Protein Chips Compatible with MALDI Mass Spectrometry Prepared by

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Protein chips compatible with MALDI mass spectrometry prepared by ambient ion landing Petr Pompach, Oldrich Benada, Michal Rosulek, Petra Darebna, Jiri Hausner, Viktor Ruzicka, Michael Volny, and Petr Novák Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01366 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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Analytical Chemistry

Protein chips compatible with MALDI mass spectrometry prepared by ambient ion landing Petr Pompach†,‡,#, Oldřich Benada†,¦, Michal Rosůlek†,‡, Petra Darebná†,‡, Jiří Hausner†,‡, Viktor Růžička§, Michael Volný*,†,# and Petr Novák*,†,‡,# †

‡

Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science, Charles ¦ University in Prague, Prague, Czech Republic; Faculty of Science, J. E. Purkyně University in Ustí nad Labem, Ústí # § nad Labem, Czech Republic; AffiPro, s.r.o., Mratin, Czech Republic; BioVendor, a.s., Brno, Czech Republic. KEYWORDS: Ambient ion landing, protein (arrays) chips, proteases, lectins, antibodies, affinity enrichment, mass spectrometry. ABSTRACT: We present a technology that allows the preparation of MALDI-compatible protein chips by ambient ion landing of proteins and successive utilization of the resulting protein chips for the development of bioanalytical assays. These assays are based on the interaction between the immobilized protein and the sampled analyte directly on the protein chip and subsequent in-situ analysis by MALDI mass spectrometry. The electrosprayed proteins are immobilized on dry metal and metal oxide surfaces, which are non-reactive under normal conditions. The ion landing of electrosprayed protein molecules is performed under atmospheric pressure by automated ion landing apparatus that can manufacture protein chips with a predefined array of sample positions, or any other geometry of choice. The protein chips prepared by this technique are fully compatible with MALDI ionization because the metal-based substrates are conductive and durable enough to be used directly as MALDI plates. Compared to other materials, the non-reactive surfaces show minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips. Three types of protein chips were used in this report to demonstrate the bioanalytical applications of ambient ion landing. The protein chips with immobilized proteolytic enzymes showed the usefulness for fast in-situ peptide MALDI sequencing; the lectin-based protein chips showed the ability to enrich glycopeptides from complex mixtures with subsequent MALDI analysis; and the protein chips with immobilized antibodies were used for a novel immunoMALDI workflow that allowed the enrichment of antigens from the serum followed by highly specific MALDI detection.

INTRODUCTION Immobilization of proteins is of fundamental interest in biochemistry, biosensor, and material science as well as bioanalytical chemistry1-5. Immobilized proteins are physically confined within a certain defined region or space with retention of their biological activity5. Protein immobilization has a long history, and the immobilization techniques are used to produce many biotechnological products in the field of diagnostics, bioaffinity chromatography, and biosensors5-7. The concept of protein chips and protein arrays originates from miniaturized immunoassays on solid substrates that were developed several decades ago using protein spotting onto nitrocellulose substrates. These membrane protein arrays exhibited better sensitivity than immunoassays carried out in microtiter plates, which were standard at that time, simply because they required smaller volumes5,8,9. The protein arrays and chips required a surface-immobilized capture reagent (such as antibody, aptamer, lectin or other protein molecules with sufficient affinity to its partner) to which the experimental sample is bound. After the wash step to remove the nonspecifically bound material, the specifically bound analyte is visualized using a suitable

technique5,9. The fluorescence detection techniques are commonly used for protein chips and arrays, but alternative detection techniques have specific advantages5,9. Mass spectrometry provides the most prominent alternative detection scheme, mainly because it is label free and simultaneously detects many channels that are created by the ability to separate individual ionized molecules by their mass-to-charge ratios (m/z). The multichannel nature of mass spectrometry is important because it allows better separation from the background signal that originates from the chip and further distinguishes different analytes that bound to the same immobilized protein as long as they differ in molecular mass. To utilize mass spectrometry detection directly from the protein chip, the surface and the substrate underneath must be compatible with the selected desorption-ionization mass spectrometry method. The current desorption-ionization method of choice for protein analysis is Matrix-assisted laser desorption/ionization (MALDI), which requires the sample substrate to be conductive, stable in vacuum, and compatible with the MALDI matrix deposition. At the same time, the protein chips must be manufactured in such a way that it allows proteins to get immobilized without disrupting the

