200-20 000 (Figure 5D-F). The low-mass region is, however, generally considered to be of more relevance regarding potential biomarkers.30 It can be concluded that [60]fullerenoacetic acid preferentially binds low molecular weight serum constituents in contrast to Cu(II)-IDA-[60]fullerene, which seems to exhibit high binding properties toward proteins of higher molecular weight (Figure 5D,F). This phenomenon of diversity in the binding nature, however, can be used for boosting the range of information obtained from biofluids. Identification of Low-Abundance, Low-Mass (Peptidic) Serum Constituents. For the comparative study in terms of the investigation of low molecular weight serum compounds, dioctadecyl methano[60]fullerene and [60]fullerenoacetic acid were studied, even if Cu(II)-IDA-[60]fullerene was found to possess the highest protein capacity. The reason for doing so is the elution conditions used in the case of IMAC. The application of imidazole requires a subsequent desalting step, associated with a nondefinable sample loss, which might distort the results obtained. In this study, a new strategy is elaborated, which enables both screening of biological samples employing MELDI and identification of serum constituents in a broad mass range (m/z 1000-30 000) after elution of bound analytes (Figure 6). Samples are selectively enriched on MELDI materials exposing different affinities. For screening (m/z 2000-30 000), direct particle irradiation can be accomplished. For identification, however, elution of adsorbed compounds is necessary. While the analysis of small molecular weight constituents (m/z 4000 have to be subjected to digestion prior to identification. As indicated in Derivatized Fullerenes as MELDI Carrier Materials, MELDI is a powerful tool for multiplexed protein profiling using derivatized [60]fullerenes. MELDI, however, can only be applied as a rapid screening method for biological samples. Recently, a discussion emerged on the incidence of potential biomarkers in the low molecular weight mass range (m/z 1000-4000).31 The quest of biomarkers in this region offers an advantage, as peptides up to 4000 Da can directly be subjected to fragmentation (MS/MS) without precedent digestion. For the evaluation of this “lower mass range approach”,


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Figure 5. Influence of fullerene derivatization on the MELDI protein profile pattern in the m/z range of 2300-6300 (A-C) and 10 00020 000 (D-F). (A and D) Dioctadecyl methano[60]fullerene; (B and E) [60]fullerenoacetic acid; (C and F) Cu(II)-IDA-[60]fullerene. Conditions, Bruker Ultraflex MALDI-TOF/TOF; each spectrum, addition of 350 shots; matrix, SA. Sample: diluted human serum.

serum-incubated fullerene derivatives were directly subjected to MELDI measurement in the m/z range of 1000-4000. As it can be depicted from Figure 7A,D, the carboxylated fullerene provided a number of signals, whereas the more hydrophobic dioctadecyl methano[60]fullerene was characterized by total absence of expedient masses. The intensity and resolution of the peptide mass signals, based on direct irradiation of the MELDI supports, were, however, found to be too low for reasonable MS/MS analysis. For the purpose of a more comprehensive study of lower mass serum constituents, adsorbed compounds were eluted from the derivatized fullerenes as described in Experimental Procedures. The resulting MALDI spectra obtained from those eluates are shown in Figure 7B,E. Even if the signal intensity slightly increased, the masses were still unemployable for effective MS/MS evaluation. It is important to mention that the direct MALDI analysis of the incubated particles, as well as the analysis of the eluate, resulted in the same peak pattern, providing strong evidence that the adsorption of serum peptides and proteins on fullerene derivatives is, at least to a great extent, reversible. µ-HPLC at this stage shed light on the abundance of low molecular weight serum compounds. Both extracts were, after the elution from the derivatized fullerenes with acetonitrile, separated on a novel monolithic poly(p-methylstyrene-co-1,2bis(p-vinylphenyl)ethane) (MSt/BVPE) styrene capillary using RP conditions. This material benefits from favorable hydrodynamic properties, enabling strong reduction of equilibration times between gradient runs, application of steeper gradients, and thus, an increase of separation speed compared to other commercially available monolithic supports. This is an essential enhancement in proteome research, since the speed of chromatographic separations is generally seen as one of the rate-

Figure 6. Introduction of the new workflow as a combination of MELDI, solvent-selective elution, and µ-LC-MALDI/MS or µ-LCESI/MS for the investigation of proteins and peptides from biofluids. Journal of Proteome Research • Vol. 6, No. 1, 2007 49

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Figure 7. Representation of the low molecular mass workflow of human serum enriched on derivatized fullerenes, (A-C) [60]fullerenoacetic acid, (D-F) dioctadecyl methano[60]fullerene. (A and D) MELDI mass spectra; (B and E) MALDI spectra of the eluate before LC-separation; (C and F) MALDI spectra after LC separation and fractionation. Chromatographic conditions, 0-70% B in 15 min, 6 µL/min, 25 °C, UV 214 nm; mass spectrometric conditions, Bruker Ultraflex MALDI-TOF/TOF; each spectrum, addition of 400 shots; matrix, HCCA; m/z, 1000-4000. Sample: diluted human serum.

