Comparative analyses of different surfactants on matrix- assisted laser desorption/ionization mass spectrometry peptide analysis

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ISSN: 1469-0667 © IM Publications LLP 2010doi: 10.1255/ejms.1097 All rights reserved

EuroPEAN JourNALofMASSSPEctroMEtry

Peptides are a fascinating family of molecules that can func-tion as antibiotics, toxins, ion-transport regulators, protein binding inhibitors, enzyme inhibitors, immune suppressants and several other functions.1,2 Due to this enormous interest in peptides, peptidomics have focused on different research fields, with new technologies and reagents being constantly necessary to improve peptide visualization. Among these chemicals, surfactants are commonly used in protein chemistry,

from assisting in protein/peptide solubilization to denaturing proteins,3–5 and frequently employed in electrophoresis. Moreover, they are also involved in protein and peptide charac-terization by mass spectrometric techniques, especially when this technique is becoming an indispensable tool for peptide structural characterization.6 Nevertheless, some problems and pitfalls have recently occurred with this technique. for example, some samples accompanying excessive amounts of

Comparative analyses of different surfactants on matrix-assisted laser desorption/ionization mass spectrometry peptide analysis

Santi M. Mandal,a Satyahari Dey,a Mahitosh Mandal,a Simone Maria-Netob and Octavio L. Francob,*

aIndian Institute of technology, Kharagpur 721 302, West Bengal, Indiabcentro de Análises Proteômicas e Bioquímicas, Pós-Graduação em ciências Genômicas e Biotecnologia ucB, Brasília-Df, Brazil. E-mail: [email protected] and [email protected]

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been extensively used for proteomics and peptidomics analysis. Nevertheless, these analyses, when focused on low molecular mass proteins, show some limitation due to background inter-ference from surfactant ions. Surfactants are routinely used as a solubilizing or denaturing agents for proteins and peptides. In this report, an evaluation and further comparison of the effects of an ionic surfactant, sodium dodecyl sulfate (SDS), and a non-ionic sur-factant, tergitol, on MALDI-MS analyses of the amphipathic peptides, angiotensin and bradykinin, were carried out. At concentrations ≥ 10 mmol L−1, SDS deteriorates the MALDI spectral quality by reducing the signal and intensity of the analyte ions. In particular, it affects the hydrophobic peptide where the signal of surfactant-interfering ions suppresses the analyte ion signal. Whereas, the non-ionic surfactant, tergitol, improves the MALDI-MS analysis of peptide mixtures or hydrophobic peptides by reducing interference from the surfactant itself in positive ion mode analysis. Three-dimensional molecular modeling of two different peptides in complex to tergitol NP-40 and SDS were conducted in order to explain the molecular effects of both agents. In summary, while SDS must be removed from the sample solution to avoid interference of ions from SDS and suppression of analyte ion signal, tergitol at low concentrations may be used as an additive with sample solution for MALDI-MS analysis of peptides. Finally, molecular modeling analyses associated with dock-ing were used in order to explain experimental biochemical data.

Keywords: matrix-assisted laser desorption/ionization mass spectrometry, sodium dodecyl sulfate, surfactant, tergitol, molecular modeling

Introduction

S.M. Mandal et al., Eur. J. Mass Spectrom. 16, 567–575 (2010)received: 27 November 2009 n revised: 25 May 2010 n Accepted: 26 May 2010 n Publication: 18 August 2010

568 Comparative Analyses of Different Surfactants on MALDI-MS Peptide Analysis

salts or surfactants suffer from a suppression of the ioniza-tion process or the adduct formation, affecting the MALDI mass spectrum and limiting its capability for high throughput analysis.7,8

Earlier studies on protein and peptide analysis by MALDI- MS showed that surfactants such as sodium dodecyl sulfate (SDS), triton-X and other surfactants interfered with MS analysis by affecting ion signals and degrading analyte mass resolution,9,10 although some contradictory results have been reported.11–13 Several articles have addressed the effects of surfactants on subsequent MALDI-MS analysis of peptides and presented various methods for the removal of surfactants to reduce signal deterioration of the analyte in the presence of surfactants.12,14,15 Here, we show the effect of common ionic and non-ionic surfactants, particularly SDS and tergitol, for MALDI analysis of both hydrophobic and hydrophilic peptide mixtures. Moreover, molecular modeling analyses of complexed peptides to surfac-tants are also provided in order to shed some light on surfactant molecular mechanism. Data reported here aim to help in a rational selection of surfactants for peptidomics analysis.

