Enhanced on-plate digestion of proteins using a MALDI-digestion chamber

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Enhanced on-plate digestion of proteins using a MALDI-digestion chamber Michael Rühl a , Vahid Golghalyani a , Günes Barka b , Ute Bahr a , Michael Karas a,∗ a b

Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, DE-60438, Germany SunChrom Wissenschaftliche Geräte GmbH, Industriestraße 27, Friedrichsdorf, DE-61381, Germany

a r t i c l e

i n f o

Article history: Received 24 June 2016 Received in revised form 4 October 2016 Accepted 10 November 2016 Available online xxx Keywords: On-plate digestion On-plate reduction and alkylation Digestion chamber Temperature and humidity control MALDI-MS

a b s t r a c t A commercially available digestion chamber (SunDigest, SunChrom) is used for on-target digestion of proteins for subsequent direct MALDI-MS analysis. Temperature and humidity in the chamber can be controlled and kept constant during the reactions. For the first time, not only the tryptic digestion but also reduction and alkylation were performed in only one ␮L of sample directly on the MALDI sample plate. Each reaction step was optimized with regard to reactant concentration, temperature and time. Under optimized conditions a significant increase in sequence coverage is obtained. Proteins such as BSA and Apo-transferrin give best results when all reaction steps are performed at 50 ◦ C, which results in a considerably reduced total time of 65 min compared to in-solution digests. Moreover, the on-target protocol is not only faster, but also superior with respect to the required protein amount; at the tested lower limit for PMF-analysis of 100 fmol the on-target procedure exhibits strongly superior sequence coverage and identification scores. Using lower protein amounts (10–50 fmol) an identification with MS/MS-analysis is possible. This newly developed protocol combines the benefits of the programmable SunDigest humidity chamber with a time-saving on-plate reduction, alkylation and digestion workflow for protein identification. © 2016 Published by Elsevier B.V.

1. Introduction The fast identification and characterization of small amounts of protein samples is a central task for mass spectrometry in bioanalysis and proteomics, protein identification of sub-␮g to ng of protein even in complex mixtures is nowadays routine. The method of choice in protein analysis and proteomic workflows is the proteolysis with trypsin. Prior to tryptic digestion several sample preparation steps like denaturation, reduction and alkylation are commonly performed. These steps are often important to unfold the protein for a higher accessibility of the protease during the digestion process [1]. Since the whole procedure is time consuming and elaborate, several accelerating methods for in-solution digestion of proteins have been reported in recent years which are either based on temperature effects (heating, IR or microwave assisted), ultrasonic energy, immobilized digestion (filter aided sample preparation, immobilized trypsin on a solid support) or sol-

∗ Corresponding author at: Max-von-Laue-Straße 9 60438 Frankfurt, Germany. E-mail addresses: [email protected] (M. Rühl), [email protected] (V. Golghalyani), [email protected] (G. Barka), [email protected] (U. Bahr), [email protected] (M. Karas).

vent effects and have been comprehensively reviewed and critically evaluated recently[2]. Using MALDI-MS for gel-free proteome analysis an acceleration of digestion can also be reached by on-plate digestion directly before mass analysis. Moreover, in this approach sample losses during sample transfer is avoided and only minimal amounts of sample are required. For the on-plate digestion, ␮L-amounts of proteins are deposited on the MALDI-target and directly mixed with trypsin for accelerated protein identification [3,4]. The combination of protein-liquid chromatography and on-plate digestion of the collected fractions has successfully been applied to the analysis of complex mixtures [5–7]. Since digestion on a MALDI target is regarded as less efficient and less reproducible compared to insolution digestion [8], different auxiliary means to support the digestion have been described such as infrared radiation [9], application of alternating current [10], sonication [11] or modification of the target by graphite [12]. Immobilization of trypsin on different supports for on-target digestion is reported to minimize digestion time due to the tolerance of higher enzyme-to-protein ratios and to avoid autolysis of the enzyme. The use of trypsin directly immobilized onto the MALDI target was already reported in 1990 [13–15]. Later, agarose beads [16], magnetic nanospheres [17] and graphene oxide [18,19] served as support for covalently bound enzymes.

http://dx.doi.org/10.1016/j.ijms.2016.11.011 1387-3806/© 2016 Published by Elsevier B.V.