determining steps in a typical proteomics workflow. The separation of the eluates from dioctadecyl methano[60]fullerene and [60]fullereneoacetic acid is presented in Figure 8, panels A and B, respectively. A high-resolution separation was achieved within 10 min employing a flow rate of 6 µL/ min. The chromatograms prove the presence of a wide variety of peptides (tR range ∼3.0-5.5 min) in the case of [60]fullerenoacetic acid, which is in accordance with direct MALDI and eluate analysis. µ-LC separation on monolithic MSt/BVPE 50


together with UV detection is thus a simple, effective, and informative method for estimation of the occurrence of lowabundance, low molecular weight serum constituents. For the purpose of further quality rating of serum separation, target spotting of chromatographic runs was performed on anchor chip targets using thin layer HCCA preparation including angiotensin I (Mr.: 1296.684) and ACTH 18-39 (Mr.: 2465.198) as internal standards.32 Overlays of the resulting mass spectra (first 100 fractions) of both elutes can be found in Figure 7C,F.


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Figure 8. µ-HPLC-UV/vis separation of the serum eluate obtained from (A) dioctadecyl methano[60]fullerene and (B) [60]fullereneoacetic acid on a monolithic MSt/BVPE capillary (80 × 0.2 mm). Conditions, 0-70% B in 15 min, 6 µL/min, 25 °C, UV 214 nm; inj., 500 nL. Sample: diluted human serum.

The overwhelming impact of separation and fractionation on the intensity and number of mass signals is convincingly demonstrated, when comparing those overlays with the MALDI evaluation of the untreated eluate. Not until this stage of the serum preparation is the entire complexity of the lowabundance peptide species revealed. Even in case of dioctadecyl methano[60]fullerene, numerous high-resoluted masses, suitable of MS/MS evaluation, appeared. This comparative study is essential and should be kept in mind when analyzing low-abundance species in biological samples. The effect of elution, separation, and fractionation of serum is, in addition,

clarified by the improvement of the isotopic resolution (R) and signal-to-noise (S/N) of one selected mass signal (m/z: 1530.702) (Figure 7A-C). At the stage of MELDI (direct particle irradiation), R and S/N were found to be 6119 and 102.5, respectively. MALDI measurement of the eluate resulted in a slight improvement (R, 6675; S/N, 139.8), but eluate fractionation implicated a conspicuous increase, namely, R ) 14516 and S/N ) 450.7. We suppose matrix suppression effects as well as disturbing interference of the laser energy with the particles to be responsible for the comparatively ineffective detection of lowmass compounds in the case of MELDI. At the stage of MALDIJournal of Proteome Research • Vol. 6, No. 1, 2007 51

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Figure 9. 2D-plots of the MALDI-MS spectra (first 100 fractions), obtained after serum eluate separation and target spotting. Chromatographic and MS conditions, see Figures 8 and 7, respectively. (A) Eluate of dioctadecyl methano[60]fullerene; (B) eluate of [60]fullereneoacetic acid. Table 2. Listing of Peptides Identified by MALDI-MS/MS Fragmentation from Human Serum after Incubation on Different Fullerene Derivatives, Elution with ACN/0.1% TFA, and µ-LC Separation observed mass [Da]

theoretical mass [Da]

mass error [ppm]

score

2069.890 2126.127 2208.110 2254.247 2080.046 1529.956 2020.199

2069.984 2126.005 2208.055 2254.100 2079.960 1529.862 2020.097

55.00 70.00 30.00 70.00 55.00 62.00 55.00

128.13 153.36 114.13 138.76 85.86 40.01 41.10

2020.219

2020.097

62.00

42.00

database

[60]Fullerenoacetic Acid Swis-Prot HGLGHGHEQQHGLGHGHKF Swis-Prot GHGLGHGHEQQHGLGHGHKF Swis-Prot KHNLGHGHKHERDQGHGHQ Swis-Prot GHGLGHGHEQQHGLGHGHKFK Swis-Prot HNLGHGHKHERDQGHGHQ Swis-Prot RPHFFFPKSRIV Swis-Prot SSKITHRIHWESASLLR Dioctadecyl Methano[60]fullerene Swis-Prot SSKITHRIHWESASLLR

