Ionization

Article pubs.acs.org/ac

In-Source Decay during Matrix-Assisted Laser Desorption/Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer Daiki Asakawa,* David Calligaris, Tyler A. Zimmerman, and Edwin De Pauw* Mass Spectrometry Laboratory, Department of Chemistry, and GIGA-Research, University of Liège, B-4000 Liège (Sart-Tilman), Belgium S Supporting Information *

ABSTRACT: The type of ions detected after in-source decay (ISD) in a MALDI source differs according to the ion source pressure and on the mass analyzer used. We present the mechanism leading to the final ISD ions for a Fourier transform-ion cyclotron resonance mass spectrometer (FTICR MS). The MALDI ion source was operated at intermediate pressure to cool the resulting ions and increase their lifetime during the long residence times in the FTICR ion optics. This condition produces not only c′, z′, and w fragments, but also a, y′, and d fragments. In particular, d ions help to identify isobaric amino acid residues present near the Nterminal amino acid. Desorbed ions collide with background gas during desorption, leading to proton mobilization from Arg residues to a less favored protonation site. As a result, in the case of ISD with MALDI FTICR, the influence of the Arg residue in ISD fragmentation is less straightforward than for TOF MS and the sequence coverage is thus improved. MALDI-ISD combined with FTICR MS appears to be a useful method for sequencing of peptides and proteins including discrimination of isobaric amino acid residues and site determination of phosphorylation. Additionally we also used new software for in silico elimination of MALDI matrix peaks from MALDI-ISD FTICR mass spectra. The combination of high resolving power of an FTICR analyzer and matrix subtraction software helps to interpret the low m/z region of MALDI-ISD spectra. Finally, several of these developed methods are applied in unison toward a MALDI ISD FTICR imaging experiment on mouse brain to achieve better results.

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protonation in classical MS/MS by collision-induced dissociation (CID), the peptide amino acid composition does influence ISD fragmentation. The N−Cα bond cleavage at the N-terminal side of cysteine residues is high compared to other amino acids,10,21 whereas the ISD ions originating from cleavage of Nterminal side of Gly, Val, and Ile are somewhat low.12,22 In contrast, a proline-rich peptide without a basic chemical group undergoes alternative peptide bond cleavage N-terminal to the first proline of the XPP motif.23 Recently, it was reported that the peptide bond cleavage in MALDI-ISD can be enhanced by addition of ammonium sulfate24 or ammonium persulfate25 in the MALDI matrix solution. The MALDI ion source is well-suited for use with axial timeof-flight (TOF) mass spectrometers fitted with ion sources working at a pressure below 10−5 mbar. Higher laser fluence increases the amount of released hydrogen radicals and therefore the yield of ISD fragments. At the same time, it may decrease the resolving power due to broadening the thermal distribution of kinetic energy of the ions. Recently, MALDI-ISD has been observed with orthogonal TOF (o-TOF) mass spectrometers.26,27 In the o-TOF configuration, the

atrix-assisted laser desorption/ionization with in-source decay (MALDI-ISD) mass spectrometry has been used for top-down proteomics,1−5 including imaging experiments.6−8 MALDI-ISD is initiated by a hydrogen radical transfer reaction from MALDI matrix to analyte,9,10 which mainly occurs in the matrix crystal before desorption.11 The choice of MALDI matrix is essential for MALDI-ISD experiments. In particular, the use of a reducing matrix, such as 2,5-dihydroxybenzoic acid (2,5-DHB),2,12 2-aminobenzamide (2-AA),13 or 1,5-diaminonaphthalene (1,5-DAN)14−16 efficiently cleaves the N−Cα bond on the peptide backbone and disulfide bond between cysteine residues by hydrogen attachment. The hydrogen attachment to a carbonyl oxygen on the peptide backbone leads to the formation of the c′/z• fragment pair via an aminoketyl radical intermediate.10 Subsequently, radical z• fragments undergo either gain of a hydrogen atom during collisions, or loss of their side chain via unimolecular dissociation in the MALDI plume. Such reactions are affected by the initial velocity of analyte ions,17 which indicates the collision rate in the MALDI plume18 and depends upon the matrix used.19,20 Therefore, our recent observations suggest that ISD fragmentation mainly occurs in the MALDI plume rapidly after desorption. Although MALDI-ISD fragmentation is less prone to specific cleavages, such as cleavages because of preferential sites of © 2013 American Chemical Society

