Surface Effects of Iron Oxide Nanoparticles on the MALDI In-Source

Jun 4, 2019 - Inorganic nanoparticles as MALDI matrices have recently been explored to study the molecular mass determination and structural analysis ...
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Article Cite This: ACS Appl. Nano Mater. 2019, 2, 3999−4008

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Surface Effects of Iron Oxide Nanoparticles on the MALDI In-Source Decay Analysis of Glycans and Peptides Angelo J. Antone,† Qiaoli Liang,‡ Jennifer A. Sherwood,† James C. Weiss,† Joseph M. Wilson,† Sanghamitra Deb,§ Carolyn J. Cassady,‡ and Yuping Bao*,† †

Department of Chemical and Biological Engineering, ‡Department of Chemistry, and §Central Analytical Facility, The University of Alabama, Tuscaloosa, Alabama 35487, United States

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ABSTRACT: Inorganic nanoparticles as MALDI matrices have recently been explored to study the molecular mass determination and structural analysis of glycans and peptides. However, the specific factors contributing to the success of the analysis are not well elucidated. In this paper, we investigated the roles of nanoparticle surface coatings and additive ions in MALDI in-source decay (ISD) analysis of model glycans and peptides. Specifically, iron oxide nanoparticles with four defined capping molecules (gluconic acid, citric acid, lactobionic acid, or glutathione) were tested, and the roles of additives (NH4OH, NaOH, LiOH, NaCl, or trifluoroacetic acid) were examined. For a model glycan, maltoheptaose, and a model peptide, substance P acid, nanoparticle capping molecules, additive cations, and additive anions altogether influenced the molecular ion sensitivity and ISD fragmentation efficiency. KEYWORDS: MALDI matrix, iron oxide nanoparticles, glycan analysis, ISD, interfacial effects, capping molecules, additives, ligand exchange

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INTRODUCTION Matrix-assisted laser desorption ionization (MALDI) is a widely used ionization technique for the analysis of molecules by mass spectrometry (MS).1 The success of a MALDI analysis is highly dependent on the choice of matrix2,3 because the matrix absorbs laser energy and transfers the absorbed energy to analytes, facilitating analyte desorption and ion formation. Besides traditional organic compounds with high UV absorption,4 nanoparticles (NPs) or nanostructures with strong UV absorbance have recently drawn increasing interests as alternatives for the analysis of small molecules and peptides,5−8 such as gold (Au),9 silver (Ag),10,11 platinum,12 quantum dots,13,14 silicon,15 titanium oxide (TiO2),5 and iron oxide16−18 NPs. The primary roles of MALD matrix are energy absorption and assisting analyte desorption/ionization. These NPs are performing such roles and are generally considered as nanoparticle MALDI matrices, although some different terminology has been used, such as “surface-assisted laser desorption”. Even though some types of nanoparticles do not have the maximum absorbance at the MALDI laser wavelength 337 nm, these nanoparticles do exhibit certain levels of absorption, which are sufficient for MALDI analysis, such as Au NPs with maximum absorbance at 520 nm, Ag NPs with maximum absorbance 410 nm and others shown in Figure S1. The key advantages of NP MALDI matrices are their clean spectral background in the low mass region and thus their suitability for the analysis of small molecules.19−22 The strong UV absorption of inorganic NPs (e.g., 5 nm Au NPs23) also © 2019 American Chemical Society

enhances ionization and fragmentation of analytes, and fragment generation is dependent on the concentration of the nanomatrix.24 In addition, the interfaces between the analytes and NPs tuned by NP sizes affect signal intensities of the analytes. Previous studies showed that smaller sized NPs generated enhanced signal intensities compared with larger sized NPs when using commercial iron oxide slurries in the analysis of small biomolecules.25 The MALDI analysis of amyloid-beta peptides using small size Ag NPs (∼3 nm) exhibited higher intensity and better sensitivity compared to larger size NPs and traditional organic matrices.26 Among all the nanomaterials, the iron oxide NP platform became highly attractive for several reasons. First, compared to the absorption properties of other NP nanomaterials (e.g., 520 nm Au NPs and 410 nm Ag NPs), iron oxide NPs have a high absorbance at the MALDI UV laser wavelength (e.g., 337 nm)17 but are less disruptive in fragmentation than TiO2 NPs.27 Second, we have established a highly reproducible approach for the surface functionalization of iron oxide NPs,28,29 allowing for effective attachment of a variety of surface capping molecules. Finally, the magnetic response of iron oxide NPs allows for fast purification and concentration of target compounds.30−32 In addition to the size effects, capping molecules are important for NP dispersion and analyte Received: May 24, 2019 Accepted: June 4, 2019 Published: June 4, 2019 3999

DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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ACS Applied Nano Materials

precursor (1.63 g) was decomposed at 280 °C for 2 min in 10 mL of 1-octodecene in the presence of trioctylphosphine oxide (0.06 g), oleic acid (0.07 mL), and oleyl alcohol (3.6 mL). Subsequently, the reaction was cooled to room temperature, and iron oxide NPs were washed with ethanol/hexane and collected by centrifugation. After complete drying under vacuum overnight, the NPs were redispersed in chloroform (0.5 mg/mL) or in hexane (1 mg/mL) as stock solutions for surface functionalization. Surface Functionalization of Iron Oxide NPs with Selected Capping Molecules. The surface functionalization of iron oxide NPs was achieved by replacing the original hydrophobic coating with the desired hydrophilic molecules (CA, GA, GSH, and LA) using our well-established ligand exchange method.28,29,44,45 Specifically, iron oxide NP stock solution was mixed with the capping molecule solution under 5 min sonication. The well-mixed solution continued to react in a shaker (45 °C, 250 rpm) overnight to facilitate the ligand exchange. The specific conditions of the capping molecules (amounts and solvents) were determined experimentally based on their solubility and molecular weights as follows: CA (2 mL of NPs at 0.5 mg/mL in chloroform mixed with 4 mL of an aqueous CA solution), GA (1 mL of NPs at 0.5 mg/mL in chloroform mixed with 2 mg of GA dissolved in 1:3 binary solution of acetone to methanol), GSH (1 mL of NPs at 1 mg/mL hexane mixed with 2 mg of aqueous GSH dissolved in 4 mL of ethanol), and LA (2 mL of NPs at 0.5 mg/ mL in chloroform mixed with 2 mg of LA dissolved in 4 mL of acetone). Once the ligand exchange was completed, the NPs coated with various capping molecules were collected and washed three times with nanopure water to remove any excess ligands. The NPs with desirable surface coatings were then dispersed in nanopure water at a concentration of 0.5mg/mL. Because the resulting solutions were acidic, the pH of the NP solutions was adjusted to neural with ammonium hydroxide. Characterization of Nanoparticles. The size and morphology of iron oxide NPs functionalized with various capping molecules were examined under a transmission electron microscope (TEM, Hitachi 7860). The high-resolution TEM images were collected on a FEI Technai F-20 TEM. The surface charges and dynamic sizes of the NPs in aqueous solution were measured using a Malvern (Malvern, UK) Zetasizer Nano series dynamic light scattering device. UV−vis spectra of these NPs in solution were collected using a Shimadzu (Kyoto, Japan) UV−vis spectrophotometer (UV-1700 series), and the Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer (Bucks, UK). Mass Spectrometry. The MALDI experiments were performed on a Bruker Daltonics Ultraflex MALDI-TOF mass spectrometer in reflectron positive mode. An LTB Lasertechnik Berlin (Berlin, Germany) MNL100 nitrogen laser with 337 nm wavelength pulsed at 150 μJ/3 ns was used to ionize and excite the samples. NP matrices (0.5 mg/mL in water) were used either directly or with the addition of NaOH, LiOH, NaCl, NH4OH, or TFA. Maltoheptaose was prepared in water at 2 mg/mL; substance P acid was prepared in water at 1 mg/mL. Samples were mixed with NP matrix at 1:1 ratio. One microliter of the analyte/matrix solution was then applied to a Bruker Anchorchip target. On target wash was performed for dried sample spots with a chloroform:methanol (3:1, v/v) solvent mixture. The purpose of this study was to investigate factors promoting ISD fragmentation when using NP matrices. The analyte concentration, NP matrix concentration, and matrix/analyte ratio were selected out of experimental simplicity. Our previous study17 used 0.1 mg/mL concentration, and the NP concentration was reduced consequently. Additionally, ISD study generally is conducted at a higher sample concentration compared to MS analysis. Electrospray ionization (ESI) experiments were performed on a Waters Xevo G2-XS QTof mass spectrometer with capillary voltage 1.3 kV, sample cone 30 V, source offset 30 V, source temperature 80 °C, desolvation temperature 175 °C, cone gas 0 L/h, and desolvation gas 400 L/h. Maltoheptaose (2 mg/mL) was mixed with 20 mM NaOH in water and diluted 100 in methanol:water (50:50, v/v) prior

