ionization mass spectrometry.

PRINCIPLES OF HYDROGEN RADICAL MEDIATED PEPTIDE/PROTEIN FRAGMENTATION DURING MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY Daiki Asakawa* Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan Received 16 April 2014; revised 30 June 2014; accepted 30 June 2014 Published online 6 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21444

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is a very easy way to obtain large sequence tags and, thereby, reliable identification of peptides and proteins. Recently discovered new matrices have enhanced the MALDIISD yield and opened new research avenues. The use of reducing and oxidizing matrices for MALDI-ISD of peptides and proteins favors the production of fragmentation pathways involving “hydrogen-abundant” and “hydrogen-deficient” radical precursors, respectively. Since an oxidizing matrix provides information on peptide/protein sequences complementary to that obtained with a reducing matrix, MALDI-ISD employing both reducing and oxidizing matrices is a potentially useful strategy for de novo peptide sequencing. Moreover, a pseudo-MS3 method provides sequence information about N- and C-terminus extremities in proteins and allows N- and C-terminal side fragments to be discriminated within the complex MALDI-ISD mass spectrum. The combination of high mass resolution of a Fourier transform-ion cyclotron resonance (FTICR) analyzer and the software suitable for MALDI-ISD facilitates the interpretation of MALDI-ISD mass spectra. A deeper understanding of the MALDI-ISD process is necessary to fully exploit this method. Thus, this review focuses first on the mechanisms underlying MALDI-ISD processes, followed by a discussion of MALDI-ISD applications in the field of proteomics. # 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:535–556, 2016

Keywords: in-source decay; redox reactions; hydrogen-abundant and hydrogen-deficient radicals; sequencing

I. INTRODUCTION Mass spectrometry (MS) has become a central method for the identification and characterization of proteins. For identification by MS, proteins are usually reduced, alkylated (e.g., Scarbamidomethylation) and finally enzymatically digested to generate peptides. These peptides are then ionized by electrospray ionization (ESI) (Fenn et al., 1989) or matrix-assisted laser desorption/ionization (MALDI) (Karas & Hillenkamp, 1988), to Contract grant sponsor: Japan Society for the Promotion of Science for Young Scientists; Contract grant number: PD: 23-10272. Correspondence to: Daiki Asakawa, Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. E-mail: [email protected]

Mass Spectrometry Reviews, 2016, 35, 535–556 # 2014 by Wiley Periodicals, Inc.

establish a peptide mass fingerprint that can be compared to “in silico” digestion of proteins from databases, with the aim of identifying the digested protein. Ionized peptides can also be fragmented using collision induced dissociation (CID) to confirm protein identification (Steen & Mann, 2004). This analytical work flow is available only if the database contains a primary sequence of the protein. In contrast, de novo sequencing by MS is essential for the identification of unknown peptides (Seidler et al., 2010). CID generally leads to peptide bond cleavage, thus de novo sequencing is conducted by interpreting mass differences between series of consecutive b and y0 ions. However, CID backbone cleavage efficiency is strongly affected by peptide sequence. Therefore, CID often yields incomplete peptide sequence information, decreasing its usefulness for de novo sequencing. Tandem MS with electron-mediated fragmentation techniques, such as electron capture dissociation (ECD) (Zubarev, Kelleher, & McLafferty, 1998) and electron transfer dissociation (ETD) (Syka et al., 2004), have been widely used for peptide sequencing (Coon, 2009). ECD/ETD is initiated by an electron association to multiply-protonated molecules, already formed by ESI, generating a fragile radical species. Several mechanisms have been proposed to explain ECD/ETD fragmentation patterns. The classical ECD mechanism involves recombination of an electron and an excess proton in a multiply-protonated molecule with subsequent hydrogen transfer to a carbonyl group at the peptide backbone (Zubarev, Kelleher, & McLafferty, 1998). Alternatively, Sobczyk et al. and Syrstad et al. proposed that electron capture occurs via a p amide orbital, producing an amide anion-radical intermediate (Sobczyk et al., 2005; Syrstad & Turecˇek, 2005). Zubarev and coworkers hypothesized that electron capture occurs at a hydrogen bond between the backbone nitrogen and carbonyl groups, resulting in the production of a negative charge on the amide nitrogen and aminoketyl radical (Patriksson et al., 2006). Despite their differences, these mechanisms all involve the formation of an aminoketyl radical intermediate, which eventually leads to the c0 /z• fragment pair. A recent review article describes in detail the processes of ECD/ETD (Turecˇek & Julian, 2013). Compared to CID, the fragmentation in ECD/ ETD is less prone to specific cleavages. Therefore, ECD/ETD is a potentially useful method for de novo peptide sequencing. Alternative to the classical protein analysis procedure with enzymatic digestion, direct mass spectrometric characterization of intact protein, named “top-down” sequencing, has been

