Smart MALDI plate for protein digest enrichment, desalting and dire

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Thermoresponsive arrays patterned via photo click chemistry: Smart MALDI plate for protein digest enrichment, desalting and direct MS analysis Xiao Meng, Junjie Hu, Zhicong Chao, Ying Liu, Huangxian Ju, and Quan Cheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13640 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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ACS Applied Materials & Interfaces

Thermoresponsive arrays patterned via photo click chemistry: Smart MALDI plate for protein digest enrichment, desalting and direct MS analysis Xiao Meng,† Junjie Hu,† Zhicong Chao,† Ying Liu,*,†,‡ Huangxian Ju,† Quan Cheng*,‡

†State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China ‡Department of Chemistry, University of California, Riverside, 92521, CA, USA

______________________________ * Corresponding author. Phone/Fax: +86-25-89681918. E-mail: [email protected] Phone/Fax: 1-951-827-2702. E-mail: [email protected]

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ABSTRACT: Sample desalting and concentrating is a crucial step prior to MALDI-MS analysis. Current sample pretreatment approaches require tedious fabrication and operation procedures, which are unamenable to high-throughput analysis and also result in sample loss. Here we report the development of a smart MALDI substrate for on-plate desalting, enrichment, and direct MS analysis of protein digests based on thermo-responsive, hydrophilic/hydrophobic transition of surface grafted poly(N-isopropylacrylamide) (PNIPAM) microarrays. Superhydrophilic 1-thioglycerol microwells are first constructed on alkyne-silane functionalized rough ITO substrates based on two sequential thiol-yne photo-click reactions, while the surrounding regions are modified with hydrophobic 1H,1H,2H,2H-perfluorodecanethiol. Surface initiated atom transfer radical polymerization (SI-ATRP) is then triggered in microwells to form PNIPAM arrays, which facilitate sample loading and enrichment of protein digests by concentrating large volume samples into small dots and achieving on-plate desalting through PNIPAM configuration change at elevated temperature. The smart MALDI plate shows high performance for mass spectrometric analysis of cytochrome c and neurotensin in the presence of 1 M urea and 100 mM NaHCO3, as well as improved detection sensitivity and high sequence coverage for α-casein and cytochrome c digests in fmol range. The work presents a versatile sample pretreatment platform with great potential for proteomic research.

KEYWORDS: PNIPAM, MALDI-MS, Photo click chenistry, Surface patterning, On-plate desalting and enrichment

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■INTRODUCTION

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has become one of the most important tools for peptide and protein analysis in proteome research.1 However, the existence of contaminants such as buffer salts seriously interfere with the matrix-analyte co-crystallization, which significantly impair the sensitivity and reproducibility in MS detection,2 especially for low abundance peptides/proteins derived from practical biological samples. Therefore, sample concentration and desalting has become a crucial step prior to MALDI-MS analysis. Numbers of off-target strategies using inorganic nanomaterials,3-7 and water dispersed nanomaterials,8-10 have been applied for sample pretreatment. However, the operation procedure is not only time consuming, but also results in unavoidable sample loss and potential contamination. On-plate desalting and enrichment protocols have become attractive to simplify operation process and improve desalting/enrichment efficiency. Controlling the surface hydrophobicity is the most effective method for on plate peptides/proteins desalting. Self-assembled octadecyltrichloro silane (OTS)11 and hydrophobic polymer12-14 coating were constructed on sample support to increase surface hydrophobicity for retaining peptides/proteins target, while salts were rinsed away during the surface washing step. However, the dense self-assembled monolayer (SAM) and compact rigid hydrophobic polymer coating prevented the sufficient access of sample droplets to the substrate, therefore limited the sample loading amount and impaired its capability in target enrichment. One approach to improve sample loading amount onto surfaces is to incorporate them within a flexible 3D matrix instead of a 2D substrate.15 Stimuli-responsive deformable polymers provide significant variations in coating thickness, porosity, and hydrophilicity upon external stimuli, therefore should be promising candidates for sample pretreatment.16 Poly(N-isopropylacrylamide) (PNIPAM) is a 3



nontoxic thermoresponsive polymer which undergoes structural transition from hydrophilic to hydrophobic upon heating above a lower critical solution temperature (LCST) at 32 oC. PNIPAM has been widely used for temperature responsive small molecule17 and protein18 encapsulation as well as cell adhesion and release.19-21 The temperature dependent adsorption of different proteins were studied on derivative of PNIPAM coated MALDI probe,22 but the MALDI-MS results were not satisfactory due to the limited surface density of polymer and lack of surface patterning, which limited the high-throughput analysis and impaired the surface enrichment effect.