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secondary or tertiary structure or otherwise losing their biological activity5,9. Most protein chips intended for fluorescence detection are made with glass slides substrates, which are coated with a substance that either passively absorbs the arrayed proteins or allows them to be cross-linked to the reactive surface5,9. Many other substrates were developed based on sorption, cross-linking, affinity or even hybridization. The overview of different types of chemical reactivity that allow protein immobilization is available in monographies5,9 and reviews 1,2. The protein chips with MALDI have been successfully coupled using gold or gold-deposited Self-Assembled Monolayers (SAM) substrates that utilize covalent cross-linking between the surface and the protein10-14 on porous silicon14 or specialized polymers15. Other substrates do not exhibit sufficient conductivity or stability for detection based on MALDI ionization, which results in poor ionization efficiency and inadequate sensitivity in mass spectrometry. Gold surfaces are capable of protein cross-linking via a covalent bond, and this has been successfully used in Ciphergen chips5,16 and different protein variants of the Surface-enhanced laser desorption/ionization (SELDI) technique17. SELDI has been proven to be a useful mass spectrometry technique, but it also suffers from certain limitations18,19. The main disadvantage of this approach is that the gold, silicon or polymer surfaces can nonspecifically bind many different proteins present in the same sample. Due to the nonspecific interactions, the gold protein chips suffer interference by other molecules from the investigated sample. This hinders the analysis, especially at ultralow concentrations of the targeted analyte. It is generally difficult to immobilize proteins directly on inert metal or metal oxide surfaces (without any soft intermediate layer) by standard techniques without the loss of their activity. One of the techniques that allow immobilization on metallic surfaces without utilizing gold cross-linking or any other wet chemistry is ion reactive landing. The vacuum version of reactive landing experiment20-22 has been developed from the original 1977 softlanding experiment performed by the Cooks lab23; the soft-landing technique has been later used in many variants to direct biomolecular ions onto bare surfaces and SAMs20-31. Notable research works include studies of ion soft landing of a nucleotide32, intact virus33 and first protein-ion landing study27. Recently, the ambient variant of the ion soft and reactive experiment was developed in which the dry ions were obtained at atmospheric pressure using electrospray ionization and rapid heating34. In this arrangement, the ions are produced by electrospray, desolvated by heating and directed toward a target surface, which is either grounded or oppositely (with respect to the electrospray polarity) biased. Ambient ion landing maintains many aspects of the vacuum variant but without the necessity to utilize vacuum instruments34, which helps to overcome the low yield limitation35a,b. Some applications of ambient ion landing have been already demonstrated. He et al. showed that covalent conjugation on reactive surfaces by ambient landing is possible under controlled conditions36, and Krasny et al. used ambient landing to prepare stable TiO2 and ZrO2 coated chips that

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have been used for LC-MALDI in fully automated regime37,38. Blacken tried atmospheric deposition of inorganic stationary phase on MALDI plates, but the results were negative39. Here, we present a method that allows the preparation of MALDI compatible protein chips by ambient ion landing of proteins on metal and metal oxide surfaces. The electrosprayed proteins are immobilized on dry inert surfaces into a predefined chip/array geometry using previously described automated ion landing apparatus33. The protein arrays prepared by this technique are fully compatible with MALDI ionization. EXPERIMENTAL SECTION Chemicals and Materials. Unless stated otherwise, all chemicals (including proteins) were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile and water were obtained from Merck (Darmstadt, Germany). Ambient ion landing. Functionalized MALDI plates were prepared as previously described38 with only a few changes in the procedure. The protein solution was delivered at a flow rate of 1 µL/min to 20 µm nanoelectrospray emitter (New Objective, Woburn, MA). A positive voltage (+1.5kV) provided by external high voltage power supply was applied to the liquid junction (metal union). Preheated nitrogen gas at 45 - 60°C was used to further improve nebulization and evaporation. The charged aerosol was dried by passing through 10 cm long desolvation tube (d= 4mm) that was kept on ground potential and externally heated to 45 - 60°C as well. The dried ions were deposited on custom-made steel plates or ITO (Indium Tin Oxide) glass slides (Bruker Daltonics, Billerica, MA) that were connected to a high voltage of the opposite polarity (-1.5 kV). The surface was masked using a sticker tape with punched holes to obtain reproducible 2 mm circular spots of landed material. Protein concentration, buffer compositions and spraying conditions varied depending on the type of modification. Trypsin was diluted with 20 mM ammonium bicarbonate buffer, pH 7.8 to a concentration of 2 µM. Pepsin, rhizopuspepsin, and a mixture of pepsin and rhizopuspepsin (1:1 molar ratio) were dissolved in 20 mM ammonium acetate, pH 4.0 to a concentration of 10 µM. Concanavalin A and Wheat Germ Agglutinin (WGA) were diluted with 20 mM ammonium bicarbonate pH 7.8, 10% acetonitrile to a concentration of 10 µM. Leptin antibody (Biovendor, Brno, Czech Republic) was diluted in 20 mM ammonium bicarbonate buffer, pH 7.8 to a concentration of 2 µM. Pepsin, rhizopuspepsin, protease mixture and anti-leptin antibody were sprayed for 5 min per position, and lectins were sprayed for 10 min per position. For the time-resolved trypsin experiment, the trypsin solution was sprayed for 30 min, 15 min, 5 min, 3 min and 1 min. The desolvation tube and nebulizer gas were heated to 60°C for proteases and 45°C for lectins and antibody. Surface washing. After the ion landing deposition, all functionalized surfaces were washed for 5 min in water, except for pepsin, rhizopuspepsin, and the protease mixture surfaces that were washed by 1.6 µM acetic acid