ExperimentalMaterialsa-cyano-4-hydroxy-cinnamic acid (cHcA), bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and angiotensin (Asp-Arg-Val-tyr-Ile-His-Pro-Phe-His-Leu) fragments used in this work were bought from Applied Biosystems, uSA. Acetonitrile, trifluoro-acetic acid (tfA), SDS and tergitol NP-9, NP-15 and NP-40 were procured from Sigma (St Louis, Mo, uSA). HPLc grade methanol and Nanopure water (18 MΩ) were used in all exper-iments.

MALDI sample preparationSurfactant stock solutions (SDS and tergitol NP-9, NP-15 and NP-40) were prepared in nanopure water and stored at −20°c until analysis. Peptide concentration in the final solution prior to MALDI analysis was 2.0 pmol mL−1. the final solution was adjusted for each surfactant at four concentra-tions: 5 mmol L−1, 10 mmol L−1, 15 mmol L−1 and 20 mmol L−1. cHcA matrix (10 mg mL−1) solution was prepared freshly with 50% acetonitrile in 0.3% tfA. Next, 4 mL of cHcA was mixed with sample surfactant mixture as matrix. then, a 1.0 mL sample was spotted onto the MALDI 100-well stain-less steel sample plate and allowed to air dry prior to the MALDI analysis.

MALDI-ToF-MSA Voyager MALDI tof mass spectrometer (Applied Biosystems, uSA) equipped with a 337 nm N2 laser and operated at an accel-erating voltage of 20 kV was used. the spectra were recorded in the positive linear ion mode as the average of 100 laser shots of random positions across a spot. reproducibility of each spec-trum was checked 20 times from duplicate samples. Samples were ionized with 100–200 shots of a 3 ns pulse width laser

light. the signal was digitized at a rate of 500 MHz and aver-aged data was presented to a standard Voyager data system for manipulation. MALDI-tof was calibrated using a Saquazyme calibration mixture (Applied Biosystems) consisting of bovine insulin (5734 Da), E. coli thioredoxin (11,674 Da) and horse apomyoglobin (16,952 Da). Each spectrometry assay was conducted in five replicates.

In silico analyses, molecular modeling and protein–ligand dockingInitially, a threading method was carried out in order to find the best template for homology modeling. the Blastp algorithm was applied to the Protein Data Bank to indicate the most similar structures to bradykinin and angiotensin sequences, and 1JJQ and 1N9u were the Protein Data Bank structures selected as their templates, respectively. A primary structure was constructed using the Modeller 9v6 program.16 the partial simulation for energy minimization was executed in the same program using 100 steps from the steepest descent in order to remove possible stereochemical disturbances. the final model was visualized using Pymol17 and Swiss-PdbViewer 4.01.18 the surfactant molecules, SDS and tergitol, were obtained from Pubchem compounds. the prediction of protein–ligand complexes was performed using flexX program 3.1.3.19 the active site was defined within a radius of 6.5 Å of bound ligand. Each surfactant molecule was separately docked into the monomer unit of each peptide in their zwitterionic form. the geometry complexes were predicted and the binding strength estimated. the best solutions out of 100 conformers were indicated by the top score. All simulations were performed on a Sun AMD opteron bio-processor workstation. ProSA II was used to choose the model showing the most favorable packing and solvent exposure characteristics. ProcHEcK was used for additional analysis of stereochemical quality. Low ProSA II scores and high ProcHEcK G-factors characterize high-quality models.