Received: April 25, 2013 Accepted: July 23, 2013 Published: July 23, 2013 7809

dx.doi.org/10.1021/ac401234q | Anal. Chem. 2013, 85, 7809−7817

Analytical Chemistry

Article

MALDI ion source is operated with an intermediate pressure ion source because of the longer detection time of the o-TOF mass spectrometer. The presence of a buffer gas in the MALDI ion source contributes to cool the generated intact peptide ions and ISD ions, which increases their lifetime. Resolving power in MALDI o-TOF mass spectrometers is independent of laser power. However, TOF mass spectrometers do not have the ability to separate nearly isobaric ISD fragments and this makes it difficult to interpret MALDI-ISD spectra. The Fourier transform-ion cyclotron resonance mass spectrometer (FTICR MS) provides high-resolving power and sub-ppm mass accuracy measurements.28 When MALDI is combined with FTICR MS, it is better to operate at intermediate pressure in the MALDI ion source just as for the o-TOF mass spectrometer. A recent study led by Calligaris et al. has shown the possibility of direct identification and localization of proteins by MALDI-ISD imaging with FTICR MS.29 The use of MALDI FTICR MS allowed the simultaneous identification of several proteins in a complex mixture within 6 ppm mass accuracy. Small ISD ions have been resolved from MALDI matrix ions, giving additional sequence information. MALDI-ISD FTICR MS could be useful for top-down proteomics, as well as for N- and C-terminal sequencing. As described earlier, the hydrogen transfer reaction occurs in the MALDI matrix crystal before desorption and is followed by peptide radical reactions occurring in the MALDI plume rapidly after desorption. The peptide radicals were probably formed independent of the ion source pressure, whereas the presence of buffer gas in the MALDI ion source would be expected to have an effect on the fragmentation and reaction processes of peptide radicals. This paper mainly focuses on the influence of the buffer gas in the ion source on the ISD processes. Additionally, we also demonstrate a top-down approach for protein identification including discrimination of isobaric Leu and Ile residues and the determination of phosphorylation sites. This paper also describes new software for in silico elimination of MALDI matrix peaks, which can be applicable to any highresolving power MALDI mass spectra. The high-resolving power of FTICR MS helps to resolve isobaric matrix−analyte and analyte−analyte doublet peaks, and MALDI matrix elimination further improves peak annotation. For MALDI ISD FTICR imaging of tissues, the high resolving power of FTICR is combined with a mechanistic understanding of the ISD process to extract more information. The in silico MALDI matrix elimination further improves peak annotation within the imaging data. Thus, the below MALDI ISD FTICR imaging experiment on mouse brain is a test case where several of the methods developed in this paper are applied in unison to achieve better results.

Table 1. Monoisotopic Mass (Mm) and Sequence of Analyte Peptides Useda analyte peptide

Mm

amyloid β-protein 1−28

3260.528

renin substrate substance P reduced calcitonin

1758.932 1346.728 3431.729

fibrinopeptide A

1535.685

sequence DAEFR HDSGY EVHHQ KLVFF AEDVG SNK DRVYI HPFHL VIHN RPKPQ QFFGL M(NH2) CSNLS TCVLG KLSQE LHKLQTYPRT NTGSG TP(NH2) ADSGE GDFLA EGGGV R

a

Basic amino acids, arginine (R), histidine (H), and lysine (K) are given in bold print. Isobaric amino acids, leucine (L) and isoleucine (I) are underlined.