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developed. For this approach, intact proteins are directly ionized by ESI and the resulting multiply-charged protein is fragmented in the mass spectrometer. Subsequently, protein identification is made by either database searching or de novo sequencing. Topdown sequencing have been performed using both CID and ECD/ETD (Reid & McLuckey, 2002). Although labile posttranslational modification (PTM) in proteins is usually lost during the CID fragmentation process, the ECD/ETD preferentially produce c0 and z• fragments without degradation of PTMs. Therefore, one of the most promising applications of top-down sequencing performed by ECD/ETD is determination of PTM locations in protein (Garcia, 2010; Zhou et al., 2012). Similarly to ECD/ETD, MALDI in-source decay (ISD) mainly leads to the N–Ca bond cleavage of the peptide backbone (Brown & Lennon, 1995) and have been utilized for top-down sequencing of intact proteins (Debois et al., 2013; Hardouin, 2007). MALDI-ISD fragmentation may proceed through the aminoketyl radical intermediates as in the case of ECD/ETD. Because electrons and hydrogen atoms are present in the MALDI plume (Scott et al., 1994; Frankevich, Knochenmuss, & Zenobi, 2002), this hydrogen-abundant radical intermediate is thought to be formed via either electron capture or hydrogen attachment. Zubarev and coworkers have focused on N–Ca bond cleavages involving an electron capture of a multiply-charged peptide (Kocher, Engstrom, & Zubarev, 2005). However, MALDI-ISD produces fragment ions as an intense signal for peptides barely yielding doubly-charged precursors. Therefore, the mechanism involving electron capture is not a dominant process in MALDI-ISD. In contrast, Takayama focused on the MALDI-ISD process involving attachment of a hydrogen atom to a peptide and the source of the hydrogen radical was investigated using a deuterium-labeled peptide and matrix (Takayama, 2001). A synthetic dodecapeptide (RLGNQWAVGDLAE) was used as a model with a focus on c0 3 and c0 4 fragments. A number of exchangeable hydrogen atoms in c0 3 and c0 4 fragments 9 and 12, ½c0 3 ðD10 Þ þ Dþ and ½c0 4 ðD13 Þ þ Dþ were also observed in the MALDI-ISD spectrum. This result suggests that the c0 fragments are produced via deuterium-abundant peptide radicals, which are formed by deuterium transfer to peptides from the matrix. In contrast, the MALDI-ISD spectrum clearly shows ½c0 3 ðD9 Þ þ Dþ and ½c0 4 ðD12 Þ þ Dþ fragments. Those products may originate from hydrogen/deuterium (H/D) back exchange, but competitive processes for c0 fragment formation are considerable. To confirm the hypothesis for c0 fragment formation, Zubarev and coworkers use a methyl esterified peptide, [sarcosine]13-proline (Kocher, Engstrom, & Zubarev, 2005). Because the peptide does not contain any exchangeable hydrogen atom at the peptide backbone, the interpretation of MALDI-ISD spectra with H/D exchange was simplified. The deuterium labeling experiment produced an intact peptide ion containing two deuterium atoms, [M(D1) þ D]þ. In contrast, the resulting c0 -series ions contained three deuterium atoms, ½c0 n ðD2 Þ þ Dþ . Although ½c0 n ðD1 Þ þ Dþ was still observed, the intensity ratio of ½c0 11 ðD1 Þ þ Dþ =½c0 11 ðD2 Þ þ Dþ corresponded to the ratio of [M þ D]þ/[M(D1) þ D]þ, indicating ½c0 11 ðD1 Þ þ Dþ and [M þ D]þ to be formed by H/D back exchange during experiment. Those results clearly indicate that hydrogen transfer from the matrix to the peptide induces N–Ca bond cleavage. As expected with this mechanism, the efficiency of the hydrogen transfer reaction during the MALDI process depends on the reducing property of the matrix molecule such that the choice of matrix is 536