Results and discussionMALDI-MS has the ability to generate high-quality mass spec-tral results of intact biomolecules, but peptide or proteomics analyses with low molecular mass range have some limitations due to background interference from the salt or surfactant ions. Here, we examined MALDI-MS performance in the presence of surfactants. In order to evaluate the suitability of surfactants, we considered the parameters: (1) surfactant background ions, (2) peptide–surfactant adduct formation and (3) suppres-sion of the peptide signal.

first, an aqueous 5 mmol L−1 SDS solution was prepared with cHcA matrix and cast onto the MALDI sample plate and allowed to dry. the resulting positive ion MALDI spec-trum, shown in figure 1, is dominated by sharp signals at m/z 598, m/z 655, m/z 732, m/z 809 and m/z 886. the mass m/z 598 corresponds to the dimer of sodium dodecyl sulfate ion (2M+Na+) and m/z 886 corresponds to the trimer of sodium

S.M. Mandal et al., Eur. J. Mass Spectrom. 16, 567–575 (2010) 569

dodecyl sulfate ion (3M+Na+). the mass differences between the peaks (m/z 655–m/z 598) and (m/z 732–m/z 655) = (m/z 809−m/z 732) = (m/z 886−m/z 809) are m/z 57 and m/z 77, respectively. the mass m/z 57 and m/z 77 correspond to c4H9

+ and c6H5

+ ions, respectively, and may be indicative of some fragmentation.

figure 2 shows the results from analysis of peptide mixture with various SDS concentrations. An addition of 5 mmol L−1 SDS to the peptide mixture makes no difference to peptide mass (m/z 904 and m/z 1296). the additional peaks with peptides came from the fragmentation ion of SDS. the increase in analyte ion signal and intensity was found to be at 10 mmol L−1 concentration of SDS. By increasing the SDS concentration more than 10 mmol L−1, the peptide ion signal drops and the SDS fragmented ion signal rises considerably. finally, at 20 mmol L−1 SDS concentration the peptide (m/z 1296) signal and intensity were very low. the results indicate that lower concentrations of SDS could not cause peptide signal suppression and adduct formation. However, SDS frag-mentation ions always appear with peptide ions and dominate at higher concentrations of SDS. these trends are similar to those reported by tummala et al.,20 which showed that at very low concentrations of SDS, the sequence coverage remains the same or increases during tryptic digestion analysis of myoglobin. However, at high concentration of SDS present in peptide solution, the spectral quality deteriorates due to the presence of excess sodium ion. recently, tummala et al.20 hypothesized that the presence of SDS micelles in peptide

solution would reduce Marangoni effects during analyte crys-tallization process.

Aiming to compare the effects of an ionic and a non-ionic surfactant, three different tergitol preparations (NP-9, NP-15 and NP-40) were also evaluated. tergitol is a common commercial non-ionic surfactant of a class known as nonylphenol ethoxylates, possessing the chains without charged groups. figure 3 shows the positive ion MALDI spec-trum of tergitol NP-40, recorded from a cHcA matrix with tfA. Similarly, we see the same two fragmentation series observed for linear mode positive ion. the highest and lowest ion series are both followed by subsequent fragments spaced by 44 u, equal to that of the ethylene oxide (Eo) repeat. the minor distribution in each spectrum is displaced from the dominant series by 16 u (as m/z 1843−m/z 1827), corre-sponding to the change of the terminal group from –H to –oH. the fragmentation pattern of ethoxylated surfactants was described in the fundamental work by Hanton et al.21 figure 4 shows the effect of tergitol NP-40 on MALDI-MS analysis of peptides. there is no interfering fragmented ion observed at a concentration of less than 15 mmol L−1 of tergitol, but at higher concentrations surfactant ions interfere with peptide ions. We also observed that analyte signal and intensity increased almost two-fold at 10 mmol L−1 concentration of tergitol NP-40. Similar data was obtained with two different tergitol NP-15 and NP-9, despite some smaller structural differences (data not shown). Earlier, rosinke et al.9 exam-ined the effects of several non-ionic, cationic and zwitterionic

Figure 1. MALDI-ToF mass spectrum of SDS in positive ion linear mode. Mass spectrum acquired from the mixture of 4 mL of CHCA (matrix) and 4 mL SDS (10 mmol L−1). The spectrum represents the dimer and trimer of SDS ion, m/z 598 (2M+Na+) and m/z 886 (3M+Na+), respectively.