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Reduction and alkylation directly on the target was reported to give only poor results [11] and in consequence these steps are omitted or performed separately before applying the sample onto the target. None of the published methods combines the digestion enhancement strategy with reduction and alkylation. Reported numbers for the protein amount required for significant results in peptide mass fingerprint analysis exhibit an extreme spread between 1 ng [19] and 600 ng on-plate [6] for the standard protein bovine serum albumin, without performing reduction and alkylation steps prior to the digestion. Turiák et al. [20] showed that a concentration by evaporation of the solvent leads to a decrease of the signal intensities in the subsequent mass spectrometric analysis, thus favoring the approach of an on-target procedure using a minimal volume in order to minimize losses and improve the limit of detection. The most important technical parameters for a successful and reproducible on-plate digestion are temperature and humidity. In almost all published data for on-plate digestion experiments selfbuilt or not well-characterized humidity chambers are used to keep the deposited droplets stable during reaction for digestion temperatures which vary from room temperature to 50 ◦ C. In this work, we use a chamber with an automated temperature and humidity control for MALDI-MS targets, which was developed for direct on-tissue digestion in MALDI-imaging approaches (SunDigest, SunChrom) [21]. We will show that under controlled conditions low amounts of protein can be reduced, alkylated and digested in short time yielding sequence information comparable or superior to more elaborated standard procedures. 2. Material and methods 2.1. Reagents Bovine serum albumin (BSA), human Apo-transferrin, bovine insulin, bovine oxidized insulin B chain, trypsin, trifluoroacetic-acid (TFA), acetonitrile (ACN), hydrochloric acid (HCl), ammonium bicarbonate (Ambic), dithiothreitol (DTT), octyl-␤-d-glucopyranoside and iodoacetamide (IAA) were purchased by Sigma Aldrich (Munich, Germany). The MALDI matrix alpha-cyano-4-hydroxy-cinnamic-acid (CHCA) was obtained from Bruker Daltonics (Bremen, Germany). Water was prepared with an in-house Purelab Ultra Genetic water purification system (ELGA, Celle, Germany). BSA and human Apo-transferrin were freshly prepared in 25 mM Ambic buffer at concentrations of 1 pmol/␮L, 500 fmol/␮L and 100 fmol/␮L. Bovine insulin and bovine oxidized insulin B chain (internal standard) were prepared in a 1 mM HCl solution and then freshly diluted with water to a concentration of 10 pmol/␮L. Trypsin was prepared with 1 mM HCl solution and then diluted with 25 mM Ambic to a final concentration of 6.75 ng/␮L. DTT and IAA were prepared at different concentrations between 0.1–30 ␮g/␮L in water. Octyl-␤-d-glucopyranoside was prepared as a 10% (w/v) stock-solution in water and then diluted to 0.1% for target coating. CHCA was freshly prepared as a 3 ␮g/␮L solution in 70% ACN, 29.9% water and 0.1% TFA. 2.2. Digestion chamber For sample pretreatment and digestion, a SunDigest system (SunChrom, Friedrichsdorf, Germany) was used. The digestion chamber (Fig. 1a) allows the heating of the sample plate holder (base temperature) as well as the cover plate (cover temperature) between 4 and 50 ◦ C. Pieces of felt below the sample supports (glass slides) are soaked with water before the chamber is closed by the cover. Two fans are installed to provide a uniform environment.