interaction. We have previously shown that glutathione (GSH)-coated iron oxide NPs were highly effective as MALDI matrices for glycan MALDI in-source decay (ISD) fragmentation.17 Citric acid (CA)-coated iron oxide NPs could be used as a general MALDI matrix for the analysis of several polymers with distinctive properties.16 Our previous studies have also demonstrated the importance of the NP surface coatings and the assistant roles of additive ions.17 Glycans and glycoconjugates with proteins and lipids play important roles in numerous biological processes associated with human diseases,33 including muscular dystrophy, diabetes, neurodegenerative diseases, and cancer.34,35 Identification of the specific structures of glycans and glycoproteins is very important to understanding of their interactions with biological systems.36 Site-specific analysis of protein glycosylation is also important for disease progression.37,38 MS has been a powerful tool for the analysis of glycoproteins because of its outstanding sensitivity, specificity, and speed, which greatly benefit the identification of the glycoproteins, glycosylation sites, and structures of glycans.38,39 Glycan structural analysis by MS generally provides two types of cleavage product ions: glycosidic cleavage and cross-ring cleavage.40,41 Glycosidic cleavages break the bonds between glucose units, generating structural information about composition and sequence of the monomers; cross-ring cleavages open up a sugar ring, yielding bond linkage information.42 We have previously showed the effectiveness of GSH-coated iron oxide NP matrices in MALDI ISD analyses for glycan structure determination by generating abundant cross-ring fragmentation.17 MALDI ISD glycan cross-ring fragmentation has also been reported for (2,5-dihydroxybenzoic acid, DHB)functionalized silane-coated HgTe nanoparticles43 and DHBfunctionalized magnetic particles.24 These studies suggested the role of DHB capping in reducing NP background signal and enhancing ion intensity,24,43 while the metal oxide NP cores also affected the ion intensity.43 However, the synergetic effects of NP capping molecules and solution additives have not been studied systematically for glycan or peptide MALDI ISD fragmentation. In this paper, we investigated the effects of iron oxide NP surface coatings and additive ions for the MALDI ISD analysis of a model glycan, maltoheptaose, and a model peptide, substance P acid. Iron oxide NPs with four different surface coatings (gluconic acid (GA), citric acid (CA), lactobionic acid (LA), or glutathione (GSH)) and five types of additives (NH4OH, NaOH, LiOH, NaCl, or trifluoroacetic acid (TFA)) were examined by using both model molecules. This study provided insights regarding the MALDI ISD fragmentation mechanism of glycan/peptide with iron oxide NP matrix and suggested that NP capping molecules, additive cations, and additive anions jointly influence ISD fragmentation efficiency.

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EXPERIMENTAL METHODS

Chemicals. The following chemicals and reagents were purchased from Thermo Fisher Scientific (Waltham, MA): ferric chloride (ACROS, 98% purity), oleic acid (OA, Fisher, 95%), sodium oleate (TCL, 95%), 1-octadecene (90%), trioctylphosphine oxide (TOPO, 90%), oleyl alcohol (OL, Alfa Aesar, 85%) citric acid (99.5%), Dgluconic acid (99%), glutathione (97% reduced), and lactobionic acid (98.8%). Maltoheptaose and substance P acid acetate salt hydrate (95%) were purchased from Sigma-Aldrich (St. Louis, MO). Synthesis of Iron Oxide Nanoparticles. Spherical iron oxide NPs of about 6 nm in diameter were synthesized by using our wellestablished modified “heating-up method”.28,29 Briefly, an iron oleate 4000

DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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ACS Applied Nano Materials Scheme 1. An Overview of the Preparation Process of Iron Oxide Nanoparticles with Desirable Functionalities

to ESI experiments. The maltoheptaose−NaOH mixing times before ESI experiments were instant, 30 min, and 10 days.