570 Comparative Analyses of Different Surfactants on MALDI-MS Peptide Analysis

surfactants on MALDI-MS analysis of membrane proteins and non-covalent complexes. they found that non-ionic deter-gents, such as triton X-100 and b-d-octylglucoside, degraded the mass spectral quality less than ionic surfactants, SDS. unlike data reported here, Gharahdaghi et al.22 found that

non-ionic surfactants would not interfere with subsequent MALDI-MS analysis when a matrix solution was prepared in high organic solvent. Amado et al.23 also found that addi-tion of excess amounts of non-ionic surfactants result in the loss of sensitivity in subsequent MALDI-MS analysis of

Figure 2. Effect of SDS on MALDI-MS analysis of the peptide mixture. Mass spectra were obtained from the mixture of peptide solution with various concentrations of SDS: (a) 5 mmol L−1 SDS, (b) 10 mmol L−1 SDS, (c) 15 mmol L−1 SDS, and (d) 20 mmol L−1 SDS.

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polypeptides, while additions of anionic surfactants improve subsequent MALDI-MS results.

In a comparison of figure 2 and figure 4, we observed that SDS strongly affects the spectral quality at higher concentra-tions and mostly affects the hydrophobic peptides, mainly those such as angiotensin (m/z 1296) which is more hydro-phobic than bradykinin (m/z 904). the signal and intensity of this hydrophobic peptide increased significantly in the pres-ence of tergitol compared with SDS. Aiming to better under-stand the molecular interactions between the peptides evalu-ated and the surfactant, molecular modeling analyses were conducted with the computational docking program flexX. using this approach, the peptide surfaces were explored to identify the most energetically favorable fields for interaction with the flexible ligand. Each docking experiment consisted of a series of 100 simulations, each producing a docking solution. the docking solutions were ranked based on the sum of inter-molecular electrostatic, van der Waals and distance restraint energies. the first ranking hit indicated the most common lowest-energy conformation.

In order to explain these data, molecular models of both peptides were conducted, following an in silico docking to

surfactants (figure 5). Indeed, the purpose here was to reach an accurate view about the potential targets of linkage of each peptide and different classes of surfactants. therefore, the docking of receptor (peptide) and ligand (surfactant) was real-ized with the 1 : 1 binding geometry. Monomers of the zwitteri-onic peptide forms were used as receptors in docking experi-ments with the intention of mimicking the real conditions of MALDI analysis. As the solutions of peptides and surfactants are prepared in pure water, the prior contact between peptide and surfactant occurs in this aqueous environment, predom-inated by zwitterionic forms. Initially, SDS molecules were complexed to angiotensin and bradikinin peptides [figures 5(a) and (b)]. As expected, all interactions between ligand and peptide were based on differences of polarity coefficient, mainly due to the high polar characteristic of ligands. the set of solutions found for the complex of SDS and angiotensin reported a most stable conformation common in 81 of the complex solutions, where we can see that the sulfate group was interacting with SDS in two short hydrogen bonds (1.86 Å and 1.87 Å) [figure 5(a)]. for the first bond, the resonance of Arg2 charge generated a moment of positive charge into a hydrogen atom, which interacted with the negatively charged

Figure 3. MALDI-ToF mass spectrum of tergitol in positive ion linear mode. The mass spectrum acquired from the mixture of 4 mL of CHCA (matrix) and 4 mL tergitol (10 mmol L−1).

572 Comparative Analyses of Different Surfactants on MALDI-MS Peptide Analysis

oxygen (hydrogen acceptor) of the sulfate group of SDS. In the second bond, the electrostatic field of nitrogen involved in the peptide bond of tyr5 results in a polar moment of related hydrogen (hydrogen donor) which maintained contact with the oxygen 1 of the SDS sulfate group. Moreover, when SDS was bound to bradikinin, three possible close interactions are presented by the SDS sulfate group and the peptide [figure 5(b)], being found in 88 predicted docking structure solu-tions. A bidentate hydrogen bond, at 1.91 Å and 1.92 Å, was formed from the nitro hydrogen (hydrogen donor) of Gly5 to the oxygen 1 and the carbonyl oxygen of SDS. Another interaction was observed between carbonyl oxygen of SDS and the nitro hydrogen (hydrogen donor) of Phe,4 at 1.9 Å. on the other hand, the docking complexes of the non-ionic surfactant tergitol and angiotensin or bradikinin reported the action of van der Waals force by the presence of hydrophobic and hydrophilic inter-actions between ligand and peptide. therefore, the tergitol