Superhydrophilic microwells spotted on a superhydrophobic substrate have been reported as a condensing-enrichment approach.23-25 Benefitting from the superwettability difference between the microwell and the surrounding regions, the analytes were enriched from the highly diluted solution and anchored onto the microwell for highly sensitive trace DNA detection23 and phosphopeptides concentration.24,25 Block copolymer containing both hydrophilic and hydrophobic domains26 and concentric circle patterns with adjacent hydrophobic-hydrophilic zones27 were developed for proteins/peptides self-purification on substrate. However, it required tedious multi-steps process for block copolymer synthesis and substrate fabrication, long time incubation, and careful operation process. The photo click coupling of thiol-ene and thiol-yne occurs at mild condition in short time, tolerates a variety of functional groups, and eliminates the participation of biotoxic metal catalyst for regular click chemistry of azide-alkyne coupling,28,29 therefore is becoming an promising approach for developing microarrays30,31 and cell surface patterning.32,33 Currently, the coupling reactants are limited to small molecules; the long polymer chains sterically hinder the functional groups and 4


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therefore are rarely used in photo-click reactions.

We report here a high-throughput on-plate protein digests desalting and enrichment approach prior to MALDI-MS analysis based on thermo-responsive polymer array grafted from photo click patterned Indium tin oxide (ITO) substrate. The combination of photo-click patterning technique and in situ growth of stimuli-responsive polymer allows better control of surface property and therefore extends its application. Taking advantage of two sequential thiol-yne photo click reactions, superhydrophilic (1-thioglycerol) microwells are constructed on alkyne-silane functionalized porous ITO substrate with superhydrophobic (1H,1H,2H,2H-Perfluorodecanethiol) modified surrounding regions. The fabrication process is facile and robust, and could be completed in seconds. The porous ITO substrate provides desirable surface roughness and conductivity, which boosts surface wettability and allows direct MALDI-MS analysis subsequently. Surface initiated atom transfer radical polymerization (SI-ATRP) of N-isopropylacrylamide (NIPAM) is then triggered in the superhydrophilic microwells to generate the thermo-responsive PNIPAM array on ITO substrate. The PNIPAM array wettability could be reversibly adjusted upon temperature change, therefore functionalizes as a smart MALDI plate with controlled surface wettability transitions during the on-plate sample pretreatment process. The presented smart MALDI plate allows direct detection of 0.8 pmol cytochrome c and 6 pmol neurotensin from 100 mM sodium bicarbonate and 1 M urea contaminated solutions respectively, as well as analysis of 40 fmol α-casein and cytochrome c digests, which demonstrates impressively improved performance compared with commercial C18 desalting tip.

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■ EXPERIMENTAL SECTION Fabrication of Superhydrophilic Patterns with Superhydrophobic surrounding Regions: The porous ITO substrates were first modified with alkyne-silane via vacuum-phase silanization.34,35 The ITO substrates were sealed into a preheated desiccator with a piece of filter paper pipetted with 100 µL O-(propargyl)-N-(triethoxysilylpropyl) carbamate (OTPC) and 0.5 g MgSO4•7H2O as water source for the hydrolysis reaction. The desiccator was then vacuumed and kept in oven at 80 oC overnight. The superhydrophilic patterns with superhydrophobic surrounding regions were fabricated via repeats “photo-click” thiol-yne reactions.31 The alkyne modified ITO substrates were wetted with THF solution containing 50 vol% 1-thioglycerol and 2 wt% DMPAP (photoinitiator), covered by a resin photomask, and irradiated under 365 nm UV light with 14.4 mW·cm-² for 20 s to fabricate hydrophilic 1-thioglycerol pattern. After removing the photomask, the substrates were washed sequentially with THF and ultrapure water, dried with N2. The second photo-click reaction was performed subsequently under 365 nm UV light with 150 mW·cm-² for 60 s with 5 vol% 1H,1H,2H,2H-perfluorodecanethiol and 2 wt% DMPAP ethanol solution to backfill the unexposed portion of the substrate with 1H,1H,2H,2H-perfluorodecanethiol coating, which rendered the residual alkyne-surface superhydrophobic. Finally, the patterned substrates were washed extensively with ethanol, ultrapure water, and dried with N2 before use (Scheme 1a). Preparation of Thermo-responsive PNIPAM Arrays: PNIPAM were grafted from superhydrophilic patterns on ITO substrate via SI-ATRP reaction. The hydroxyl groups of 1-thioglycerol were first converted to surface initiators via reacting with a DMF solution of 0.08 M BIBB and 0.1M TEA for 30 minutes, followed by DMF and ultrapure water rinsing and N2 stream dry.36 The SI-ATRP was then conducted by incubating the slides in an aqueous solution of 2 M 6