CA, sugar hydroxyl and carboxylic acid groups for GA and LA, and amino and carboxylic acid groups for GSH. A comparison between LA and GA also allows for the study of size differences in capping molecules. The surface coatings of NPs are critically important as MALDI matrices because the surface capping molecules are in direct contact with the analytes. The surface capping molecules also assist in the process of energy transfer to the analyte, analyte desorption, and ionization. In addition, the NPs can be dispersed in different solvents depending on the surface coatings, allowing for flexible choices of additives, offering selective interfaces between NPs and analytes. Figure 2 shows the typical TEM images of spherical iron oxide NPs coated with CA, LA, GA, and GSH, namely NPCA, NPLA, NPGA, and NPGSH. The histograms of the TEM image analysis suggested that all the nanoparticles were around 6−7 nm (Figure S2). The NPs functionalized with all four capping molecules generated well-dispersed NPs in solution and free of aggregation. The high-resolution TEM images of all four samples (Figure S2) indicated their crystalline structures. The DLS plots of these NPs in aqueous solution showed their hydrodynamic sizes peaked around 20 nm with slight variation (NPCA: 18 nm; NPGA: 23 nm; NPGSH: 18 nm; and NPLA: 21 nm), as shown in Figure S4a. The single peaks of all samples suggested free of NP aggregation in solution. The presence of the different capping molecules on iron oxide NP surfaces was verified by FT-IR spectroscopy (Figure 3a). All the four capping molecules have carboxylic acid groups either bonded to the NP surfaces or ionized. Therefore, all four samples exhibited a characteristic −COO− asymmetric stretch around 1630 cm−1 and −COO− symmetric stretch around 1370 cm−1. However, the bands for the NPCA were more distinct because of the higher number of −COOH groups within one molecule. The bands for NPGSH were merged with the amide II band (N−H bending vibration) around 1580 cm−1 and amide III bands around 1350 cm−1, leading to much broader bands in those regions. The band around 1082 cm−1 for NPCA was assigned to the −C−O vibration. The band around 1020 cm−1 for NPGSH is from the −C−N stretch. The −C−O−C− stretching bands around 1029 cm−1 of the NPLA and NPGA were assigned to sugar molecules. Regardless of the capping molecules, all the samples showed the Fe−O vibrations at the tetrahedral units around 620 cm−1 and octahedral site vibration around 440 cm−1, indicating that the capping molecules did not affect the iron oxide core. All four capping molecules generated negatively charged surfaces for iron oxide NPs with (CA, −39 mV; GSH, −35 mV; GA, −33 mV; and LA, −32 mV), as shown by the zetapotential plots of the NPs (Figure 3b). The carboxylic groups from the various molecules were hypothesized to attach onto the iron oxide surfaces based on the well-documented interaction mechanisms;47,48 the remaining functional groups of the capping molecules then contributed to their unique

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RESULTS AND DISCUSSION NP Synthesis and Characterization. The synthesis and surface functionalization of iron oxide NPs were achieved by using our well-established methods,28,29,44,45 which produced iron oxide NPs with a narrow size distribution and controlled surface functionality. The preparation process of iron oxide NPs with desirable surface functionalities is illustrated in Scheme 1. Generally, the surface-to-volume ratio of NPs increases as NP size decreases (e.g., 50% surface atoms for 4 nm NPs). As MALDI matrices, the surface areas of NPs directly affect the interfaces between matrices and analytes and subsequently the effectiveness of NP matrices. Previous studies suggested that the size of NPs mainly affects their surface area and magnetic property46 but has little effects on their UV absorption.17 The NP surface area greatly affects the interfacing area between the NP matrices and the analyte molecules, which subsequently affects the performance of NP matrices. Several studies have shown that the smaller sized NPs had better sensitivity as a MALDI matrix than the same type of NPs of larger size, such as Au,9 Ag,26 and iron oxide.25 Generally, the smaller the NPs, the higher the interfacial area between NPs and analytes and the better the uniformity of the sample and interactions between NPs and analytes. However, as NPs get smaller, isolation from aqueous solution during washing steps becomes a challenge. To balance the preparation process and potentialsize-dependent performance, NPs of ∼6 nm were chosen for this study. The as-synthesized iron oxide NPs were coated with hydrophobic oleic acid molecules, which were subsequently replaced with several carefully selected capping molecules, including CA, GA, LA, and GSH (Figure 1). The rationales for selecting these molecules are that they all potentially generate negatively charged surfaces but at the same time offer chemical variation, such as a high number of carboxylic acid groups for

Figure 1. Iron oxide NP capping molecules studied. 4001

DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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Figure 2. TEM images of iron oxide NPs coated with selective capping molecules: (a) CA, (b) GA, (c) LA, and (d) GSH.