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aqueous solution (pH=4), and let dry at room temperature. Scanning electron microscopy. Dry surfaces with landed proteins were sputter-coated with 20 nm of gold in a Polaron SC5100 sputter coater (Quorum Technologies Ltd., Lewes, United Kingdom). The samples were examined by Tescan Vega LSU scanning electron microscope (Tescan, Brno, Czech Republic) at 10 kV using a secondary electrons detector. In-situ protein digestion on proteolytic chips. Five picomols of myoglobin in 20 mM ammonium bicarbonate buffer (pH 7.8) were applied on the trypsin chip and incubated in the humidity chamber at 37°C for 2 hours. After the incubation, 1 μl of α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (water/acetonitrile/saturated HCCA in methanol in ratio 1:1:1) was added to each spot, and the samples were analyzed using MALDI FT-ICR mass spectrometer. The mass values of tryptic peptides were searched using MASCOT (Matrix Science, version: 2.2.07) against the SwissProt database (taxonomy mammals, updated). Five picomols of myoglobin in 20 mM glycine buffer (pH 2.5) were applied on the chip with immobilized proteolytic enzyme and incubated for 2 min at room temperature. The enzymatic reaction was quenched by the addition of 1 µL of the HCCA matrix. The samples were analyzed by MALDI FTICR mass spectrometer, and masses of peptides were searched against the myoglobin sequence with no enzymatic preference. For confirmation of correct matches, peptides were fragmented using MALDI-TOF/TOF mass spectrometer. The sequence coverage was visualized using the MS Tools software40. Glycopeptides enrichment on lectins chips. The glycopeptides enrichment efficiency of lectin chips was demonstrated using tryptic digest of β Nacetylhexosaminidase from Aspergillus oryzae, isolated by the procedure described previously41. Ten micrograms of purified enzyme were re-suspended in ammonium bicarbonate buffer, pH 7.8, reduced by DTT and alkylated with iodoacetamide. After overnight incubation with trypsin (Promega, Madison, WI), the protease was denatured by heating at 95°C for 5 min. Ten picomols of the peptide mixture was applied on surface modified by Concanavalin A. After 1 hour incubation at room temperature in the humidity chamber, the chip was washed 3 × 5 min with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), and 1 µl of 2,5-dihydroxybenzoic acid (DHB) matrix (50 mg/mL of DHB in 50% acetonitrile) was added to each spot. Mass spectrometric detection was performed by MALDI FT-ICR mass spectrometer. The spectra were semi-manually curated using GPMAW 7.0 software (Lighthouse data, Denmark), and ion signals corresponding to glycopeptides were assigned. The sample of IgG isolated from human serum was treated according to the same protocol. Ten picomols of the tryptic digest of the IgG1 and IgG2 mixture was digested as described above and spotted on the chips functionalized with WGA lectin. Incubation and further procedure were the same as for Concanavalin A.