Sensors for humidity and temperature (inner temperature) are located in the cover and in the heating plate. Heating and humidity are regulated and controlled by the SunDigest 1.18 Software. The software either regulates the humidity and the inner temperature by changing the parameters base and cover temperature and the fan speed automatically. For our purposes, the SunChrom glass slide sample plate holders on the heating plate were replaced by an in-house manufactured MALDI target holder (Fig. 1b). A mat of felt cut in the size of the heating-plate was placed onto the SunDigest heating-plate. (4 mm thick felt was obtained from the JP Stoff Export (Mörfelden-Walldorf, Germany). A plastic spacer of about 1 cm was placed on top of the felt to prevent direct contact of the MALDI-target with the wet felt. Before each run, the felt was fully soaked with distilled water. After each run, condensed water was carefully removed from the cover, the area around the humidity sensor and the area around the fans. Time, temperature and humidity were edited in the so called SMART mode of the software for each step of the sample preparation and the digestion. All temperature and humidity adjustment cycles were edited for 5 min. Using the cycle restriction function of the software, the total preparation or digestion time could be set by repeating each cycle as often as intended. The cooldown parameters could be set in the cooldown window of the SunDigest software. The readouts of each parameter were stored as txt-files. 2.3. On-plate sample pretreatment and digestion Octyl-␤-d-glucopyranoside was used as detergent for enhancement of trypsin- digestion efficiency and signal intensities in subsequent MALDI-MS [22,23]. 1 ␮L of a 0.1% (w/v) Octyl-␤d-glucopyranoside-solution was deposited on each spot and dispensed to cover the whole spot area and dried. Unless otherwise mentioned in the text, all reactions were performed with protein concentrations of 1 pmol/␮L according to a standard protocol which comprised following steps: reduction at 50 ◦ C for 60 min, alkylation at 37 ◦ C for 60 min and digestion at 37 ◦ C for 60 min. Reduction: 1 ␮L of each DTT-solution (0.1, 0.2, 1, 2, 10, 20 ␮g/␮L) was mixed with the protein-solution directly on the MALDI-target before the target was placed into the SunDigest chamber and the cover closed. Following parameters were adjusted in the SunDigest program: base temperature: 50 ◦ C, cover temperature: 46 ◦ C, fan speed 0%. The resulting inner temperature and humidity were recorded. Alkylation: Directly after reduction the chamber was opened and the target removed from the heating plate. The SunDigest cooled down to alkylation temperature. 1 ␮L of each IAA-solutions (0.15, 0.3, 1.5, 3, 15, 30 ␮g/␮L) was added to the reduced protein solutions directly on the MALDI-target and the target was replaced into the SunDigest. The parameters for alkylation were adjusted in the remote program as follows: base temperature: 36 ◦ C, cover temperature: 33 ◦ C, fan speed 0%. Digestion: Again the digestion chamber was opened and the target removed from the heating plate. 2 ␮L Trypsin-solution with a concentration of 6.75 ng/␮L was immediately mixed with the protein and peptide solutions directly on the MALDI-target before it was transferred to the SunDigest. The settings were the same as for alkylation. Reduction, alkylation and digestion times were set using the cycle restriction function. Prior to MS-measurement the resulting peptides and reaction products were mixed with 0.5 ␮L of the matrix-solution. 2.4. Sample preparation and digestion in-solution The proteins were diluted in water or 25 mM Ambic solution to different concentrations. DTT was added to the protein solution

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Fig. 1. SunDigest device. (a) standard set-up for MALDI-imaging, using glass slides for tissue analysis. (b) modified device for the on-plate digestion.