ammonium bicarbonate aqueous solution at 56 °C. The reduced calcitonin was purified by ZipTip before MALDIISD analysis. The 1,5-DAN was dissolved in water/acetonitrile (1/1, v/v) with 0.1% formic acid for a saturated solution. Subsequently, a saturated solution of 1,5-DAN was mixed with the same solvent (water/acetonitrile (1/1, v/v) with 0.1% formic acid) at 3/1 ratio (v/v). One microliter of analyte and 0.5 μL of matrix solutions were deposited on the MALDI plate. Tissue Preparation. A six-month-old Balb C mouse was provided by Central Animal Housing at the University of Liège. During intraperitoneal anesthesia with 60 mg/kg pentobarbital, the mouse was dissected to extract the brain, which was then snap frozen by immersion in precooled isopentane at −80 °C for 1 min. The brain was then kept at −80 °C until required. All further manipulations of tissues were done on dry ice to minimize localized tissue warming. The mouse brain was placed at −20 °C for 1 h before use. Tissue sections were then prepared on a Microm HM 500 O microtome (Microm, Heidelberg, Germany) with the microtome chamber chilled at −20 °C with the specimen holder at −25 °C. Several 14 μm thick horizontal sections were created and thaw mounted onto ITO-coated microscope slides (Sigma Aldrich, Steinheim, Germany). Following the thaw mounting of tissue sections onto MALDI target slides, these were allowed to dry for 1 h in a desiccator. Then, the tissue slices were washed six times using a previously described protocol:30 70% ethanol, 100% ethanol, Carnoy’s fluid (60% ethanol, 30% chloroform, and 10% acetic acid), 100% ethanol, H2O, and 100% ethanol. All rinse steps were carried out for 30 s, except for the step using Carnoy’s solution, which was for 2 min. Following washing, ITO slides were then dried in a desiccator for 1 h before MALDI matrix deposition. 1,5-DAN (5 mg/mL solution in 1/1 ACN/0.2% trifluoroacetic acid (TFA)) was sprayed onto the tissue surface by an ImagePrep automated sprayer device equipped with a new spray head (Bruker Daltonics GmbH, Bremen, Germany) as described previously.6 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. MALDI-ISD mass spectra were recorded using a 9.4 T SolariX FTICR MS equipped with an ESI/ MALDI dual-ion source and with a Smartbeam-II laser (wavelength, 355 nm; focus setting, “minimum”; repetition rate, 1000 Hz) and an UltraFlex II TOF/TOF MS (Bruker Daltonics, Germany). MALDI-ISD spectra were externally calibrated using a peptide mix calibration standard. A frequency-tripled Nd:YAG laser (355 nm) was used for the MALDI experiment in both instruments. For MALDI FTICR analysis, a single MALDI-ISD spectrum was acquired from 800 summed laser shots and total MALDI-

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EXPERIMENTAL SECTION Materials. Reducing matrices (2,5-DHB, 2-AA, and 1,5DAN) and proteins (myoglobin and bovine β-casein) were purchased from Sigma-Aldrich (Steinheim, Germany). All peptides were purchased from Bachem (Weil am Rhein, Germany). All the solvents used were HPLC grade quality. All reagents were used without further purification except for water, which was purified through a Milli-Q water purification system (Millipore, Billerica, MA, U.S.A.). Sample Preparation. Analyte peptides and proteins were dissolved in water at concentration of 20 and 40 pmol/μL, respectively. The sequences of peptides used are summarized in Table 1. Calcitonin was reduced with 10 mM DTT in 50 mM 7810



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Figure 1. MALDI-ISD spectra of amyloid β protein 1−28 obtained by (A, C) TOF MS in reflectron mode and (B, D) FTICR MS. 1,5-DAN was used as the matrix.

Method” command of DataAnalysis 4.0 (Bruker Daltonics), and the VB script is available in the Supporting Information. Default peak picking parameters of the FTICR MS method in DataAnalysis were also used, with all thresholds set to 0.0 other than the absolute intensity threshold at 105 counts. A code was written in the Java programming language (www. oracle.com, JDK version 1.6.0_22) to subtract MALDI matrix peaks from MS imaging data, where the first step is to read the imaging peak list data into the Java heap space for subsequent manipulation. The peak list of matrix ions of 1,5-DAN is read in a similar manner. The code then calculates if the m/z values from the imaging data match any of the known MALDI matrix peaks within a user-defined mass tolerance, followed by subtraction of the identified peaks. Another user-defined constraint for identifying MALDI matrix peaks is a threshold of the percentage of positions over which a peak occurs. If the peak occurrence percentage is high, it is likely to be a ubiquitous MALDI matrix peak. The peaks identified as matrix are then deleted from the imaging data, and the refined peak lists are output into text files. The software then creates a total ion count (TIC) peak list by summing intensities over all refined peak lists within 0.01 Da bins over a defined mass range. This TIC spectrum visualizes the results of an entire imaging data set in a single spectrum, and is used to visually confirm the removal of MALDI matrix peaks. An additional Java code creates 2D ion images that correspond to a tissue surface at selected m/z ratios (within a user-defined tolerance). The code plots the refined peak lists to produce ion images that are free from MALDI matrix interference. The intensity values of the ion images are scaled to the maximum intensity observed over the entire imaging data set within the selected m/z range. The Java source codes for plotting ion images and matrix peak subtraction are in the Supporting Information.