surface chemical characteristics. For instance, the remaining carboxylic groups of CA molecules generated highly negatively charged surfaces (−39 mV) for NPCA; the extra carboxylic groups of GSH also led to negatively charged surfaces (−35 mV) of NPGSH, which were likely balanced by the positive charges from amino groups. Only one carboxylic group exists in a LA or GA molecule for NP surface attachment; the deprotonated hydroxyl groups could unlikely generate such negatively charged surfaces of NPGA or NPLA. It was hypothesized that the negatively charged surfaces likely resulted from the second layer binding of excess GA and LA molecules to NPGA and NPLA via hydrogen bonds between sugar hydroxyl groups, as illustrated in Figure S3. To verify this hypothesis, heating experiments were performed using NPGA as a model system by heating NP solutions at 65 °C for 30 min and then precipitating NPs out of the hot solution quickly with hot ethanol. The thermal treatment was used to break down the H-bonds between sugar molecules. Then, the DLS and zeta-potential of NPGA were measured after heating (Figure S4c,d). After heating, the hydrodynamic size of NPGA increased slightly to ∼29 nm, likely due to the interactions between NPs (Figure S4c). However, a significant difference in

Figure 3. Iron oxide nanoparticles with four surface coatings: (a) FTIR spectra, (b) zeta-potential plots, and (c) UV−vis absorption spectra.

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DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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ACS Applied Nano Materials zeta-potential of ∼10 mV more positive was observed, suggesting that the heating treatment likely removed the major part of the second layer sugar molecules, leaving a hydroxyl group capping surface (Figure S4d). The zetapotential change directly supported the double-layer hypothesis. The highly negatively charged surfaces of NPs also contribute to the colloidal stability and dispersity of these NPs in solution. The UV−vis absorbance of the NPs was not affected by the surface coatings, as indicated by the UV−vis absorption spectra of NPs with four different coatings (Figure 3c). The UV−vis spectra were not normalized with concentration. The slight variation in the signal intensities of the spectra likely resulted from the concentration variation of the NPs. The iron oxide NPs with all four coatings showed a strong absorption of below 400 nm, which is well-suited for the 337 nm wavelength of the MALDI laser. NP Capping and Additive Effect on Glycan MALDI ISD Analysis. These NPs with the four types of capping molecules were subsequently used as MALDI matrices for the analysis of the model glycan and peptide. The major advantage of using NPs for glycan MALDI analysis is the clean spectral background in the low mass region. Prior to analyzing model molecules, MALDI spectra were collected using NP solutions only with and without additive ions (Figures S5 and S6). The NP background mass spectra were similar regardless of capping molecule variation, with or without NaOH. This observation suggested that the observed background masses (mostly below m/z 200, not interfering with glycan/peptide ISD spectra quality) were not related to the capping molecules. Therefore, laser ablation unlikely caused significant capping molecule dissociation from NPs. Figure 4 shows the MALDI/TOF ISD mass spectra of maltoheptaose acquired by using iron oxide NPs with the four selected surface capping molecules without any additive and with 7 mM NaOH. Comparisons of analyte ion signal intensities and ISD fragmentation were made among these coatings. For all four types of MALDI NP matrices, the primary signals were from the sodiated molecular ion, [M + Na]+. However, the ion signal intensities varied in the order of NPGA > NPCA > NPGSH > NPLA. The signal intensities from MALDI are related to the efficiency of analyte desorption and ionization, which are influenced by the matrix−analyte interaction. Therefore, the higher ion signal intensities from NPCA and NPGA were likely from the facilitation of analyte desorption and ionization by the multiple carboxylic groups of CA surfaces or/and the second layer of GA molecules on NPGA surfaces. The lowest ion signal from NPLA was likely due to the bulky chemical structure of LA along with second layer attachment, which might minimize the energy transfer. Maltoheptaose is a linear α-D-glucosyl sugar with seven glucose units, and the main bond is α-1.4 linkage. The glycan fragmentation can take place between the residues (Bn, Cn) and across a glucose ring (2,4An, 0,2An), with cross-ring cleavages providing more informative sequence specific structural information. Without NaOH additives, glycosidic cleavage (Bn, Cn/Yn) fragments of maltoheptaose were mainly observed when using NPGA and NPGSH as matrices while fragment ion signals of maltoheptaose were not evident when using NPCA or NPLA matrices. With addition of 7 mM NaOH (Figure 4b), the four types of NPs showed quite similar spectra, where sodiated molecular ion intensity decreased and abundant cross-ring cleavages (2,4An with one, two, or three