MS analysis of the unseparated eluate, suppression of low concentrated species at the expense of high-abundance masses might be responsible for lack in intensity and signal diversity. All those unfavorable effects are diminished by high-resolution separation and simultaneous fractionation in terms of target spotting. Figure 9 demonstrates the overlaid spectra given in Figure 7C,F by plotting the fraction number against the detected m/z values (1000-4000). It has to be emphasized that signal intensity is not considered in this figure, since every mass signal that is above a given threshold is represented by a dot. Generally, the signals derived from [60]fullerenoacetc acid (Figure 9B) are more intense that those from dioctadecyl methano[60]fullerene (Figure 9A). Nevertheless, it can clearly be seen that the carboxylated support exceeds the RP carrier in terms of number of found masses. Further confirmation is given by fragmentation of the separated and fractionated eluates employing MALDI-MS/MS. Table 2 summarizes all peptides that could get identified by MS/MS spectra, followed by database searching in Mascot against the Swiss-Prot database. This includes Kininogen-1 precursor, a protein belonging to the cystain super family, responsible for the proteinase inhibition, as well as the Clusterin precursor also known as 52

sequence


access key

protein

P01042 P01042 P01042 P01042 P01042 P10909 P01024

Kininogen-1 precursor Kininogen-1 precursor Kininogen-1 precursor Kininogen-1 precursor Kininogen-1 precursor Clusterin precursor Complement C3 precursor

P01024

Complement C3 precursor

apolipoprotein J, a glycoprotein which is highly accumulated in inner ear fluids and cerebrospinal fluids. The latter named protein has been implicated in neurodegenerative disorders.

Conclusions In this contribution, a new strategy for the identification of low molecular weight serum constituents by combination of MELDI and elution of bound compounds followed by µ-LC separation and MALDI evaluation of the fractions (alternatively by µ-LC-ESI-MS) is presented. Because of the relatively high protein binding capacity and the different derivatizations, for the first time, fullerenes were successfully employed for selective enrichment of peptides and proteins. The workflow enables rapid protein profiling as well as MS/MS identification using selective enrichment on newly introduced fullerene derivatives followed by elution and fractionation of bound compounds. High-resolution µ-LC separation of the enriched serum samples employing monolithic MSt/BVPE capillaries tremendously increased the number of detectable mass signals in the m/z range of 1000-4000 and improved the mass resolution and the S/N ratio by ∼140% and 200%, respectively. It has, however, to be kept in mind that the success of this novel technique significantly depends on the adsorbent nature used for serum

research articles


enrichment, as it has been shown that the carboxylated fullerene clearly exceeded the amount of low molecular weight compounds bound to the reversed-phase fullerene. While we have illustrated that the approach presents a precious tool for peptide identification from biological samples, continuative studies are required for further optimization. This includes the introduction of a broader variety of adsorbents and optimization of elution, as well as the separation conditions and optimization of the spotting rate and on-target matrix preparation.

Acknowledgment. This work was supported by the Austrian Science Foundation (FWF), SFB-Project 021 (Vienna, Austria), the Genome Research in Austria (GEN-AU) (Federal Ministry for Education, Science and Culture, Vienna, Austria), and by the West Austrian Initiative for Nano Networking (WINN). The authors thank Dr. C. Sar (Department of Organic and Pharmacological Chemistry, University of Pecs) for elemental analysis. Prof. G. Bartsch (Department of Urology, Medical University of Innsbruck) is gratefully acknowledged for providing human serum samples. References (1) Marko-Varga, G.; Fehniger, T. J. Proteome Res. 2004, 3, 167-178. (2) Bogdanov, B.; Smith, R. D. Mass Spectrom. Rev. 2005, 24, 168200. (3) Alaiya, A.; Al-Mohanna, M.; Linder, S. J. Proteome Res. 2005, 4, 1213-1222. (4) Hutchens, T. W.; Yip, T. T. Rapid. Commun. Mass Spectrom. 1993, 7, 576-580. (5) Merchant, M.; Weinberger, S. R. Electrophoresis 2000, 21, 11641177. (6) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (7) Feuerstein, I.; Rainer, M.; Bernardo, K.; Stecher, G.; Huck, C. W.; Kofler, K.; Pelzer, A.; Horninger, W.; Klocker, H.; Bartsch, G.; Bonn, G. K. J. Proteome. Res. 2005, 6, 2320-2326. (8) Trojer, L.; Stecher, G.; Feuerstein, I.; Bonn, G. K. Rapid Commun. Mass Spectrom. 2005, 19, 3398-3404. (9) Najam-ul-Haq, M.; Rainer, M.; Huck, C. W.; Stecher, G.; Feuerstein, I.; Steinmu ¨ ller, D; Bonn, G. K. Curr. Nanosci. 2006, 2, 1-7. (10) Najam-ul-Haq, M.; Rainer, M.; Schwarzenauer, T.; Huck, C. W.;

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PR060347M

Journal of Proteome Research • Vol. 6, No. 1, 2007 53

A New Analytical Material-Enhanced Laser Desorption Ionization

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