Immuno enrichment on antibody chips. Leptin (BioVendor, Brno, Czech Republic, Lot: AP-13-017P1) was resuspended at different concentrations in the dilution buffer provided in the commercially available ELISA leptin detection kit (BioVendor, Brno, Czech Republic). One microliter of each leptin solution was applied on the surface with immobilized polyclonal human anti-leptin antibody and incubated for 30 min at room temperature in the humidity chamber. After incubation, the chip was washed 3 times for 10 min in PBS buffer and rinsed with water. The spots were covered with 1 µl of sinapinic acid (SA) matrix (saturated solution of SA in 50% acetonitrile, 0.1% TFA), and leptin was detected by mass spectrometric measurements using the MALDI-TOF/TOF operating in linear mode. Chemiluminescence detection. For chemiluminescence detection, a parallel chip with the leptin antibody sample set was washed with PBS and further incubated with secondary antibody (conjugation solution from the ELISA leptin detection kit, BioVendor) for 1 h. After another washing by PBS buffer (3 × 10 min), the chip was incubated for 1 min in enhanced chemiluminescence (ECL) solution (ThermoFisher Scientific, Waltham, MA) and visualized by western blot imaging system ChemiDoc (Bio-Rad, Hercules, CA). MALDI-Time-of-flight Mass Spectrometry (TOF-MS). Mass spectra of peptides were acquired in positive reflector mode using an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with SmartBeam laser (200Hz). The MS data were acquired in the m/z range between 500 and 4500 by averaging signals from 2000 consecutive laser shots using target randomwalk movement. Prior to each data acquisition, the external calibration was conducted using a peptide calibration standard (Bruker Daltonics, Bremen, Germany). Intact proteins were acquired in a positive linear mode by the accumulation of 1000 laser shots in the mass range between 7000 and 19000. The mass spectrometer was calibrated externally by using protein calibration mixture I (Bruker Daltonics, Bremen, Germany). The FlexAnalysis 3.0 program (Bruker Daltonics, Bremen, Germany) was used to process the data. MALDI Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). High-resolution mass spectra were acquired using solariX 12T mass spectrometer equipped with dual ESI/MALDI ion source (Bruker Daltonics, Billerica, MA). Mass spectra were acquired in a positive ion mode with 2048k data points at m/z 150 using a SmartBeam II laser MALDI source (1k Hz, 25 laser shots, 4 acquired scans). The results were analyzed using Data Analysis 4.0 (Bruker Daltonics, Bremen, Germany). RESULTS AND DISCUSSIONS For a multiply-charged protein ion to survive the collision with a surface without the loss of biological activity, the kinetic energy must dissipate during the collision process, without breaking the covalent bonds or without unfolding into an inactive protein conformation. The energy



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Figure 1. Top left panels: Images of stainless steel MALDI chip with landed trypsin visualized by scanning electron microscope. Panel A before washing; Panel B after washing; Spectrum Panels: MALDI-MS spectra of myoglobin digested for 2 hours at 37°C on MALDI chip functionalized with different amounts of landed trypsin. The identified peptides are labeled with stars. The insets represent the primary amino acid sequence of myoglobin with identified peptides highlighted in red. must, therefore, be distributed into rotational-vibrational and electronic degrees of freedom of landed ionized molecule and the substrate. The excited degrees of freedom must not exceed a dissociation or irreversible refolding threshold. To avoid loss of activity due to the above reasons, the very first protein ion landing studies were performed using surfaces with liquid interlayer that efficiently protected the soft-landed proteins27,42. The energetics of soft and reactive landing on metal oxides and SAMs has been studied but only for a limited number of systems20,21,25,26,28,43. The dependence on projectile’s kinetic energy on the ion-landing experiment is much better understood than the surface properties that influence the survival yield. This study combines the ion soft and reactive landing methodology with the experiences obtained from ion technologies that operate at atmospheric pressure. The kinetic energy of ions in experiments performed at atmospheric pressure is very low and is close to thermal, which protects their structural integrity upon collision with the surface. This is different from the conditions experienced by the ion beams in vacuum, but we discovered that the ion reactive landing phenomenon still exists even in the atmospheric pressure environment, where constant collisions with the air molecules do not allow the projectile ion to achieve higher kinetic energies. Our experiments with ambient reactive landing on dry metal and metal oxide surfaces are in agreement with the recent reports about ambient ion landing of small molecules on SAMs34,36. The fact that it is possible to reactively land and

permanently immobilize protein ions on a dry conductive surface without the loss of bioactivity was not obvious; the technique based on this phenomenon allows efficient utilization of ion landing for targeted modification of surfaces at atmospheric pressure. Such modified surfaces obtain new functionalities that are given by the biological activity of the attached protein. In this work, we demonstrated that MALDI plates can be easily modified by proteins using ambient ion landing. The protein-modified surfaces withstood washing with common buffers and solvents and could be used as MALDI compatible protein analytical chips. To demonstrate the utility of the presented technique, we focused our ambient ion landing experiments on the design of MALDI protein chips modified by immobilization of three different types of proteins: enzymes, lectins, and antibodies. Enzymes. Studies have shown that trypsin (and nonenzyme protein streptavidin) can be immobilized by reactive ion landing on a dry metal/metal oxide surface with retention of their activity20. Both trypsin and streptavidin monomer are robust, compact globular proteins. Trypsin’s internal structure is further reinforced by six disulfide bridges, and its survival rates in vacuum reactive landing experiments were thus high20. Based on these previous results, we first selected trypsin and other similarly durable proteolytic enzymes as probes for MALDI compatible protein chips. In general, the stability of any enzyme upon immobilization might increase or decrease depending on whether the surface provides a denaturing