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NIPAM monomer and catalyst (9 mM CuBr/18 mM 2,2’-bipyridine) for 30 min under a dry nitrogen atmosphere. The monomer and catalyst solution was purged with a stream of purified nitrogen for 30 min before reaction to reduce the amount of O2 present. The resulting smart MALDI plates were rinsed with copious amounts of ultrapure water to remove any unbound polymers and other unreacted reagents and dried with N2 (Scheme 1b). On-plate Enrichment and Desalting of Protein and Protein Digests. Cytochrome c, myoglobin, and neurotensin with 100 mM sodium bicarbonate and various concentrations of cytochrome c or α-casein digests were loaded on PNIPAM arrays at room temperature followed by 30 min incubation at 60 oC to dry the sample droplet. The dried sample spots and the adjacent area were then rinsed by pipetting/expelling 60 oC ultrapure water for 3 times to remove salts and contaminants. The expelled water was discarded each time. Then 1 µL of matrix CHCA aqueous solution (10 mg/mL, 50% acetonitrile, and 0.1% TFA) was pipetted on sample loading patterns at room temperature and leave in 4oC refrigerator for solvent evaporation and cocrystallization (Scheme 2). MALDI-TOF MS. MALDI-TOF MS experiments were performed in a reflection mode on a 4800Plus MALDI TOF/TOF Analyzer (AB Sciex, U.S.A.) with the Nd:YAG laser at 355 nm, a repetition rate of 200 Hz and an acceleration voltage of 20 kV. MALDI-MS was directly performed on matrix deposited patterns of the smart MALDI plate. For each spectrum, 50 shots from different positions of the target spot were collected and analyzed. All data analysis was performed with a Data Explorer TM Software from AB Sciex (U.S.A.).

■RESULTS AND DISCUSSION Patterning of Superhydrophilic Array via Photo-click Chemistry. The ITO substrate with 7



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desirable roughness was fabricated via a layer-by-layer (LBL) assembly technique and subsequent calcination to enhance surface porosity (Scheme 1a).24,37 In contrast to the flat ITO substrate (Figure S1a), micrometer islands and clusters with high roughness and porosity were formed on ITO substrate after calcination (Figure 1a) while the ITO substrate remained transparent, which is important for the subsequent photo patterning process. Modification with alkyne terminus silane OTPC increased the surface contact angle from ~10o to ~68o for porous ITO substrate (Figure S1b,c). The superhydrophilic patterns were created through two times continuous thiol-yne “photo-click” reactions under photomask.31,32,38,39 Compared with common surface patterning methods, the facile “photo-click” reaction provides obvious advantages such as mild reaction conditions, free of toxic catalyst, short reaction period, patterning accuracy with millimeter-micrometer scale resolution, and the practicability for patterning in large areas. Two continuous photo-click reactions were performed on alkyne-surface to generate 1-thioglycerolsuperhydrophilic patterns with contact angle of ~5o in 1H,1H,2H,2H-perfluorodecanethiol superhydrophobic background with contact angle of ~144o (Figure

1b).

Compared

with

the

wettability

of

1-thioglycerol

(~34o)

and

1H,1H,2H,2H-perfluorodecanethiol (~112o) modified flat ITO substrate (Figure S2), the nanoscale roughness of calcinated ITO substrate effectively improved the intrinsic wettability of the surface modified molecules.32,40 Figure 1c showed the photograph of water droplets patterning on a 11 x 4 1-thioglycerol array with superhydrophobic surrounding regions. A 10 µL water droplet was well anchored within 1x1 mm 1-thioglycerolsquare spot (inset, Figure 1c), which is favorable for confining high volume sample solution in the small area and would benefit high-throughput analysis. PNIPAM Graft from Superhydrophilic Array. SI-ATRP has been used to graft polymer 8


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brushes on solid supports and yield polymers with high molecular weight, low polydispersity, and impressive capacity.41 Both hydroxyl groups of 1-thioglycerol from superhydrophilic patterns were converted to initiators via reacting with BIBB, and SI-ATRP was triggered subsequently in the superhydrophilic regions to fabricate thermo-responsive PNIPAM polymer patterns from porous ITO substrate (Scheme 1b). Surface plasmon resonance spectroscopy (SPR) was used to monitor PNIPAM growth in real time at room temperature and 60 oC respectively (Figure S3).To make the reaction condition comparable to porous ITO substrate, we fabricated a porous calcinated coating on Au substrate with similar method as on the ITO substrate. PNIPAM growth at room temperature was demonstrated in Figure S3a, the hydrophilic polymer took the coil configuration and well extended into aqueous solution. Water can easily penetrate into the hollow structure of stretched PNIPAM chain and resulted limited SPR resonance angle change (∆R) of 0.13 degree. In comparison, the hydrophobic PNIPAM polymer chain collapsed on surface at 60 oC, and the SPR resonance angle change was enhanced to 0.69 degree due to the polymer’s compact configuration and enhanced surface packing density (Figure S3b). There was no measurable SPR signal change at both temperatures in the absence of catalyst, demonstrated the specificity of polymer growth and resistance of nonspecific adsorption from the monomer NIPAM. XPS and FT-IR investigations were also carried out to characterize the grafted PNIPAM polymer patterns. The characteristic signals for silicon (Si2s at 155.1 eV, Si2p at 103.2 eV), oxygen (O1s at 533.1 eV, O2s at 23.1 eV), carbon (C1s at 285.0 eV), indium (In3d3/2 at444.5 eV, In3d5/2 at 451.7 eV) and stannum (Sn3d3/2 at 486.5 eV, Sn3d5/2 at 495.1 eV)42 were observed in the XPS wide scan spectrum of alkyne silane modified ITO substrate. Signals for In, Sn and O decreased after PNIPAM polymer grafting with the appearance of nitrogen peak (N1s at 398 eV) (Figure 2a), and the molar ratio of carbon:nitrogen is 6.26:1, which was close to the theoretical ratio of 6:1 for PNIPAM.42,43 Three peaks were discerned in the high 9