ISD spectra were accumulated from 25 single MALDI-ISD spectra. The laser power was optimized to obtain MALDI-ISD mass spectra that have high signal-to-noise ratios (S/N) for the ISD ion peaks. Ion cooling time and time-of-flight values were set to 20 and 2 ms, respectively. The pressure in MALDI ion source was adjusted to 4.2 mbar. For MALDI TOF analysis, mass spectra were acquired in positive reflectron mode. The ion accelerating voltage was 25 kV with the voltages of the electrodes 1 and 2 set at 21.7 kV and 9.5 kV, respectively to carry out the pulsed ion extraction. The time delay before the ion extraction and laser fluence were set at 30 ns and 75%, respectively. With regard to the MALDIISD spectra, 2000 laser shots were acquired. Pressure in the ion source was adjusted to 3 × 10−6 mbar. For MALDI-ISD FTICR imaging, the pixel step size for surface rastering was set at 120 μm in the FlexImaging software (Bruker Daltonics) that was interfaced with SolariXcontrol to collect from 3023 positions on the tissue surface. At each position, the MALDI-ISD spectra accumulated from 300 laser shots in positive ion mode. We employed herein the unambiguous notation of Zubarev when naming the fragment ions.31 Unless noted otherwise, all assigned peaks represent singly protonated molecules. For each MALDI-ISD spectrum, monoisotopic masses of an acquired spectrum were labeled using DataAnalysis 4.0 (Bruker Daltonics) with the SNAP peak picking algorithm. Spectra were then permanently assigned by BioTools 3.2 software. The default approach assigned peaks using a mass tolerance of 10 ppm. For all proteins, the sequence was transferred from Sequence Editor 3.2, and sequence tags were automatically determined and annotated on the spectrum, except for d and w fragment ions. Those fragments were manually assigned. MALDI Matrix Peak Subtraction Software. Peak picking was done on all the spectra from the imaging data set to produce 3023 peak lists in individual text files. Each peak list was labeled with its X−Y position on the tissue surface as its file name. This batch production was enabled by an in-house written Visual Basic script executed under the “ProcessWith-

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RESULTS AND DISCUSSION Proton Mobilization during MALDI-ISD Combined with the Collisional Process. We tested three different reducing matrices, 1,5-DAN, 2-AA, and 2,5-DHB for MALDI 7811



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Figure 2. MALDI-ISD spectra of (A) renin substrate and (B) substance P obtained with FTICR MS. 1,5-DAN was used as the matrix. Asterisks indicate matrix adduct on z fragment.

Figure 3. MALDI-ISD spectra of (A) reduced calcitonin and (B) fibrinopeptide A obtained with FTICR MS. Enlarged spectra for (C) c′12 and z′12 and (D) c20 ′ and z20 ′ obtained by FTICR MS in narrow band mode. 1,5-DAN was used as the matrix. Asterisks indicate matrix adduct on z fragment.

FTICR MS experiments. 1,5-DAN was found to give better ISD signals than 2-AA and 2,5-DHB for the analysis of peptides and proteins and was used for MALDI-ISD FTICR MS experiments. Figure 1 shows the comparison of MALDI-ISD mass spectra of amyloid β protein 1−28 obtained by two different mass

spectrometers, UltraFlex II TOF MS and SolariX FTICR MS. First, we focused on the resolving power in MALDI-ISD spectra obtained with TOF and FTICR mass spectrometers. The monoisotopic masses of protonated c′17 and z′18 are 2066.98 and 2069.04, respectively. Figure 1C and 1D show enlarged MALDI-ISD spectra for those fragments region. Although a 7812