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or stabilizing microenvironment. Inactivation due to selfdigestion of proteolytic enzymes should be reduced after surface immobilization because of the protection of the enzyme molecules from mutual digestion. It has also been reported that in some cases enzymes coupled to inorganic carriers exhibited improvement in stability44. Immobilized enzymes are common in biochemistry and biotechnology because keeping enzymes in place throughout the reaction allows easier localization of products and their further separation from reaction mixture or detection. The goal of our work was analogous - to utilize reactive landing immobilization for preparing proteolytic MALDI chips that would allow retention of cleaved peptides in a defined location. Scanning electron microscopy was used to visualize the surface modification by ambient ion landing. Figures 1A and B show metal surface with landed

trypsin before and after washing. Both figures show contours of structures that were formed by large trypsin domains formed after the landing on the surface. The washing step removed the loosely bound soft-landed layers and only the trypsin molecules, which achieved binding interaction with the surface, stayed immobilized on the surface after the washing. The protein to be proteolytically cleaved and subsequently sequenced by MALDI peptide mass fingerprinting was sampled directly on the proteolytic MALDI chip with landed and washed enzyme (such surface corresponds to electron micrograph in Figure 1B). After the completion of the proteolytic enzyme reaction (see protocol in Experimental), the MALDI matrix was deposited, and the standard MALDI ionization was performed

Figure 2. Panel A Mass spectrum of myoglobin digested for 2 minutes at room temperature on ITO glass MALDI proteolytic chip with immobilized pepsin. The stars indicate assigned peptides. Sequence maps of myoglobin in-situ digested by pepsin (Panel B), rhizopuspepsin (Panel C) and pepsin/rhizopuspepsin mixture (Panel D) on the MALDI chip.



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in-situ. We tested ambient ion landing of the following proteolytic enzymes: trypsin, pepsin, and rhizopuspepsin (rhizopus acid protease). All the above proteases maintained the enzymatic activity after the ambient reactive landing immobilization and transferred the proteolytic functionality to the MALDI chip surface. The spectra in Figure 1C document the digestion of myoglobin on the proteolytic MALDI chips prepared by trypsin landing at different deposition times. As expected, longer deposition time resulted in a larger amount of landed trypsin on the surface. Enzymatic digestion of the myoglobin sample created several overlapping peptides that covered 100% of its sequence. The spectra in Figure 1 show that the smallest amount of electrosprayed and landed trypsin immobilized on the MALDI chip, which still produced 100% coverage of the myoglobin sequence, was 0.1 µg. However, the overall ion intensities decreased by two orders of magnitude compared to the spectra obtained from positions with higher amounts of deposited trypsin. A surface with only 0.04 ug of deposited trypsin did not achieve 100% sequence coverage. In further experiments, other proteolytic enzymes were reactively landed on the protein chip surface, and their activities were evaluated. Figure 2 shows mass spectrum and corresponding sequence coverage maps of myoglobin digested by surface-landed pepsin, rhizopuspepsin, and their mixture. As expected, pepsin provided a higher number of cleaved peptides than trypsin, as seen in the myoglobin sequence coverage diagrams in Figures 2B. Rhizopuspensin provided 100% sequence coverage of myoglobin, but the number of overlapping peptides was much lower compared to other proteases

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(Figure 2C). Ambient reactive landing allows simple coimmobilization of different molecules, as long as they are compatible in solution and it is possible to ionize them by electrospray together. Interestingly, the myoglobin digestion could be further adjusted using combinations of proteolytic enzymes (Figure 2D). This combination of proteolytic enzymes provided the best results with 100% sequence coverage and the highest number of overlapping peptides. Since the results described above exhibited a sufficient spatial resolution within the single myoglobin sequence, it can be expected that these MALDI proteolytic chips will be advantageous for hydrogen-deuterium exchange applications. Lectins. Figure 3 shows the analytical performance of MALDI chips prepared by ambient ion landing of Concanavalin A (ConA), a lectin from the legume lectin family, which binds mainly to internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups. The solution of trypsinated hexosaminidase was sampled on the MALDI chip with immobilized ConA and washed according to the enrichment protocol described in the Experimental section. The measurements performed by the highresolution MALDI FT-ICR mass spectrometer allowed accurate glycopeptides assignment. Three unique glycopeptides were detected by using the enrichment procedure followed by exact mass fingerprinting. Panel 3A shows the results of the enrichment experiment focused on O-glycopeptides in the m/z region 2000-3500. Several glycoforms of the WVPAATEAPISSFEPFPTPTAGAS and