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resolution C1s spectrum of PNIPAM-grafted substrate: C-C/C-H (282.0 eV), N-C (283.2 eV) and N-C=O (284.9 eV) with the area ratio 4.6:1.1:1 (Figure 2b), which was close to the theoretical ratio of 4:1:1 of PNIPAM.43,44 Fourier transforms infrared (FT-IR) spectrum showed the characteristic bands at 3446 cm-1 for N-H, 2977 cm-1 for -CH3, 2926 cm-1 for -CH2, 1640 cm-1 for C=O stretching vibrations, and double bands at 1395 and 1385 cm-1 for symmetric deformation of isopropyl -CH3 (Figure S4), which was in accordance with the previous literature reports,45,46 confirming the successful growth of PNIPAM on the rough ITO substrate. Controlling Wettability of PNIPAM Array upon Temperature Change. The grafted PNIPAM brushes demonstrated hydrophilicity-hydrophobicity transition in response to temperature change, and showed significant morphologic variation between a chain stretching state and shrinking state at the LCST. SPR is very sensitive to surface reactions and adsorption process,47 and was used for the first time here for characterization of surface grafted PNIPAM configuration transition in real time upon temperature change. After PNIPAM polymer growth and PBS rinsing to get rid of nonspecific adsorption at 60 oC, SPR signal was observed to increase at first due to water refractive index increase with temperature decreased to room temperature, followed by a sustained drop due to the configuration transition of PNIPAM polymer from hydrophobic state to hydrophilic state. SPR signal decreased further during this process until PNIPAM polymer chain well extended into solution, which resulted about 0.16 degree of SPR resonance angle change compared with the initial state at room temperature (Black line, Figure 2c). The SPR resonance angle change of PNIPAM grown at 60oC with subsequent cooling to room temperature was very close to the SPR resonance angle change of PNIPAM grown at room temperature (0.13 degree, Figure S3a), demonstrating the complete transition of PNIPAM from hydrophobic state to hydrophilic state when cooling to room 10


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temperature. In the control experiment, SPR signal was increasing during temperature decrease and returned to the initial level at the room temperature in the absence of catalyst (Red line, Figure 2c). The conformation changes of surface grafted PNIPAM brushes at different temperatures were further confirmed by AFM and SEM after freeze-drying and heat-drying treatments respectively, which allow the thermosensitive brushes remain stretched or shrunk conformation on the dried surface.48 The freeze-drying PNIPAM demonstrated needle liked structure, and the heat-drying PNIPAM demonstrated mound liked structure, which confirmed the “stretching-shrinking” change of the thermoresponsive brushes (Figure 2d,e, Figure S5). The water droplet spread fast on hydrophilic PNIPAM at room temperature with the contact angle of ~18o. In comparison, ~54o of contact angle was achieved when pipetted a droplet of 60 oC water onto hot PNIPAM grafted substrate (Figure 2f). The appropriate hydrophobicity provided satisfactory control over protein/peptide adsorption/release, which not only assisted protein/peptide surface desalting and enrichment, but also benefited the formation of homogeneous crystals on top of the polymer layer for direct MALDI-MS analysis. To compare the capability of PNIPAM for target retention at different temperatures, SPR was used again to characterize the adsorption of cytochrome c and neurotensin on the PNIPAM grafted Au substrate at different temperatures. 1 mg/mL cytochrome c was well retained on hydrophobic PNIPAM polymer chain when incubated for 30 min at 60 oC, resulting in a 0.28 degree of angle change (Figure 3a). In comparison, same amount cytochrome c only generated 0.03 degree of SPR signal on hydrophilic PNIPAM at room temperature (Figure 3b), demonstrated the capability of complete release of captured target when PNIPAM extended to brush configuration. PNIPAM also demonstrated similar retention difference in response to temperature change for 1 mg/mL 11