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monoisotopic signal of z′18 was overlapped with a c′17 fragment, this contained two 13C atoms (m/z 2068.99) in the TOF mass spectrum (Figure 1A), but FTICR with a narrow band measurement allows the detection of both ISD ions (Figure 1B). Next we examined the MALDI-ISD fragmentation behavior obtained by TOF and FTICR mass spectrometers. Amyloid β protein 1−28 contains an Arg residue near the N-terminus, which favors protonation of the N-terminal side of ISD fragments. As a result, MALDI TOF measurement of amyloid β protein 1−28 preferentially gave c′ ions by ISD (Figure 1C). The intensity of observed ISD ions in MALDI TOF MS strongly depends on the position of the Arg residue. This result is in good agreement with previous reports.32 MALDI mainly produces singly charged analytes, and the presence of an Arg residue contributes to enhance the yield of protonation.33 This phenomenon strongly supports the fact that a proton is localized on the Arg residue in the protonated peptides formed by MALDI. The Arg residue is therefore required to observe an intense signal for these peptides in the MALDI-ISD mass spectrum. In contrast to MALDI TOF MS, ISD observed in MALDI FTICR MS favors the formation of z′ ions from amyloid β protein 1−28 (Figure 1D) compared with that in MALDI TOF MS (Figure 1C). Figure 2 shows MALDI-ISD FTICR spectra of renin substrate and substance P that contain an Arg residue near the N-terminus. Although the MALDI-ISD TOF spectrum of renin substrate shows only z′9 ions as Cterminal fragments,17 several z′ ions are observed in the MALDI-ISD FTICR spectrum (Figure 2A). In the case of substance P, only z9′ ions were observed in the MALDI-ISD FTICR spectrum (Figure 2B). The z′8 and z′10 ions were absent due to the presence of Pro residues, and instead, y′8 and y′10 were observed. In contrast to FTICR results, the MALDI-ISD TOF spectrum of substance P did not show any z′ and y′ ions (data not shown). It should be noted that Figure 2 shows a doubly protonated molecule, [M + 2H]2+, which is absent in the TOF spectrum. However, the relative intensity of the doubly protonated molecule to singly protonated one is below 1%. It does not contribute to generating the abundant z′ ions in Figure 2. Therefore, those results indicate that protonated ISD fragments without an Arg residue observed in MALDI FTICR MS would be formed by proton transfer from the Arg residue to a less favored protonation site with subsequent N−Cα bond cleavage. The major difference between the MALDI ion sources in TOF MS and FTICR MS is pressure. During TOF MS measurements, resulting ions are transferred to the flight tube without collision with background gas. For FTICR measurements, the MALDI ion source is operated at intermediate pressure. The intermediate pressure ion source causes desorbed ions to collide with background gas during the desorption process. The collision between analyte ions and background gas contributes to increase their lifetime by internal energy transfer from peptide ions to background gas.26,27 So, depending on collision energy and pressure, ions are not only cooled but can also be activated. According to the FTICR results, the proton is mobilized from the Arg residue to less favored protonation sites. It is well-known that low-energy collision activation of protonated peptides leads to various isomers containing different protonation sites.34 After the formation of the c′/z• fragments pair, proton transfer between c′ and z• fragments is impossible. Therefore, proton mobilization occurs before or at the same time as c′/z• fragments pair formation, which

generates rapidly after desorption. During the early step of the desorption, the presence of buffer gas probably contributes to activation of the generated ions. Figure 3 shows MALDI-ISD mass spectra of reduced calcitonin and fibrinopeptide A obtained by FTICR MS, which allows separating nearly isobaric ISD fragments generated from reduced calcitonin. These peptides contain an Arg residue near their C-terminus that favors protonation at the C-terminal side. As expected from the results of Figures 1 and 2, intermediated pressure MALDI-ISD (FTICR MS) not only produced C-terminal side fragment ions but also c′ ions. The presence of a basic amino acid is not necessary for the observation of ISD fragments in an intermediate pressure ion source. As an example, c′ ions of fibrinopeptide A are observed starting at the c′10 ion which does not contain any basic amino acid. In this case, the favored site of protonation in these ISD ions is the N-terminal amino group. It has been previously reported that the N-terminal amino group is not sufficient for the observation of c′ fragments by high-vacuum MALDI-ISD measurement (TOF MS).17,32 For MALDI-ISD operated in an intermediate pressure ion source (FTICR MS), the influence of the basic amino acids in ISD fragmentation is less straightforward than in the high-vacuum ion source (TOF MS). This phenomenon can be explained by collisional activation of generated ions during the early desorption process, increasing proton mobility. The proton mobilization also mediates the cleavage of the CO−NH bond that is weakened by the protonation of the backbone amide nitrogen.34−36 Although y′ ions are observed in Figures 1−3, their counterpart b ions are absent. This suggests that peptide bond cleavage is less likely to occur during intermediated pressure MALDI-ISD. A higher energy is needed to localize the proton on backbone amide nitrogen which is an energetically less favored protonation site compared with the backbone carboxyl oxygen and the N-terminal amino group.37 The intermediate pressure MALDI-ISD provides enough internal energy to mobilize the proton via backbone carboxyl oxygen and basic side chain, whereas protonation of the backbone amide nitrogen does not occur. Therefore, the observed y′ fragments in intermediate pressure MALDI-ISD spectra (FTICR MS) were formed via hydrogen attachment as well as the c′/z• fragments pair formation. The details of this formation mechanism are described below. Mechanism of MALDI-ISD Combined with the Collisional Process. Figures 1−3 show not only c′ and z′ ions, but also a and y′ ions. The yield of a and y′ ions increases with the increasing of internal energy of peptide ions which depends on the matrix proton affinity.32 Recently, we have proposed the formation mechanism of a and y′ fragments via the formation of the a•/x′ fragments pair (Scheme 1).11 Those fragments did not often appear in the high-vacuum MALDI-ISD experiment with 1,5-DAN.17,32 This indicates that the use of 1,5-DAN did not provide enough internal energy to form O-centered peptide radicals which is an intermediate of a and y′ fragments formation. In contrast, when intermediate pressure MALDIISD was used, this radical could be formed by hydrogen attachment followed by collisional activation that then leads to the formation of a and y′ fragments. The w and d ions are informative fragments to discriminate isobaric amino acid residues, Leu and Ile (Scheme 2). The use of 1,5-DAN in MALDI-ISD with TOF MS highly favors the formation of w ions, which forms from α-cleavage of z• fragments.17 Therefore, MALDI-ISD with 1,5-DAN is a useful 7813