critical importance (Demeure et al., 2007; Sakakura & Takayama, 2010). One of the such matrix molecule, 2,5dihydroxybenzoic acid (2,5-DHB) is widely used for MALDIISD experiments (Hardouin, 2007). Recently, several new matrices, including 1,5-diaminonaphthalene (1,5-DAN) (Demeure et al., 2007), 5-aminosalicylic acid (5-ASA) (Sakakura & Takayama, 2010), 5,1-aminonaphthol (5,1-ANL) (Osaka, Sakai, & Takayama, 2013), 1,5-dihydroxylnaphthalene (1,5-DHN) (Osaka, Sakai, & Takayama, 2013), 2-aminobenzoic acid (2-AA) (Smargiasso, Quinton, & De Pauw, 2012), and 2-aminobenzamide (2-AB) (Smargiasso, Quinton, & De Pauw, 2012), have been developed. These release hydrogen radicals upon laser irradiation, thereby increasing the yield of ISD fragments. The most impressive of these new matrices is 1,5-DAN, which efficiently produces fragment ions by MALDI (Demeure et al., 2007; Quinton et al., 2007). The structure of a reducing matrix is shown in Scheme 1a. The mechanism of MALDI-ISD has been also investigated by using selective deuterium-labeled peptides (Bache et al., 2008). Since a very low level of H/D scrambling is observed during MALDI-ISD, the N–Ca bond cleavage occurs with limited vibrational excitation. MALDI-ISD coupled with H/D exchange has also been applied to the characterization of protein structure dynamics (Rand et al., 2011). MALDI-ISD provides deuterium contents of the proteins under investigation at a spatial resolution approaching single-residue resolution. Moreover, the observed the deuterium contents in ISD fragments are in good agreement with previously reported amide hydrogen exchange rates obtained by nuclear magnetic resonance studies. This result indicates that protein structures and dynamics in solution are accurately reflected in the deuterium contents of ISD fragments. Additionally, MALDI-ISD has been found to be an easy-to-operate method allowing detailed analysis of the deuterium content of the backbone amide.

SCHEME 1. Molecular structure of matrix for MALDI-ISD.

Mass Spectrometry Reviews DOI 10.1002/mas

HYDROGEN RADICAL MEDIATED PEPTIDE/PROTEIN FRAGMENTATION DURING MALDI-MS

Recently, alternative cleavage at the Ca–C bonds in the peptide backbone was found to occur when MALDI-ISD was performed with an oxidizing matrix, such as nitrosalicylic acid (NSA) isomers (Asakawa & Takayama, 2011a; Asakawa, Sakakura, & Takayama, 2013c) and 7,7,8,8-tetracyanoquinodimethane (TCNQ) derivatives (Asakawa & Takayama, 2012a). MALDI-ISD with an oxidizing matrix is initiated by hydrogen abstraction from the amide portion of the peptide backbone onto the matrix, leading to a hydrogen-deficient peptide radical containing a radical site on the amide nitrogen. Subsequently, the resulting radical principally generates a and x ions, indicating cleavage of the Ca–C bonds in the peptide backbone to be occurred. The structures of oxidizing matrices are shown in Scheme 1b. Notably, the use of 5-formylsalicylic acid (5-FSA) induces the cleavages of both N–Ca and Ca–C bonds, since the formyl functional group has both reducing and oxidizing properties (Asakawa & Takayama, 2011a). The cleavage of both N–Ca and Ca–C bonds in the peptide backbone occurs without degradation of the labile post-translational modifications, providing evidence for limited vibrational excitation in the MALDI-ISD process. Consequently, MALDI-ISD may be able to be used to identify sites with post-translational modifications, such as phosphorylation (Lennon & Walsh, 1999) and glycosylation (Hanisch, 2011). In addition, structural information of isolated glycans could be obtained by MALDI-ISD (Asakawa, Smargiasso, & De Pauw, 2012b). This review focuses on the fundamental aspects of MALDIISD and its recent applications in the field of proteomics. A mechanistic understanding of the MALDI-ISD process allows more information to be obtained from MALDI-ISD mass spectra. In addition, the pseudo-MS3 method, the development of software and the use of high resolution mass spectrometer can enhance the interpretation of MALDI-ISD mass spectra. The goal of this review is to explore the applications of MALDI-ISD for proteomics. Herein, Zubarev’s unambiguous notation was adopted for peptide fragment ions (Zubarev, 2003). According to this notation system, homolytic Ca–C, CO–NH, and N–Ca bond cleavage yields the radical fragments a•/x•, b•/y•, and c•/z•, respectively. The abstraction of a hydrogen atom from a• or x• fragment produces a or x fragment, respectively. The addition of a hydrogen atom to a• or x• fragment produces a0 or x0 fragment, respectively. Unless noted otherwise, all assigned peaks represent singly protonated and deprotonated species in positive ion mode and negative ion mode, respectively.