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neurotensin, where 1 hour incubation was applied at 60 oC to ensure sufficient binding. SPR signal kept increasing during incubation and resulted in a 0.06 degree of resonance angle change (Figure 3c), much less compared to cytochrome c adsorption due to the higher hydrophilicity of neurotensin as well as its smaller molecular mass.49,50 In comparison, little signal change was observed for same concentration of neurotensin incubation on hydrophilic PNIPAM at room temperature (Figure 3d). Performance of Smart MALDI Plate for On-Plate Desalting and Enrichment. The smart MALDI plate was then applied to on-plate desalting and enrichment of target protein/peptide from complex biological samples using the “hydrophilic-hydrophobic” transition property upon temperature change. As it demonstrated in Scheme 2, the sample loading was performed at room temperature, and the sample droplets were confined and enriched on the hydrophilic PNIPAM patterns. With temperature increasing above the LCST, the target analytes were retained in hydrophobic PNIPAM while major contaminants and salts were selectively removed by hot water rinsing. MALDI matrix was added at room temperature, the PNIPAM polymer chain returned to hydrophilic and released the retained analytes to cocrystallize evenly with matrix. The desalting effect of PNIPAM patterns was first demonstrated with 10 ng cytochrome c and 10 ng neurotensin in the presence of 1M urea, and both cytochrome c M+ and M2+ peaks and neurotensin M+ peak were well recognized from the background in mass spectra after the surface desalting process (Figure 4a,c). In the control experiment, a layer of salt precipitate was formed after sample drying on conventional MALDI plate, which impeded the UV absorption/ionization process and thus reduced the target signals (Figure 4b,d).51 The desalting effect was also investigated with 10 ng cytochrome c (Figure S6a-d) and 10 ng neurotensin (Figure S7a-d) in the presence of 100 mM NaHCO3 and mixture of 100 mM NaHCO3 and 1 M urea solution respectively, and the performance were as good 12


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as that in 1 M urea. Buffers are usually present in biological samples to stabilize their molecular structure and maintain their activities.11 Desalting of cytochrome c (Figure S6e-j) and neurotensin (Figure S7e-j) in PBS, HEPES and Tris-HCl buffer were also performed respectively with the smart MALDI plate, which all demonstrated very obvious target peaks in mass spectra after the surface desalting processes. The protein extraction and desalting efficiency of the smart MALDI plate was further evaluated with 10 ng myoglobin in the presence of 100 mM NaHCO3 solution, which has similar molecular mass with cytochrome c but with higher hydrophobicity. Myoglobin M+ peak was also well recognized from the background in mass spectrum after desalting on smart MALDI plate (Figure S8a). In contrast, background noise occupied the mass spectrum with direct analysis on conventional MALDI plate. The scanning electronic microscope (SEM) was also used to directly characterize the surface features for the cocrystals of matrix CHCA and 500 mM NaCl contaminated cytochrome c on PNIPAM patterns before and after on-plate desalting (Figure S9). The salt crystals occupied the whole sample spot during the matrix drying and co-crystallization process with an obvious Na and Cl peak in EDX spectrum without on-plate desalting (Figure S9a,c), while the pellet-like CHCA-protein cocrystals were formed after on-plate desalting with little Na and Cl peak observed in EDX spectrum (Figure S9b,d). The intensities of Si peak remain constant before and after hot water rinsing, while C, O peaks increased due to the contribution of cytochrome c, which is in accordance with the previous report.27 Compared with other on-plate desalting techniques relied on amphiprotic block copolymer26 and hydrophobic-hydrophilic interval concentric circles,27 PNIPAM patterned substrate has provided controllable surface wettability, which not only simplified the fabrication process, but also eliminated target loss by protecting targets in condensed polymer chain during hot water rinsing step. It also generated effective matrix co-crystallization by avoiding the trap of targets in hydrophobic polymer brushes during matrix addition step. The rinsing solution 13



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were collected during the hot water rinsing process for 10 ng cytochrome c and 10 ng neurotensin samples in presence of 1 M urea, and demonstrated little cytochrome c nor neurotensin peak in the MALDI mass spectra (Figure S10). The UV-Vis absorption spectra were measured for cytochrome c and neurotensin loading samples and rinsing solutions collected from the smart MALDI plate to further demonstrate the high loading capacity of PNIPAM patterned substrate. The characteristic absorption peak at 280 nm indicated the existence of protein and peptide in sample loading solutions, which were barely observed for all the rinsing solutions (Figure S11a,b), demonstrating almost all the protein and peptide targets were extracted from loading solutions to the PNIPAM spots. In comparison,