Figure 3. MS spectra of tryptic glycopeptides after enrichment (top red spectrum) using MALDI chip functionalized with Concanavalin A and without enrichment (bottom flipped black spectra). Panel A) MS spectrum of enriched glycopeptides WVPAATEAPISSFEPFPTPTAGAS (pep) and its truncated form WVPAATEAPISSFEPFPTPTAGA (pep*) bearing different combinations of hexoses. Panel B) MS spectrum of glycopeptide ELSDIFPDHWFHVGGDEIQPNCFNFSTHVTK bearing high mannose glycans (green circle - mannose, blue square - N-acetylglucosamine).


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the WVPAATEAPISSFEPFPTPTAGA peptides bearing up to six hexoses were observed but only after the sample enrichment on a MALDI chip. Figure 3B shows the enrichment of N-glycopeptides (from the same sample) in the spectrum region above m/z 4000. The detected ions correspond to the tryptic peptide ELSDIFPDHWFHVGGDEIQPNCFNFSTHVTK bearing high mannose glycans (Man5, Man6 and Man7) at m/z 4890.2513, m/z 5052.2867 and m/z 5214.2200. The blackflipped spectrum was obtained from the same sample in the same m/z region but without the in-situ enrichment on the MALDI chip. The spectra in Figures 3A and 3B clearly show that unlike the protein chip, the glycopeptides identification by standard MALDI peptide mass fingerprinting (without enrichment or another purification step) is not possible because the glycopeptide ionization is suppressed by the presence of other tryptic peptides. Both results correspond to previously published data that described propeptide O-glycosylation and catalytic subunit N-glycosylation of fungal hexosaminidase45. The efficiency of in-situ enrichment of glycopeptides was further demonstrated using MALDI chip prepared by ion landing immobilization of WGA. WGA is an agglutinin protein that binds mainly to N-acetyl-D-glucosamine and sialic acid, and it is, thus, useful for affinity-based detection of peptides bearing complex N-glycans. Figure 4 shows the MALDI spectrum of the trypsinated mixture of human IgG antibodies (IgG1 and IgG2) enriched on a WGA MALDI chip. Two glycopeptides, the TKPREEQYNSTYR from IgG1 and the TKPREEQFNSTFR from IgG2, both bearing complex N-glycans with core fucose, were clearly identified in the enriched spectrum. These glycosylations are in agreement with previously described glycoforms motives on IgG molecules46. Besides practical aspects, the presented results of experiments with lectin immobilization contributed to the fundamental aspects of protein ion landing as well. It was shown previously that streptavidin, a noncovalently bound tetrameric protein, exhibits affinity towards biotin even after immobilization by vacuum reactive landing20. This was not surprising because individual streptavidin monomers still exhibit some affinity towards biotin. However, lectins only function as noncovalent multimers. Our results, thus, demonstrated that noncovalent structures exist on the modified MALDI chips because either the lectin multimers survived the ionization and ion landing processes or they were reassembled on the surface after landing. Antibodies. A mass spectrometry technique based on the application of enrichment on immobilized antibodies in combination with MALDI-TOF detection has been previously described in the literature as “immunoMALDI” or “iMALDI”47-49. Using examples of the clinically relevant antibody-antigen systems, we demonstrated that ambient ion landing can be used for the modification of MALDI substrates by antibodies, and the same immunoMALDI workflow can be performed in-situ on the MALDI protein chips. Supplemental Figure S1 shows MALDI-TOF spectrum of commercially available leptin standard. Leptin is

Figure 4. MS spectrum of enriched IgG1 glycopeptide TKPREEQYNSTYR (pep.1) and IgG2 glycopeptide TKPREEQFNSTFR (pep.2) bearing complex fucosylated glycans (green circle - mannose, blue square - Nacetylglucosamine, red triangle – fucose, yellow circle galactose) using MALDI chip functionalized with WGA lectin. The flipped black spectrum was obtained from the same sample without the in-situ enrichment. a 16.1 kDa protein of 167 amino acids and an important hormone that regulates metabolism and feelings of satiety in mammals. The spectrum is dominated by the wellresolved leptin peak at m/z = 16 100. While it is easy to detect pure recombinant leptin standard by MALDI ionization, leptin could not be detected directly from a spiked synthetic serum using MALDI ionization (Supplemental Figure 2, flipped spectrum in bottom panel). This was due to the strong serum matrix effects such as the presence of other proteins or ionization suppression due to salts and small molecules. Matrix interference is a known and common problem in desorption ionization mass spectrometry when any biofluid sample is analyzed directly without sufficient sample pretreatment and clean-up. However, when the same serum sample was applied on the MALDI chip with anti-leptin antibody deposited by ambient landing, and the proper in-situ enrichment procedure was performed (see Experimental), the leptin peak at m/z = 16 100 was clearly detected in MALDI-TOF spectrum (Supplemental Figure S2, top panel). In the next step, we focused on the determination of limit of detection as well as the sensitivity and dynamic linear range. Figure 5 shows the calibration experiment in which 1 µL of leptin spiked artificial sera samples of different concentrations were pipetted on different positions of the antileptin protein chip. Subsequently, the sample positions were washed, and the enriched samples were analyzed by