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intense than w ions because of the low abundance of a• radical fragments compared to z• radical fragments. In addition, the formation of a•/x′ fragment pairs is a minor pathway even in the intermediate pressure MALDI-ISD source. Nevertheless, all isobaric amino acid residues in the tested peptides could be discriminated by MALDI-ISD combined with FTICR MS (Inset of Figures 1−3). In contrast, for renin substrate that contains three isobaric residues, only Ile10 can be assigned by high-vacuum MALDI-ISD (TOF MS).17 Therefore, intermediate pressure MALDI-ISD (FTICR MS) is a better method for discrimination of Leu and Ile compared to high-vacuum MALDI-ISD (TOF MS). Top-Down Sequencing of Proteins Including Leu/Ile Discrimination and Localization of Phosphorylation Sites. Small proteins, myoglobin (17 kDa), and bovine βcasein (24 kDa) were used to test the applicability of MALDIISD combined with FTICR MS for top−down protein sequencing (Figure 4). In high-vacuum MALDI-ISD measurement (TOF MS), c′ ions of myoglobin were detected from c′16, which is the first c′ fragment to contain the Lys residue.17 In contrast, c′ ions in Figure 4A were detected from c10 ′ , which is in agreement with results obtained for reduced calcitonin and fibrinopeptide A. The isobaric residues at position 135, 137, and 142 are assigned as Leu residues just as in high-vacuum MALDI-ISD measurement.17 Unfortunately, Leu and Ile residues could not be directly discriminated in the near Nterminal side of proteins because of the absence of a and d ions. In order to increase the yield of a• and d fragments, we used oxidizing matrices, 5-nitroslicylic acid, and 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane, which induce specific Cα−C bond cleavages that lead to the a•/x fragments pair.38,39 However, MALDI-ISD with those oxidizing matrices did not provide enough useful information for Leu and Ile discrimination because of fragmentation efficiency. So, matrix choice can dramatically affect the quality of MALDI mass spectrum. It will be necessary to find better oxidizing matrix that allows discriminating Leu and Ile residues near the Nterminus. The site determination of post-translational modification (PTM) such as phosphorylation40 and glycosylation41 is also an important application of MALDI-ISD, because the peptide backbone cleavage occurs without degradation of PTMs during the high-vacuum MALDI-ISD process. In the case of

Scheme 1. Proposed Mechanism of MALDI-ISD with Hydrogen Attachment

Scheme 2. Side-Chain Loss from d• and z• Radicals at Leu and Ile Residues

method for the discrimination of Leu and Ile near the Cterminus.17 In contrast, d fragments which are generated by side-chain loss from a• fragments, are rarely observed during high-vacuum MALDI-ISD (TOF MS).17,32 As shown in Figures 1−3, intermediate pressure MALDI-ISD (FTICR MS) shows both d and w fragments, which were formed by radical-induced cleavage at the Cβ−Cγ bond of a• and z• radical fragments, respectively (Scheme 2). The observation of d ions is also evidence for the existence of a• radicals which are considered as intermediates of d fragments. However, d ions were less