same

amounts

of

cytochrome

c

and

neurotensin

were

loaded

on

1H,1H,2H,2H-Perfluorodecanethiol modified ITO substrate, and 280 nm absorption peaks were clearly observed from the 1st and 2nd times rinsing solutions, indicating sample loss to some extents from a non-thermo-responsive hydrophobic 2D surface (Figure S11c,d). α-casein was then introduced as the internal standard for quantifying the amount of cytochrome c retained on PNIPAM patterns after on-plate pretreatment precess.52 1 mg/mL α-casein was added into 1 mg/mL of cytochrome c aqueous solution in the absence of salt as standard solution. The mixture solution was co-crystallized with CHCA on smart MALDI plate, and the internal standard/analyte (i/a) ratios was determined as 1.70 by comparing the intensities of internal standard peak [α-casein]2+ at m/z 11866 and analyte peak [cyt-c]+ at m/z 12320 (Figure S12a). After on plate desalting for 100 mM NaHCO3 contaminated cytochrome c, 1 mg/mL α-casein was mixed with CHCA and added on cytochrome c loaded PNIPAM spot for co-crystallization with CHCA and MALDI-MS analysis, the i/a ratio became 1.54 (Figure S12b), which was close to the i/a ratio of standard solution and demonstrated little target lost during hot water rinsing step, as well as the complete release of retained sample during matrix addition and co-crystallization steps. 14


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Protein identification in various biological samples is an important component of MS-based proteomics, which relies on the mass spectra of digested peptides and the interpretation of the MS data using database-supported search engines.53 However, a complete detection of low-abundance protein digests is challenging due to the interference from the introduced contaminants during digestion.54 Commercial

desalting

pretreatment

kits

normally

relied

on

reverse-phase

chromatography materials, such as ZipTip (Millipore, Bedfold, MA), HyperSep (ThermoScientific, Rockford, IL), Protein chip arrays (BIO_RAD H50, H4, and SEND ID),which require hundred times pipetting during operation process including tip precondition, sample binding, sample washing and sample release. The performance of smart MALDI plate was compared with C18 HyperSep tip for protein digests desalting with different amounts of α-casein and cytochrome c digests, and their sequence and positions were summarized in Table S1 and Table S2. As it demonstrated in Figure 5, abundant matrix ions were yielded at lower molecular mass range for C18 tip desalting method, while clean spectra for peptides were achieved with improved signal-to-noise (S/N) ratio for smart MALDI plate desalting method for α-casein digests. 20 peptides were directly detected in the MS spectrum for 4 pmol α-casein digests after desalting on smart MALDI plate (Figure 5a), while only 7 peptides were detected from same amount α-casein digests after C18 tip desalting (Figure 5b). The advantages of smart MALDI plate over C18 tip is obvious with the amount of α-casein continued to decrease due to the remarkable sample loss during multiple pipetting for low abundant targets.24 There were only 5 peptide peaks identified from 400 fmol α-casein digest sample after C18 tip desalting (Figure 5d), and only matrix cluster in lower molecular mass range was recognized for 40 fmol α-casein digest (Figure 5f). In comparison, 8 peptides were recognized from 400 fmol α-casein digested sample (Figure 5c), and the signal-to-noise ratio for the five peptide peaks identified in Figure 5d were determined as 67.1 (m/z: 971.5), 38.1 (m/z: 1267.7), 28.4 (m/z: 1386.6), 261.5 (m/z: 15



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1759.9), and 60.0 (m/z: 1952.0), which were 2.2, 2.2, 3.3, 8.8 and 8.3 times respectively compared with C18 tip desalting method. When α-casein amount continued decrease to 40 fmol, peptide peaks at m/z 1267.7, 1386.6, and 1759.9 were still identified for PNIPAM pattern on smart MALDI plate (Triangle labeled peak, Figure 5e) with some polymer background interference (Star labeled peak, Figure 5e). The desalting efficiency of the smart MALDI plate was further demonstrated by the analysis of a series of cytochrome c digests (Figure 6). 11 (Figure 6a) and 8 peptide peaks (Figure 6c) were clearly identified respectively from MS spectra of 4pmol and 400 fmol cytochrome c digests after desalting on smart MALDI plate, while only 7 (Figure 6b) and 5 peptides peaks (Figure 6d) were identified respectively with much less signal intensities for same amount cytochrome c digests after C18 tip desalting. The signal-to-noise ratio for peptide peaks at m/z 955.4, 1065.4, 1193.4, 1456.5, and 1584.6 in Figure 6c were determined as 199.4, 23.2,78.2, 119.1, and 93.1 respectively, which were 7.1, 1.5, 3.4, 6.2, and 11.6 times higher compared with the corresponding peaks with C18 tip desalting method (Figure 6d). When the amount of cytochrome c decreased to 40 fmol, no assignable signal could be observed with C18 tip desalting method (Figure 6f), while 6 peptides could be clearly detected using the smart plate desalting method (Figure 6e).The smart MALDI plate provided higher mass spectra quality with amino acid sequence coverage score of 67.3% for cytochrome c digest, which was much better than C18 tip desalting method (36.5%) and comparable to the previously reported desalting methods based on magnetic nanomaterial with off-line votex assist mixture and centrifugation assist separation before MALDI analysis.55,56 The smart MALDI plate was also used for the analysis of 150 fmol bovine serum albumin (BSA) digests. There were 37 peptides peaks identified from MS spectra with amino acid sequence coverage score of 55% (Figure 16