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MALDI and immunochemical detection. The concentrations of leptin in artificial serum samples were selected to cover a range of absolute sampling amount from 30 pg to 80 ng (2 fmol – 5 pmol). Figure 5A shows that the clearly visible peak of leptin signal was detected even at 0.16 ng of the sampled leptin, which corresponds to 0.16 µg/mL leptin concentration in the serum. The parallel-prepared protein chip with bound leptin samples was scanned, and the chemiluminescent signal was measured from each sample position (see Experimental section). The luminescence measurements confirmed the MALDI-MS quantitative information, as can be seen in Figure 5B.

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applications for the development of MALDI chip-based immunoassays. For example, we have recently reported the development of immunoMALDI assay for the determination of haptoglobin phenotype44. In this assay, the MALDI chip was constructed by ambient ion landing of anti-haptoglobin antibody, and haptoglobin was then enriched directly from human serum. Isoforms of haptoglobin have an affinity to the same antibody, but they have different molecular mass. Thus, MALDI ionization directly from the protein chip allowed distinguishing between the two haptoglobin isoforms and direct determination of the phenotype. This assay has been successfully tested on the cohort of 116 patient sera50. CONCLUSION

Figure 5. Panel A MALDI-TOF spectra of different amounts of leptin (m/z = 16 100) sampled and enriched on the ITO glass MALDI chip functionalized with polyclonal anti-human leptin antibody. Panel B The parallel independent chemiluminescence detection of leptin enriched on the same substrate surface. Supplemental figure S3 shows the linear range of the calibration dependency experiment (based on the MALDI results). The linearity exists only between 10-1000 fmol (0.16-16 ng). The demonstrated linear dynamic range over the two orders of magnitude is expected for a MALDITOF/TOF instrument, and despite the lack of internal standard the dependency, it is linear with R=0.98. This demonstrated the semi-quantitative nature of the method. In a separate experiment, we found that similar calibration results and practically the same limit of detection were obtained for leptin standard by regular MALDI without enrichment. This indicates that the analytical figures of merit are mainly given by the performance of the MALDI-TOF/TOF instrument, and the in-situ enrichment on the protein chip is efficient and represents no additional limitation or significant sample loss. Since all antibodies are chemically similar, they are also likely to exhibit comparable behavior during electrospray ionization and ion landing. It is thus reasonable to conclude that ambient ion landing is a usable technique for immobilization of any antibody with retention of its immunoaffinity towards the antigen. This offers many potential

We have developed a technique that allows the modification of MALDI surfaces by ambient ion landing of electrosprayed proteins. The modified MALDI surfaces obtained the protein functionality of the landed proteins and were used as MALDI compatible analytical protein chips. The presented method for in-situ immunoenrichment or enzymatic treatment is based on interactions between the analyte and the protein chip combined with the detection by MALDI mass spectrometry. This method is rapid, does not rely on external purification steps, and can be automated. The MALDI protein chips prepared by ambient ion landing are compatible with different MALDI source designs: the standard high-vacuum MALDI source used in TOF instruments with pulsed ion extraction (delayed extraction), as well as the MALDI source on FT-ICRMS that use elevated pressure and subsequent refocusing of ions in electrodynamic ion optics elements (e.g. ion funnels). The ambient ion landing technique consumes low amounts of proteins to construct the protein chips, which are comparable with that of other bioanalytical techniques based on immobilized proteins, such as standard ELISA. We demonstrated the usefulness by preparing MALDI protein chips based on proteolytic enzymes, lectins, and antibodies. In all experiments, the landed protein always maintained its biological activity and was able to impose on the surface upon immobilization. The technology has the potential to be used for immobilization of any protein molecules that can survive electrospray ionization and ambient ion landing without a significant loss of activity. The success of the technique for preparation of MALDI compatible protein chips is based on the fact that ambient ion landing allows immobilization directly on a dry metal or metal oxide surface. The absence of any interlayer between conductive MALDI surface modified by ion landing and antibody affinity molecules reduces the non-specific interactions of other proteins in the sample and maintains the original conductivity of the MALDI plate, which provides efficient ionization.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Mass spectra of recombinant leptin standard, immune-affinity en-


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riched and non-enriched spiked human sera. The linear range of the calibration dependency experiment. Control experiments.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] and [email protected]. Tel.: +420 325 873 610 (PN) and +420-734-492-970 or +1-206366-5746 (MV).