and kept at 57 ◦ C for 1 h. After the reduction step IAA was added. After reduction and alkylation, trypsin solution was added to the mixture and kept at 37 ◦ C for 1 h and 18 h, respectively. All reactions were performed in a total volume of 5 ␮L [8]. The resulting peptide solution was mixed with 0.5 ␮L of the matrix-solution for MS analysis. This use of the whole sample volume is facilitated by preconditioning the metal target with the detergent solution. 2.5. MS-measurement and data analysis All peptide spectra were acquired with an LTQ-Orbitrap XL combined with a MALDI-Duo system (Thermo Fisher, Bremen, Germany). MALDI targets were Thermo Fisher 394-well plates for the MALDI-Duo system. All measurements were performed as triplicates on different spots under otherwise identical conditions. For each spectrum thirty spectra were accumulated with the mass resolution set to 30,000 from m/z 800–4000. For the acquisition of the mass spectra the crystal positioning system (survey CPS) was turned on and the laser energy was set to 10–12 ␮J. Laser energy was adjusted for every run to achieve the best signal intensities. The spectra were recorded automatically using the XcaliburTM software (Thermo Fisher, Bremen, Germany) for the MALDI-Orbitrap. All spectra were acquired as RAW files. The spectra were signal

processed by mMass integrated into the KNIME analytics platform. Generated peak lists were submitted to Mascot [24] concurrently to perform a PMF search. The in-house KNIME workflow used several self-written and open-source programs for data processing and analysis [25,26]. The resulted peptide or protein lists were exported to excel for further data analysis. For database search up to one missed tryptic cleavage was allowed, carbamidomethylation on cysteine-residues was considered as a fixed modification and oxidation on methionine residues was considered as a variable modification. The mass tolerance for peptide mass fingerprinting analysis was set to 15 ppm. MODDE software was used for creating full factorial experimental design [27] and the data-analysis. 3. Results and discussion 3.1. Humidity and temperature adjustment The SunDigest system, which had been designed for the direct on-tissue digestion of proteins for MALDI-imaging, was modified for on-plate in-solution digestion. For both purposes constant humidity and temperature are important to assure significant and reproducible results, however, for ␮L droplets on MALDI metal targets the conditions to prevent drying or spreading are more critical

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Fig. 2. Temperature and humidity time curves for the imaging and the modified digestion chamber. Temperature gradients for 37 ◦ C (a) and 50 ◦ C (b). Humidity curves for 37 ◦ C (c) and for 50 ◦ C (d). gray lines show the modified digestion chamber values 䊏 black lines show the imaging digestion chamber values.

than for tiny droplets on biological thin sections. The computer controlled system adjusts and reads both parameters with a frequency of 5 Hz. Time to reach constant temperature values of 37 ◦ C and 50 ◦ C, respectively and high air moisture was measured in the modified and unmodified chamber. Both fans are switched off to avoid an accelerated shrinking of the droplets. Under these conditions volumes of 2 ␮L (1 ␮L of sample and 1 ␮L of reagent) stay stable up to 3 h (37 ◦ C) and 1.5 h (50 ◦ C), respectively. Fig. 2 shows the temperature and humidity gradients over 15 min. As start points for temperature adjustment a value above room temperature was chosen to avoid condensation of water on the metal sample plate. The temperature settings of the modified chamber are comparable to the MALDI-imaging device. In both cases the desired values were reached after about 8 min and stayed constant over the remaining time. The standard deviation after 8 min determined from three measurements was less than 1%. Comparing the measured relative humidity, the modified chamber reaches 100% at 37 ◦ C already after 2 min and at 50 ◦ C after 4–5 min while under standard imaging conditions saturation is only attained after 8 min independent of the temperature. The fast adjustment of the humidity is more important for on-plate digestion than for on-tissue digestion, presumably because tissue samples provide a humid surface and do not dry as fast as small volumes on a stainless steel surface. Obviously, the larger wet felt area in the modified chamber is responsible for faster humidity adjustment. In summary, the modified device shows a significantly faster humidity adjustment, a constant humidity and temperature over time and leads to stable droplets over the entire reaction process and is therefore favorable for on-plate digestion.