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S13) (amino acid sequences and positions summarized in Table S3), which demonstrated better performance compared with the non-thermo-responsive 2D material patterned commercial MALDI plate57 due to the improved loading capacity of 3D PNIPAM matrix. Reduction and alkylation of proteins are sometimes required prior to proteolysis, therefore the practicability of the smart MALDI plate was further verified with tryptic digested 4 pmol cytochrome c in presence of DL-dithiothreitol (DTT) and iodoacetamide (IAA). All the merely tryptic digested peptide peaks (Figure 6a) were clearly identified in the mass spectrum with comparable signal intensities (Figure S11), and two new groups of peptide peaks were newly appeared at 1267.0 and 1761.9 with 58 molecular mass gap inside each group (Cross labeled peak, Figure S14), indicating the alkylation of cystein and histidine residues in their respective amino acids sequences. This result demonstrated the practicability of smart MALDI plate in more complicated protein digest conditions, indicating their potential application in peptide mass finger printing. The spot-to-spot reproducibility of smart MALDI plate for surface desalting and enrichment was investigated with 10 ng neurotensin in 1 M urea and 10 random spots. The relative standard deviations (RSDs) of ion intensity for the peptide peak was 12.64% (n = 10), demonstrating a good spot-to-spot reproducibility of the smart MALDI plate. The long term stability of smart MALDI plate was also verified via comparing its desalting effect for 10 ng neurotensin in 1 M urea solution at different times, which barely demonstrated difference in target signal intensities for freshly fabricated smart MALDI plate and those stored in desiccator at room temperature for three days, five days, and one week (Figure S15), indicating the satisfactory storage stability of the smart MALDI plate.

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In addition to on-plate desalting, smart MALDI plate showed enrichment effect for low abundance targets with the unique configuration of wettability controllable spots surrounded by hydrophobic regions. 10 µL water droplets could be confined in a 1x1 mm square spot and patterned in a 11x4 array on PNIPAM smart plate at room temperature (Figure S16). Though the hydrophilicity of the PNIPAM pattern areas decreased slightly compared with 1-thioglycerolpatterns (Figure 1c), the patterned PNIPAM array still demonstrated marked enrichment effect. When the loading volume expanded to 10 µL on 1x1 mm PNIPAM spot, the sample “stain” size was still comparable to that from typical loading volume of 1 µL (Figure 7a,b). The large volume enrichment effect on PNIPAM spots was further investigated using cytochrome c digests with 40 nM and 400 nM. Increasing sample loading volume per unit area directly on substrate has the same effect as increasing sample concentration for MALDI analysis, and simplifies the operation process as well as avoids contamination by eliminating sample pretreatment step. 8 peptide peaks were identified with similar ion intensities on mass spectra for both 400 nM cytochrome c digests with 1µL loading volume (400 fmol) (Figure 7c) and 40 nM cytochrome c digests with 10 µL loading volume (400 fmol) (Figure 7d), indicating the on-plate sample enrichment with smart MALDI plate was an effective approach to preconcentrate low-abundant analytes. The enrichment efficiency of smart MALDI plate was further compared with traditional MALDI plate with the analysis of 10 µL 80 fmol/µL cytochrome c and 10 µL 60 fmol/µL neurotensin. Both the protein and peptide peaks could be detected with stronger signals on smart MALDI plate. The S/N ratios for [cytochrome c]2+ signal and [neurotensin]+ signal were 387.7 and 307.8 respectively (Figure S17a, c), which were about 12.1-fold and 8.1-fold enhanced compared with traditional MALDI plate (S/N ratio of 32.1 and 37.8 for [cytochrome c]2+ signal and [neurotensin]+ signal respectively) (Figure S17b,d). Serial dilution tests demonstrated that the detection limits for cytochrome c and neurotensin were 8 amol/µL and 60 18


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amol/µL respectively for smart MALDI plate, while there was no signal detected with same concentration cytochrome c and neurotensin from traditional MALDI plate (Figure S18). The hydrophilic pattern with hydrophobic surroundings configuration of smart MALDI plate as well as the high loading capacity of PNIPAM 3D matrix contributed to its higher enrichment efficiency for the analysis of low abundance proteins/peptides.