Figure 4. MALDI/TOF ISD mass spectra of maltoheptaose acquired using NPGA, NPGSH, NPLA, and NPCA matrices without additives (a) and with 7 mM NaOH (b). All cleavage products retain one sodium ion (labeled black), or two sodium ions (labeled blue), or three sodium ions (labeled light blue).

sodium ions) dominated over glycosidic cleavages. Further increase in NaOH concentration (20 mM) did not alter the signal-to-noise ratios (S/N) of the spectra significantly for NPGA, but the signal intensity of the cross-ring cleavage decreased greatly for NPGSH, NPLA, and NPCA (Figure S7). This observation of ion signal suppression due to salt concentration is commonly observed in MALDI analysis.49 Our studies with both 7 and 20 mM NaOH suggested that additive ions played a critical role in the MALDI ionization/ fragmentation behavior of glycans. The observation of glycan fragmentation is also consistent with our previous study employing iron oxide NP matrices on glycans.17 Because of the high MS sensitivity and minimal effects of additive concentrations, the NPGA matrix was used to study the effects of different types of additive ions. Four additives (NH4OH, NaCl, NaOH, and LiOH) at a concentration of 20 or 40 mM were examined to compare the cation and anion effects on glycan ISD fragmentation (Figure 5). For the cation effect, the spectrum comparison was made with addition of 20 mM NH4OH, NaOH, or LiOH. Addition of 20 mM NH4OH did not change the quasi-molecular ion species ([M + H]+, [M + Na]+, and [M + K]+). In contrast, the quasi-molecular ions were almost eliminated with addition of 20 mM NaOH due to energetic cross-ring cleavages. Both [M + Na]+ and [M + Li]+ 4003

DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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were different from the exclusive cross-ring fragment (2,4An) ions observed in the MALDI/TOF experiments (Figure 5d). As discussed previously, the as-prepared NPGA and NPLA were likely coated with a second layer of excess sugar molecules; therefore, the MALDI spectra of model molecules were collected by using thermally treated NPGA (Figure S9). After heating, the NPGA matrix detection sensitivity dropped compared to NPGA without heating. In addition, MALDI sample spots prepared with thermally treated NPGA were less homogeneous and the spectra quality was less reproducible. Without NaOH, thermally treated NPGA matrix produced cross-ring fragmentation, different from the NPGA without heating where molecular ions and glycosidic cleavages dominated. Addition of 10 mM NaOH further reduced the detection sensitivity of thermally treated NPGA matrix. The interaction between the capping molecule and analyte could be through hydrogen bonding, Na+ bridging, or Na+− OH−−Na+ salt bridging as illustrated in Figure 6. The Na+ ion can link one site of OH or COOH groups of the capping molecule with another site of the OH group of the glycan, or one Na+ ion bound to the capping molecule can be connected with another Na+ ion bound to the analyte through electrostatic interaction with a mutual counterion OH−. Our study showed that the presence of both Na+ and OH− ions promoted intense glycan ISD fragmentation, supporting the hypothesis that the Na+−OH−−Na+ salt bridge interaction facilitates energy transfer from NP matrix to analyte (Figure 6a). The mechanism of sodiated glycan gas phase fragmentation by collision-induced dissociation (CID) was investigated by the experimental MS and computational method.50−52 Generally, sodium cationized glycans produce glycosidic and cross-ring fragmentations during the CID process.51,52 The sodiated glycan gas phase fragmentation mechanism in MALDI ISD could share some similar route to the CID process, as ISD is believed to be a combination of the thermal (similar to CID) and radical driven fragmentation process.17 The role of OH− in the glycan fragmentation mechanism is not clearly understood, but the excited OH− ions trapped in between the NP matrix capping surface and analyte surface could possibly form OH radicals53 and further induce glycan ISD fragmentation with a preference on cross-ring fragmentations. The proposed mechanisms for Na+ and OH− ion involvement in glycan fragmentation are shown in Figures 6b and 6c. The cross-ring cleavage of glycan molecules more likely followed a radical driven pathway,51,52 while the glycosidic bond may be directly cleaved, yielding glycosidic cleavage.51 NP Capping and Additive Effect on Peptide MALDI ISD Analysis. Similar to the studies performed on the model glycan molecule, the effects of NP capping molecules and choices of additive ions were also studied by using a model peptide, substance P acid, with a focus on the effects of ionization and ISD fragmentation pattern. Figure 7 shows the MALDI/TOF mass spectra of substance P acid acquired by using iron oxide NPs with the four selected surface capping molecules without any additive. The spectra obtained by using NPCA, NPGA, and NPLA as matrices were very similar, all with abundant ISD fragments and adducted molecular ions, including [M + H]+, [M + Na]+, and [M + K]+. The best S/N was obtained with the NPCA matrix. The peptide signal intensity obtained with NPGSH was the lowest, likely due to inefficient desorption. Because of the sensitivity, the NPCA