3.2. On-target reaction 3.2.1. Reduction and alkylation In a first experiment, the extent of reduction and alkylation in dependence of the concentrations of DTT and IAA were analyzed while keeping temperature and reaction time constant. Bovine insulin was chosen as model protein because it possesses two disulfide bridges and dissociates after reduction into the insulin A and B chains which masses can be analyzed directly in the MALDI Orbitrap without the necessity of proteolysis. DTT-concentrations between 0.1 and 20 ␮g/␮L were tested for insulin by following the formation of the insulin B chain on-target and in-solution (volume of 20 ␮L). Since the mass of the intact insulin is outside the accessible mass range of the Orbitrap, oxidized insulin B was used as an internal standard for a relative quantification. Fig. 3 shows the intensity (peak height) ratio of the protonated molecules of insulin B (m/z = 3398.7) to the oxidized form as internal standard (m/z = 3494.6) with a constant concentration. At low DTT concentrations the amount of the reduction product increases for both on-target and in-solution reduction. The differences between both methods are not significant (p < 0.05). The higher standard deviation of the on-plate digestion is probably due to the higher volume error when pipetting 1 ␮L compared 10 ␮L. Above 10 ␮g/␮L DTT disturbs the crystallization of the matrix/analyte mixture and apparently reduces the ratio because of the lower spectra quality. Although 10 ␮g/␮L DTT gives higher reduction yields, we chose the concentration of 2 ␮g/␮L DTT for all further experiments for two reasons. Firstly, 2 ␮g/␮L gives sufficient reduction performance under the given conditions and is

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Fig. 3. Reduction of insulin in dependence of the DTT concentration. The intensity ratios of the reduction product chain B to the oxidized insulin B as internal standard are plotted for in-solution reduction (gray line) and on-plate reduction (black line) 䊏.

also the recommended concentration in most elaborated proteomic protocols. Secondly, the addition of further reagents for alkylation and digestion induces problems with matrix crystallization and decreases the trypsin activity and thus the digestion results. The extent of alkylation of insulin with IAA was measured after reduction of insulin with 2 ␮g DTT and then mixing the formed insulin chains with an IAA serial dilution from 0.15 to 30 ␮g/␮L. The resulting spectra showed masses for none, single or double alkylation. Fig. 4 shows the extent of alkylation for the different IAA concentration for the reactions in solution in a total volume of 30 ␮L and on-target. The alkylation extent was calculated using the following formula: Alkylation extent (%) =

Intensity doubly alkylated + Intensity singly alkylated ∗ 100 Intensity doubly + Intensity singly , alkylated + Intensity unalkylated

For IAA concentrations of 3 ␮g/␮L and higher the alkylation extent is close to 100%. In comparison to on-plate alkylation the reaction in solution shows no significant difference (p < 0.05). The higher standard deviation of the on-plate method is mainly due to inaccuracies in pipetting a volume of 1 ␮L. In a second experiment, reduction and alkylation were repeated for the standard protein BSA which Exhibits 17 disulfide bridges. A digestion with trypsin followed and the sequence coverage as well as the number of matched peptides was used for evaluation. First of all, the protein was reduced with varying DTT concentrations and subsequently alkylated with a constant concentration of 3 ␮g/␮L IAA and then digested with trypsin with an enzyme to protein ratio of 1:5 (w/w), respectively 1:2 (mol/mol) directly on the MALDI sample plate. In a second experiment the protein was reduced with the standard concentration of 2 ␮g/␮L DTT and a serial dilution of IAA was used for alkylation. The modified protein was than digested with trypsin directly on the MALDI sample plate. The resulting sequence coverage was plotted against the DTT or IAA concentration (see Supplementary data). The results confirm our findings, that there is an optimal concentration for on-plate sample preparation at about 2–3 ␮g/␮L for both reagents. Higher concentrations of the reduction or alkylation reagents deteriorate the crystallization of the sample spots and lead to a decrease of the number of matched peptides.