molecule was complexed to the angiotensin structure and a common complex conformation was reported in 83 complex solutions, which is shown in figure 5(c). In general, a nitro hydrogen (hydrogen donor) of Leu10 interacted with the oxygen 3 of SDS (1.79 Å), while a double hydrogen bond resulted from the nitro hydrogen (hydrogen donor) of His9 to the same oxygen 3 and the oxygen 2 of SDS. furthermore, the aromatic ring of Phe8 appeared close to carbon 1 and carbon 9 at 4.45 Å and 4.89 Å, respectively, establishing two hydrophobic interactions with strong and stable attraction between ligand and peptide. In contrast, the docking complex of tergitol and bradikinin resulted in a set of solutions with three hydrogen bonds and a hydrophobic interaction, which are both common for 98 complex solutions observed [figure 5(d)]. A double hydrogen bond was formed from nitro hydrogen (hydrogen donor) of Arg1 both to oxygen 1 (2.37 Å) and oxygen 2 (2.15 Å). Another hydrogen bond extends from hydroxyl hydrogen of SDS to

Figure 4. Effect of tergitol on MALDI-MS analysis of the peptide mixture. Mass spectra were obtained from the mixture of peptide solution with various concentrations of tergitol NP-40: (a) 5 mmol L−1 tergitol, (b) 10 mmol L−1 tergitol, (c) 15 mmol L−1 tergitol, and (d) 20 mmol L−1 tergitol.

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carbonyl oxygen (hydrogen acceptor) of Pro2 at 1.74 Å. In addi-tion, a very stable hydrophobic contact of approximately 4.31 Å occurred between carbon 9 of SDS and the aromatic ring of Phe4.

Simulations reported here could help to explain the inter-actions of peptides to surfactants, and thus explain the reduction of signal quality. the general signal degradation found for high concentrations of all the surfactants may be because the coating of matrix crystals with surfactants diminishes energy transfer and the efficiency of the desorp-tion process.23 Earlier, yonath et al.24 explained that a stable salt bridge was formed between positively charged amino acids with sulfate groups of SDS and more hydrophobic sequences would penetrate into the hydrophobic micellar core of SDS. However, SDS micelles show an a of 0.3 and are able to transfer the negative charge to proteins at a rate of 1.4 g of SDS per gram of proteins mass. therefore, the increase in its concentration interferes in the positive ion mode analysis.25 Although the critical micelle concentration (cMc) of SDS is approximately 8 mmol L−1 in pure water at 25°c, in the experimental condition, taking in account the presence of peptide mixture and the matrix in acetonitrile solution, the cMc has not been determined. However, it was expected that at 10 mmol L−1 concentration of SDS, where the increase in analyte ion signal and intensity was observed, the cMc of SDS was not reached or at least not outreached far from it, where changes in physicochemical properties occur and solute ions of SDS currently aggregate to form units of micelles.25 Non-ionic surfactants have been shown to interact with sufficiently hydrophobic peptides and induce

an increased amount of a-helix conformation.26 Here, no salt bridge was observed. However, these same effects could be caused by a combination of hydrophilic hydrogen bonds and hydrophobic van der Waals interactions.

ConclusionsIn summary, the effects of an anionic surfactant, SDS, and non-ionic surfactant, tergitol, have been investigated as additives for MALDI-MS analysis of both hydrophilic and hydrophobic peptide mixtures. It was found that the non-ionic surfactant, tergitol, was the best surfactant additive because it improves the signal and intensity of peptide (both hydro-phobic and hydrophilic) molecular ions, no adduct formation was observed and there were no interferences in the positive ion mode analysis from the surfactant itself. In contrast, the occurrence of more interfering ions and deleterious spec-tral effects have been assigned to SDS.27,28 In summary, our data indicate that strong anionic detergents, particularly SDS, must be completely removed from the peptide sample solution prior to drying, particularly when SDS prohibits the acquisition of useful low mass range mass spectrometric data.

Acknowledgmentsthis report was supported by cNPq, cAPES, fAPDf, ucB and fAPEMIG.

Figure 5. Molecular docking complexes of (a) angiotensin and (b) bradikinin to SDS, and (c) angiotensin and (d) bradikinin to tergitol. The three-dimensional protein structure and the small ligand SDS were docked using FlexX software. Dashed lines indicate non-cova-lent interactions between protein and ligand. Side chain residues, presented as ball and stick, indicate amino acids directly involved in peptide–surfactant interaction.

574 Comparative Analyses of Different Surfactants on MALDI-MS Peptide Analysis

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