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes Patent applied for in 2014 by PP, VR, MV and PN. The authors declare no competing financial interest.

ACKNOWLEDGMENT This work has been supported by the Institutional Research Concept of the Institute of Microbiology (RVO61388971), the Ministry of Education Youth and Sports of the Czech Republic(LH15010, LD15089, LQ1604 and LO1509), European Regional Development Funds (CZ.1.05/1.1.00/02.0109 BIOCEV), Charles University in Prague (project UNCE 204025/2012 and GAUK 932316), and the Czech Science Foundation (1624309S). Access to instruments and other facilities was supported by the EU (Operational Program Prague – Competitiveness project CZ.2.16/3.1.00/24023).

ABBREVIATIONS MALDI, Matrix-assisted laser desorption/ionization; ESI, electrospray ionization; SAM, self-assisted monolayer; FTICR, Fourier-transformed ion cyclotron resonance; TOF, time of flight; MS, mass spectrometry; ELISA, enzyme-linked immunosorbent assay; WGA, wheat germ agglutinin; DHB, 2,5-dihydroxy-benzoic acid, HCCA, α-Cyano-4hydroxycinnamic acid; ITO, indium tin oxide; PBS, phosphate buffered saline; SA, sinapinic acid; ECL, enhanced chemiluminescence; ConA, concanavalin A; SELDI, surfaceenhanced laser desorption ionization

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Figure 1. Top left panels: Images of stainless steel MALDI chip with landed trypsin visualized by scanning electron microscope. Panel A before washing; Panel B after washing; Spectrum Panels: MALDI-MS spectra of myoglobin digested for 2 hours at 37°C on MALDI chip functionalized with different amounts of landed trypsin. The identified peptides are labeled with stars. The insets represent the primary amino acid sequence of myoglobin with identified peptides highlighted in red. Figure 1. 100x56mm (300 x 300 DPI)


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Figure 2. Panel A Mass spectrum of myoglobin digested for 2 minutes at room temperature on ITO glass MALDI proteolytic chip with immobilized pepsin. The stars indicate assigned peptides. Sequence maps of myoglobin in-situ digested by pepsin (Panel B), rhizopuspepsin (Panel C) and pepsin/rhizopuspepsin mixture (Panel D) on the MALDI chip. Figure 2. 165x164mm (300 x 300 DPI)



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Figure 3. MS spectra of tryptic glycopeptides after enrichment (top red spectrum) using MALDI chip functionalized with Concanavalin A and without enrichment (bottom flipped black spectra). Panel A) MS spectrum of enriched glycopeptides WVPAATEAPISSFEPFPTPTAGAS (pep) and its truncated form WVPAATEAPISSFEPFPTPTAGA (pep*) bearing different combinations of hexoses. Panel B) MS spectrum of glycopeptide ELSDIFPDHWFHVGGDEIQPNCFNFSTHVTK bearing high mannose glycans (green circle mannose, blue square - N-acetylglucosamine). Figure 3. 154x86mm (300 x 300 DPI)


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Figure 4. MS spectrum of enriched IgG1 glycopeptide TKPREEQYNSTYR (pep.1) and IgG2 glycopeptide TKPREEQFNSTFR (pep.2) bearing complex fucosylated glycans (green circle - mannose, blue square - Nacetylglucosamine, red triangle – fucose, yellow circle - galactose) using MALDI chip functionalized with WGA lectin. The flipped black spectrum was obtained from the same sample without the in-situ enrichment. Figure 4. 84x93mm (300 x 300 DPI)



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Figure 5. Panel A MALDI-TOF spectra of different amounts of leptin (m/z = 16 100) sampled and enriched on the ITO glass MALDI chip functionalized with polyclonal anti-human leptin antibody. Panel B The parallel independent chemiluminescence detection of leptin enriched on the same substrate surface. Figure 5. 139x115mm (300 x 300 DPI)


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Protein Chips Compatible with MALDI Mass Spectrometry Prepared by

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