3.2.2. Influence of reduction and alkylation on sequence coverage To explore the influence of reduction and alkylation for ontarget digestion of proteins on the sequence coverage we compared differently prepared protein samples. The samples on the MALDI plate were either digested unmodified without reduction and alkylation, after reduction, after reduction and alkylation and compared to the 18 h in- solution protein digest after reduction and alkylation. Fig. 5 shows the results for the standard proteins BSA and Apo-transferrin (19 disulfide bridges). The sequence coverage for unmodified proteins (48% for BSA and 40% for Apo-transferrin, respectively) was considerably lower. Reduction and alkylation enhances the sequence coverage by 25–30%. This can be easily explained by the fact, that the reduction process with DTT denatures the protein and which is than is more accessible for the protease. One hour of digestion on the MALDI target yields comparable sequence coverage for BSA than the 18 h standard in-solution digestion. For Apo-transferrin the 18 h in-solution digestion shows decreased sequence coverage in comparison to the on-target reaction probably due to unspecific cleavage of the tryptic peptides. In many publications focusing on on-target digestion of proteins, it was decided to omit reduction and alkylation to speed up analysis [6,10–12] or, to perform these initial steps in solution with higher protein amounts and larger volumes [5,9,18]. Our findings are in accordance with the general finding that a considerable increase in matched peptides and sequence coverage is observed after reduction and alkylation. However, both reactions are now carried out directly on the target which is strongly preferable, since sample volumes, sample losses and reaction times are minimized. 3.3. Digestion with trypsin 3.3.1. Optimization of duration and temperature for reduction, alkylation and digestion For the optimization of the digestion of BSA and Apo-transferrin, the reduction, alkylation and digestion times were varied as well as the reaction temperatures by using a full-factorial screening experiment designed by MODDE (please refer to Supplementary information for details). The sequence coverage was used to evaluate the effect of time and temperature on the on-plate digestion. As a result of the full-factorial screening experiment, 9 different protocols including the standard on-plate digestion protocol used in

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Fig. 4. Alkylation of insulin B in dependence of the IAA concentration. Alkylation extent for in-solution (gray line)

and on-plate alkylation (black line) 䊏.

Fig. 5. Influence of reduction and alkylation for on-plate digestion on the sequence coverage. For two standard proteins, bovine serum albumin and apo-transferrin the influence of reduction and alkylation on the sequence coverage is shown and compared to an 18 h in-solution digestion * after reduction and alkylation.

the sections before were tested. The results from reduction times of 30 and 60 min, alkylation times of 5, 30 and 60 min and digestion times of 30 and 60 min, all steps performed at 37 ◦ C or 50 ◦ C, respectively are shown in Fig. 6. Under standard conditions very high sequence coverages can be obtained in an overall time of 3 h. These high values can be reached in a shorter period of time only when an elevated temperature of 50 ◦ C is used. The optimal time course was found to be 30 min for reduction, 5 min for alkylation and 30 min for digestion (Fig. 6, bar 30/5/30). Long alkylation times lead to a decrease in sequence coverage. The best results at 37 ◦ C can be achieved with longer incubation times for each step (Fig. 6, bar 60/60/60) but in none of the applied time courses the high sequence coverage observed at 50 ◦ C was reached. The effect of digestion time was also addressed and is summarized in Fig. 7. Already after 5 min a sequence coverage of 20% for BSA and 30% for Apo-transferrin was obtained. The lower sequence coverage after 2 h of digestion at 50 ◦ C could be a result of the digestion of peptides with 1 missed tryptic cleavage to peptides with masses below the lower m/z limit for MALDI-MS thus a more complete digestion induces losses in sequence information. The loss in the sequence coverage could also be a result of unspecific tryptic cleavage or unspecific hydrolysis due to the high temperature.