Figure 4. MALDI-ISD spectra of (A) myoglobin and (B) bovine β-casein obtained with FTICR MS. 1,5-DAN was used as the matrix. Asterisks indicate matrix adduct on z fragment. 7814



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Figure 5. MALDI-ISD FTICR imaging spectrum taken from mouse brain, annotated by Biotools software with the amino acid sequence for myelin basic protein. The inset shows a close-up of an unresolved matrix-analyte peak pair at m/z 630. BioTools misidentifies the y′5 ion (m/z 630.351) as the c5′ ion (m/z 630.358).

Figure 6. Total ion count spectra from MALDI ISD FTICR imaging of mouse brain, showing before (A) and after (B) removal of 1,5-DAN peaks. The insets show how the y′5 ion was obscured my a more intense 1,5-DAN peak that was removed. A mouse brain slice (C) shows corresponding ion images at m/z 630 both before (D) and after (E) MALDI matrix elimination. Scale bar is 1.0 mm.

mobilization, which requires higher energy than for phosphate group loss, is comparatively less likely to occur. This is in good agreement with this dissociation model described above. Additionally, isobaric residues Ile187, Leu191, Leu192, and Leu198 could be discriminated by mass difference between z′ and w ions. Identification of Small ISD Ion Signals using FTICR MS with Matrix Subtraction Software. Low intensity ISD signals from less abundant ion series are often hidden by isobaric MALDI matrix peaks. Identification and removal of interfering peaks is necessary to uncover small ISD signals and to increase sequence coverage with FTICR MS. For lower mass resolution of TOF MS, such peak doublets might be impossible to resolve. However, for FTICR MS our observations show that semi-resolved peak doublets that combine ISD and MALDI matrix occur frequently, 30−50 times even within a single FTICR MS spectrum using 1,5-DAN. One example is given in Figure 5, which is a MALDI-ISD FTICR MS spectrum taken from mouse brain, annotated by Biotools software with the amino acid sequence for myelin. For ISD sequencing, automated methods already exist to annotate ISD sequences

phosphorylation, collisional activation of phosphopeptides results in dominant loss of phosphate acid.42 This phenomenon through the collisional activation is not suited for determining the location of phosphorylated sites. As described earlier, intermediate pressure MALDI-ISD also leads to the collisional activation of analyte ions. To examine the applicability of intermediate pressure MALDI-ISD for phosphoproteins, we used a bovine β-casein that contains five phosphorylation sites (Ser15, Ser17, Ser18, Ser19, and Ser35) as a model. Intermediate pressure MALDI-ISD of β-casein shows c′ and z′ ions originating from the cleavage at the N−Cα bond without degradation of the phosphate group (Figure 4B), thereby allowing the location of the phosphorylation sites to be determined just as in high-vacuum MALDI-ISD measurement. The three residues, Ser15, Ser17, and Ser18 could be assigned as the phosphorylation sites from Figure 4B. It is known that the phosphate group of a phosphopeptide is relatively labile providing a low-energy pathway that competes with backbone fragmentation.42 Nevertheless, MALDI-ISD with a collisional process does not provide enough internal energy for loss of a phosphate group. Therefore, peptide bond cleavage via proton 7815



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CONCLUSION We investigated the influence of the ion source pressure on the MALDI-ISD process. The intermediate pressure MALDI source produced not only c′ and z′ fragments but also a, y′, and d fragments. In this configuration, the influence of the amino acid residue on observed ISD fragments is less straightforward than in high-vacuum MALDI-ISD. These differences can be explained by collisional activation of the protonated peptide, which leads to the formation of a•/y′ fragment pairs and increases the mobility of the proton. Furthermore, it is known that the presence of a buffer gas in the MALDI ion source contributes to increase the lifetime of the generated ions. At intermediate pressure our results suggest that collisional activation occurs at an early desorption step in the intermediate pressure MALDI source. This collisional activation during desorption process contributes to increase the sequence coverage of peptides and proteins. Additionally, MALDI matrix subtraction from MALDI FTICR mass spectra will further simplify the data to aid in peak discovery resulting from other ion series or unexpected fragmentations, and of mass shifts caused by PTMs. We envision that the combination of high resolving power of FTICR, mechanistic understanding of the ISD process and MALDI matrix subtraction method could further improve the ability of MALDI-ISD mass spectrometry.