■CONCLUSION In summary, we have demonstrated a smart, high performing MALDI plate as a highly effective platform for sample pretreatment before mass spectrometry. Benefiting from the thiol-yne photo-click reactions, well defined and precise hydrophilic patterns can be directly generated on the rough ITO substrate with hydrophobic surrounding regions. The thermoresponsive PNIPAM patterns are grafted from the hydrophilic patterns via SI-ATRP, which offers good control over surface wettability and corresponding functions in response to temperature change. The fabrication process is robust and straightforward, completed in mild condition within 1 hour. We have tested the performance of smart MALDI plate for on-plate desalting and enrichment with salt contaminated peptide neurotensin, protein cytochrome c, myoglobin, and α-casein and cytochrome c digests, which generate truly compelling results with efficient operation process. We believe the smart MALDI plate will open new avenues for on-plate pretreatment for various applications, and have great potential in proteomic research.

■ASSOCIATED CONTENT Supporting Information Available: 19



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Detailed characterization of LBL calcinated porous surface, contact angle measurements of modified ITO substrate, characterization PNIPAM growth, PNIPAM grafting on the ITO slide, characterization of on-plate desalting, sample enrichment, and MALDI-MS analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Authors *Phone/Fax:+86-25-89681918. E-mail: [email protected]. *Phone/Fax: 951-215-8702. E-mail: [email protected] Notes The authors declare no competing financial interest.

■ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (21605083, 21635005), Natural Science Foundation of Jiangsu Province (BK20160644), and the National Research Foundation for Thousand Youth Talents Plan of China. QC acknowledges financial support from the National Science Foundation (CHE-1413449).

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Scheme 1. (A)The fabrication of superhydrophilic patterns with surrounding superhydrophobic regions on porous ITO substrate viaphoto-click reactions, and (B) PNIPAM growth from hydrophilicpatterns via SI-ATRP reaction.

Figure 1.(A)SEM image of porousITO substrate.(B) Water droplet on a 1-thioglycerol modified surface (top) and 1H,1H,2H,2H-perfluorodecanethiol modified surface (bottom), and(C) Photograph of water droplets confining in a 11 x 4 superhydrophilic array patterned via repeated photo-click reactions.Inset: A zoomed photograph of a 10 µL droplet on a square pattern with 1 mm on each side. Figure 2.(A) XPS survey spectra of silanized ITO substrate (blue) and PNIPAM grafted ITO substrate (red). (B) High resolution C1s spectrum of PNIPAM grafted ITO substrate with fitted curve components. (C) SPR sensorgram of PNIPAM polymer configuration changes in response to temperature (black) and control experiment in the absence of catalyst (red). Atomic force microscope 3D images of PNIPAM pattern in waterat (D) room temperature and (E) 60 oC, and (F) Water droplet on PNIPAM pattern at room temperature (Top) and 60 oC (Bottom).

Figure 3.SPR sensorgrams for the adsorption of (A) cytochrome c and (C) neurotensin on PNIPAM at 60 oC and (B) cytochrome c and (D) neurotensin at room temperature.

Scheme 2.Schematic illustration of on-plate desalting and peptide enrichment based on the wettability change of PNIPAM patterns on rough ITO substrate at different temperature.

Figure 4.Mass spectra of 10 ng cytochrome c in the presence of 1 M urea solution (A) after on-plate 28


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desalting on smart MALDI plate and (B) on conventional MALDI plate; and 10 ng neurotensin in the presence of 1 M urea solution (C) after on-plate desalting on smart MALDI plate and (D) on conventional MALDI plate.

Figure 5.Mass spectra of a series of α-casein digests with smart MALDI plate desalting and C18 HyperSep tip desalting: 4 pmol digest with (A) smart MALDI plate desalting and (B) C18 HyperSep tip desalting; 400 fmol digest with (C) smart MALDI plate desalting and (D) C18 HyperSep tip desalting; and 40 fmoldigest with (E) smart MALDI plate desalting and (F) C18 HyperSep tip desalting. Triangles represent peptide peaks from α-casein digest, circles represent peptide peaks from β-casein digest, and asterisks represent interference from polymer background. Figure 6.Mass spectra of a series amount of cytochrome c digest with smart MALDI plate desalting and C18 HyperSep tip desalting:4 pmol digest with (A) smart MALDI plate and (B) C18 HyperSep tip desalting; 400 fmol digest with (C) smart MALDI plate desalting and (D) C18 HyperSep tip desalting; and 40 fmoldigest with (E) smart MALDI plate desalting and (F) C18 HyperSep tip desalting. Triangles represent peptide peaks from cytochrome c digest. Figure 7.Photographs of droplets with 1 µL 400 nM cytochrome c digest (left) and 10 µL 40 nM cytochrome c digest (right) sample solution on 1x1 mm PNIPAM spots at smart MALDI plate (A) before and (B) after evaporation, and the corresponding mass spectrafor the (C) left droplet, and (D) right droplet.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Scheme 2

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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For Table of ContentsOnly

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Smart MALDI plate for protein digest enrichment, desalting and dire

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