With these results, we could reduce the entire sample preparation protocol to 65 min in total instead of 180 min and could show that shorter digestion times at 50 ◦ C are favorable for on-plate digestion. 3.3.2. Influence of protein amount on sequence coverage The reduction and alkylation on the MALDI-sample plate was performed according to the standard on-plate method described above for 1000 fmol of protein. By stepwise decrease of the protein amount to 100 fmol, we addressed the applicability of the protocol for the smaller protein amounts. Because the protocols were kept constant for smaller sample protein concentrations, the molar enzyme-to-protein ratio changes respectively to higher values. Fig. 8 shows the dependence of the sequence coverage on the concentration of BSA (Fig. 8a) and Apo-transferrin (Fig. 8b) for in-solution and on-plate digestion. 1000 fmol and 500 fmol show no significant differences for the sequence coverage between in-solution and on-plate digestion for both proteins. When only 100 fmol of protein is used the sequence coverage from on-plate digestion of Apo-transferrin is 30% instead of 15% from in-solution digestion. Although Apo-transferrin can be identified with both methods, the in-solution method has a higher variability. BSA can-

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Fig. 6. Comparison of varying time/temperature protocols on the sequence coverage of on-plate digested proteins. (a) BSA and (b) Apo-transferrin. The numbers on the x-axis indicate the time in minutes applied for the steps: reduction/alkylation/digestion (reduction[min]/alkylation[min]/digestion[min]) gray bars: all steps were carried out at 37 ◦ C 䊐 white bars all steps were carried out at 50 ◦ C. For comparison the results from 䊐 standard on-plate digestion protocol are given: 60 min reduction at 50 ◦ C, 60 min alkylation at 37 ◦ C, 60 min digestion at 37 ◦ C.

Fig. 7. Time course of digestion time vs. sequence coverage. The values of the sequence coverages plotted against digestion time for Apo-Transferrin (black points) 䊏 and BSA (gray points) .

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Fig. 8. Comparison of on-plate and in-solution digestion with different protein amounts. Sequence coverage for BSA (a) and Apo-transferrin (b). gray bars show on-plate digestion results and 䊐 white bars in-solution digestion results. (c) shows the spectrum of a 1000 fmol on-plate BSA digest (d) shows the spectrum of a 100 fmol on-plate BSA digest. Peaks are annotated with the amino acid sequence belonging to it. Peaks annotated with asterisks (*) were identified as trypsin autolysis peaks.

not be successfully identified in solution while on-target about 20% of the protein sequence was found. The differences in the sequence coverage can also be seen by comparing the spectrum of a 1000 fmol BSA digest (Fig. 8 c) and the spectrum of a 100 fmol BSA digest (Fig. 8d). Lower protein amounts of 50 and 10 fmol were also tested with our protocol. The signals derived from the proteins BSA and Apo-transferrin, that occurred in the spectra of this low concentration are the signals belonging to the most abundant peptide signals of the particular protein (for spectra of 50 and 10fmol BSA digest please refer to Supplementary data.) An identification of the proteins was possible, but only after a MS/MS-measurement of the occurring peptides, but a protein-characterization with this protein concentration is hard to perform. 4. Conclusion We could show that an optimized commercial digestion chamber is a helpful and easy to handle device for MALDI on-plate digestion and on-plate sample preparation for gel-free proteomics and protein analysis. Temperature and humidity can quickly and accurately be adjusted and read out with the software of this

device. Both parameters have a variability of less than 1% during the complete reaction time. Not only digestion but also reduction and alkylation for disulfide-bridge containing proteins can be performed on-plate and lead to an absolute increase in sequence coverage of 25–30%. The sequence coverage is comparably high than that from a standard in-solution digestion over 18 h. For the reduction, alkylation and digestion only 65 min are needed. When only small amounts of protein samples are available this method is favorable because only 1 ␮L is needed for the complete procedure. Moreover, substance losses from multiple transfers of the samples are minimized. 100 fmol of protein are sufficient to uniquely identify a protein by peptide mass fingerprinting. For automation the SunDigest device can be combined with a fraction collector for analysis of complex samples after HPLC separation. The reagents such as DTT, IAA, trypsin and matrix can be added automatically which further reduces the analysis time. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijms.2016.11.011.

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Please cite this article in press as: M. Rühl, et al., Enhanced on-plate digestion of proteins using a MALDI-digestion chamber, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.011