Figure 5. MALDI/TOF ISD mass spectra of maltoheptaose acquired using NPGA with (a) no additive, (b) 20 mM NH4OH, (c) 20 mM NaCl, (d) 20 mM NaOH, (e) 20 mM LiOH, and (f) 40 mM LiOH. Fragments retaining one sodium ion are labeled in black, two sodium ions are labeled in dark blue, and a lithium ion is labeled in orange.

quasi-molecular ions and related fragment ions were observed with 20 mM LiOH addition. In addition, NaOH additives only caused cross-ring cleavages 2,4An, while spectra obtained with NH4OH and LiOH showed a mixture of cross-ring (2,4An, 0,2 An) and glycosidic cleavages (Bn, Cn). Increasing the LiOH concentration to 40 mM did not change the glycan fragmentation pattern but suppressed ISD ion signals (Figure 5e,f). The comparison of spectra obtained with NH4OH, NaOH, and LiOH at 20 mM concentration (Figure 5b,d,e) suggested that Na+ was more effective than Li+ or NH4+ in promoting glycan cross-ring fragmentation. For the anion effect, the spectrum comparison was made with addition of 20 mM NaCl and NaOH. Addition of 20 mM NaCl enhanced the [M + Na]+ ion signal intensity. The comparison of spectra generated from NaCl and NaOH (Figure 5c,d) suggested OH− to be more effective than Cl− in promoting cross-ring fragmentation. The cross-ring fragmentation enhancement by OH− was unlikely due to glycan degrading in solution, which was confirmed by ESI experiments. ESI is a softer ionization technique than MALDI and generally does not cause fragmentation of analyte molecules. ESI spectra of maltoheptaose mixed with 20 mM NaOH instantly or after 30 min (the time scale in the MALDI experiments for the sample spot to dry on the target) did not show any fragment ions. After 10 days of mixing, dominant glycosidic cleavage (Cn) ions were observed from maltoheptaose hydrolysis (Figure S8), which 4004

DOI: 10.1021/acsanm.9b00988 ACS Appl. Nano Mater. 2019, 2, 3999−4008

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Figure 6. Illustration of proposed binding and fragmentation mechanisms of glycan analyze with NP matrix, (a) binding between NPGA matrix and glycan analytes through Na+ and OH− additive ions, (b) OH radical-initiated cross-ring fragmentation of sodiated glycan, and (c) glycosidic fragmentation of sodiated glycan.

Figure 7. MALDI/TOF ISD mass spectra of substance P acid acquired by using NPCA, NPLA, NPGA, and NPGSH matrices without additive.

Figure 8. MALDI/TOF ISD mass spectra of substance P acid acquired by using NPCA with (a) no additive, (b) 10 mM NaOH, (c) 10 mM NH4OH, and (d) 0.1% TFA.

matrix was subsequently used to study the effects of additive ions. Figures 8 and 9 (which is an expansion of Figure 8a,b with detailed ion labeling) show the MALDI/TOF ISD mass spectra of substance P acid obtained by using NPCA matrices with no additives and with three additives (10 mM NaOH, 10 mM NH4OH, and 0.1% TFA). Compared to the spectrum without additive (Figure 8a), no or negative effects on peptide

ISD fragmentation ion intensity were observed with addition of 10 mM NH4OH or 0.1% TFA (Figure 8c,d), while the quasimolecular ion species remained similar ([M + H]+, [M + Na]+, and [M + K]+). In contrast, addition of 10 mM NaOH shifted the quasi-molecular ion species to [M + Na]+, [M + 2Na]+, and [M + Na + K]+, where [M + 2Na]+ ions were the most intense, and the resulting ISD fragment ion species were simplified (Figures 8b and 9b). Without additive, abundant an+ 4005

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Figure 9. MALDI/TOF ISD mass spectra of substance P acid acquired using NPCA with (a) no additive and (b) 10 mM NaOH. (c) Structure and fragment diagram of substance P acid. In the mass spectra, fragments labeled black originated from [M + H]+, blue from [M + Na]+, and orange from [M + K]+. In the structure and fragmentation diagram, color choice was based on fragmentation ion series types.