on the highest intensity ion series (usually c′ ions or z′ ions). Myelin basic protein (MBP), isoform 8 sequence with Met-1 removed and N-acetylalanine modification at position 2, was identified from the c′ ions. The Figure 5 inset shows a close-up of an unresolved matrix-analyte peak pair at m/z 630. However, the peak at m/z 630 cannot be the c′5 ion (m/z 630.358) because the ISD process does not cleave N-terminally to a proline residue. Instead, the m/z 630 matches the theoretical mass of the y5′ ion (m/z 630.351) of MBP. Although the y′ ions are not so intense in high vacuum MALDI-ISD with 1,5-DAN, collision between buffer gas and desorbed analytes occurring in the intermediate pressure MALDI source contributes to their increased yield. Therefore, understanding of the MALDI-ISD process is important for better interpretation of those spectra. We observe that semi-resolved FTICR MS peaks, as in Figure 5, often confuse peak picking algorithms. While removal of interfering peaks from ISD spectra can be done manually, we aid this process with an in-house written software that automates peak removal from larger profiling or imaging data sets (source code in Supporting Information). To demonstrate this software, the spectra in Figure 6A and 6B are TIC spectra at 0.01 Da resolution that visualize a MALDI-ISD-FTICR MS imaging data set before and after MALDI matrix removal. MALDI matrix elimination was done with a 0.02 Da tolerance. It was found that this tolerance appropriately accounts for most of the mass drift seen over the course of an FTICR imaging experiment. Thus, less matrix peaks are present in the TIC after MALDI matrix subtraction, Figure 6A, than before in Figure 6B. Not all the matrix clusters are removed in Figure 6B, as the 0.02 Da tolerance was chosen to be small enough not to remove potential analyte peaks, and this causes some massdrifted matrix peaks to fall just outside the elimination window a certain percentage of the time. Thus, intensities of these peaks are greatly reduced instead of completely eliminated in the TIC, but reduced peaks are acceptable for sequencing applications that use appropriate intensity thresholds. In fact, the software successfully subtracted a large 1,5-DAN peak (m/z 632.32) that was nearly isobaric to an analyte peak of MBP (m/ z 632.35). This elimination is shown in Figures 6A−B. Peak picking using appropriate parameters had helped the peak pair to become semi-resolved in Figure 6A (inset), but the MALDI matrix subtraction software is able to completely eliminate the interfering matrix peak. The resulting peak in Figure 6D (inset) is completely free from MALDI matrix interference. Besides peak discovery and ISD sequencing, another process improved by MALDI matrix subtraction is the generation of ion images. To demonstrate improved ion images, Figure 6C shows an optical image of a mouse brain slice that was imaged using MALDI-ISD-FTICR MS, accompanied by two ion images. The ion images were plotted at the same m/z value (630.32) and with the same plotting tolerance of 0.1 Da. The resulting difference in distributions is because the ion image in Figure 6D was created before matrix elimination, and Figure 6E was created afterward. In fact, the m/z value of these ion images corresponds to the same unresolved peak pair that was shown in Figures 6A and B. Thus, only after MALDI matrix removal can a clear ion distribution be seen for MBP isoform 8 in Figure 6F that matches previously reported distributions in the corpus callosum,43 and that would have been missed in the ion image before matrix subtraction (Figure 6D). MALDI matrix subtraction would therefore simplify the discovery other interesting ion distributions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional material as described in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (D.A.); e.depauw@ulg. ac.be (E.D.P.). Present Addresses

Daiki Asakawa: Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan David Calligaris: Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, U.S.A. Tyler A. Zimmerman: Department of Chemistry, Northwestern University, Chicago, IL, U.S.A. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Daiki Asakawa acknowledges the research fellowship from the Japan Society for the Promotion of Science for Young Scientists (23-10272). David Calligaris acknowledges a postdoctoral fellowship from the University of Liège research council under the ARC REFRACT project (Actions de Recherche Concertées, Belgium). Tyler Zimmerman acknowledges the postdoctoral fellowship Chargé de Recherches from the Fonds National de la Recherche Scientifique (FNRS), Belgium.

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REFERENCES

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