and dn+ ions originating from three types of molecular ions ([M + H]+, [M + Na]+, and [M + K]+) were all observed (Figure 9a). Addition of 10 mM NaOH simplified the spectra by minimizing ISD product ion series originated from [M + H]+ and [M + K]+, leaving sodium adduct ions as the dominant ion series. Furthermore, in addition to an+ and dn+ ions (n = 3−8, 10), cn+ (n = 2, 4−10) ions were also observed (Figure 9b), which improves peptide sequencing coverage. Without additive, only an+ and dn+ ions (n = 2−8, 10−11) were observed, with a9+ ion missing. The peptide fragmentation ion labeling followed the Roepstorff and Fohlman nomenclature as modified by Biemann.54 The observation of additional cn+ (n = 2, 4−10) ion series with 10 mM NaOH addition was interesting. Organic matrices for peptide MALDI ISD are generally categorized as reducing matrix (e.g., 1, 5-diaminonaphthalene55) or oxidizing matrix (e.g., 3-hydroxy-4-nitrobenzoic acid56). The reducing matrix generates cn/zn ions while the oxidizing matrix produces an/xn ions generally.57 The NPCA matrix without additive generated an and dn peptide ions, similar to an oxidizing matrix. The dn ions are side chain loss from an ions, useful for isobaric leucine/ isoleucine discrimination in peptide sequencing.56 With NaOH additive, NPCA matrix produced an, dn, and cn peptide ion series, functioning as both oxidizing and reducing matrix. The shift in ISD fragmentation pattern upon addition of NaOH could be related to Na+ or OH− effects. OH− ion was likely involved in assisting the hydrogen abundant reducing matrix

fragmentation pathway by generating OH or H radicals. The NH4OH additive was not as effective as the NaOH additive for ISD fragmentation enhancement. One possible explanation is that the OH− ion may be more effective when the ion is in close contact with the iron oxide NP capping molecules and analytes via NP capping−Na+−OH−−Na+−analyte bridging. The disodium adduct molecular ion [M+ − 2Na]+ was only observed with the NaOH additive, which could also contribute to the generation of cn ion series. Figure 9c shows the detailed structure and fragment diagram of substance P acid to indicate the fragments, where color choices were based on fragmentation ion series types.

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CONCLUSION In summary, for NP matrices, additive cations, additive anions, and surface capping molecules altogether affected molecule ionization and ISD fragmentation pathway for the selected model glycan and peptide. NPGA was the best capping molecule for the model glycan, maltoheptaose, while NPCA worked more effectively for the model peptide, substance P acid. For maltoheptaose, both additive cation and anion influenced glycan ISD fragmentation, with Na+ and OH− being the most effective in promoting cross-ring fragmentation, compared with NH4+, Li+, or Cl− ions. For the model peptide, substance P acid, addition of 10 mM NaOH simplified the spectra by reducing fragmentation ion series complexity and introduced additional cn+ ions to improve peptide sequence 4006

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ACS Applied Nano Materials coverage. These results suggested that Na+ and OH− ions were possibly involved in the ionization and fragmentation mechanisms in the analysis of glycans and peptides using iron oxide NP matrices. All the reported studies in this paper were in positive ion mode; our preliminary tests using iron oxide NP matrices in negative ion mode did not generate analyte ions. Future studies will be focused on tuning the NP parameters to verify their effectiveness as MALDI matrices in negative ion modes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00988. TEM histograms and high-resolution TEM images of NPGA, NPGSH, NPLA, and NPC Amatrices; illustration of second layer attachment of GA molecules on NPGA surfaces; DLS plots, zeta-potential measurement of NPGA, NPGSH, NPLA, and NPCA matrices and DLS plots and zeta-potential measurements of NPGA before and after thermal treatment; MALDI/TOF mass spectra of NP matrices only with and without additives; MALDI/TOF mass spectra of maltoheptaose acquired using NPGA, NPGSH, NPLA, and NPCA matrices with 20 mM NaOH and ESI mass spectra of maltoheptaose acquired after mixing with 20 mM NaOH in water with a brief description in the caption; MALDI/TOF mass spectra of model molecules using NPGA after thermal treatment (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; phone (205) 348-9869; fax (205) 348-7558. ORCID

Carolyn J. Cassady: 0000-0001-6208-8747 Yuping Bao: 0000-0003-2829-4082 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Science Foundation (DMR1149931). We acknowledge the University of Alabama Department of Biological Sciences for the use of TEM.

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