II. PRINCIPLE OF MALDI-ISD WITH REDUCTION A. Cleavage of Disulfide Bond Disulfide bond formation between the cysteine residues is a frequent post-translational modification, critical to protein folding and structural stability. Reducing matrices can be used to achieve the reduction of disulfide bonds during the MALDI process due to the high hydrogen atom affinity of sulfur atoms. For the cleavage of each intramolecular disulfide bond, two hydrogen radicals are added to the bond, resulting in the 2 Da shift in the peptide mass (Scheme 2a) (Fukuyama, Iwamoto, & Tanaka, 2006; Quinton et al., 2007). Indeed, when peptides containing intramolecular disulfide bonds are analyzed with a reducing matrix, isotopic patterns of the expected protonated Mass Spectrometry Reviews DOI 10.1002/mas

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SCHEME 2. (a) Intra- and (b) inter-molecular disulfide bond cleavages by MALDI-ISD.

molecules are deformed as a result of the partial reduction of disulfide bonds. In other words, MALDI with a reducing matrix results in an increased intensity of [M þ 2H þ H]þ and [M þ 4H þ H]þ, if the peptide contains two intramolecular disulfide bonds (Quinton et al., 2007). MALDI with a reducing matrix can then be used not only to detect peptides that contain disulfides in their structures but also to count the number of intramolecular disulfide bonds in the peptides. MALDI MS2 analysis with post-source decay (PSD) or CID provides information useful for peptide sequencing. However, the presence of intramolecular disulfide bonds in peptides contributes to decreased sequence coverage, since the b and y0 ions are absent when the fragments themselves remain connected via a disulfide bond. In contrast, reduction of the intramolecular disulfide bond sin a peptide by MALDI when a reducing matrix is employed followed by CID significantly improves sequence coverage of the peptide (Fukuyama, Iwamoto, & Tanaka, 2006). The use of a reducing matrix opens the possibility of performing rapid sequencing of peptides with intramolecular disulfide bonds by tandem MS. Additionally, performing MALDI with a reducing matrix also allows the analysis of peptides that are cross-linked by a disulfide bond (Fukuyama, Iwamoto, & Tanaka, 2006). These peptides are present in the protein digest with no previous reduction. This type of experiment is mainly performed to identify the cross-linked sites in native protein. Although the use of a conventional matrix, such as a-cyano-4-hydroxycinnamic acid (CHCA) yields only one signal corresponding to the crosslinked peptide, two main signals corresponding to the two free peptides constituting the cross-linked peptide can be observed in MALDI mass spectra obtained with a reducing matrix (Scheme 2b). Comparisons of MALDI mass spectra obtained with conventional and reducing matrices clearly provide interesting information on the nature of the disulfide bonds in peptides. The choice of MALDI matrix dramatically affects disulfide bond cleavage efficiency. The order of disulfide bond reduction capacities is 1,5-DAN > 2-AB 5-ASA > 2-AA > 2,5-DHB >CHCA (Demeure, Gabelica, & De Pauw, 2010; Sakakura & Takayama, 2010; Smargiasso, Quinton, & De Pauw, 2012). 1,5DAN, a reducing chemical, is a suitable matrix for disulfide bond cleavage by the MALDI process (Quinton et al., 2007). However, our recent study suggested that disulfide bond 537

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TABLE 1. Ratios of the signal intensity of reduced calcitonin [M þ 2H þ H]þ to those of non-reduced ions [M þ H]þ of peptides (%)

2,5-DHB 16.4 ± 7.8

1,5-DAN 91.6 ± 2.4

2,5-DHB/1,5-DAN

2,5-DHB/1,5-DAN

2,5-DHB/1,5-DAN

(1/1), crystal

(1/1), amorphous

(2/1), ionic liquid

29.1 ± 2.9

7.1 ± 2.9

8.7 ± 2.3

Values were taken from Asakawa et al. (2013e).

reduction efficiency is also dependent on the physical state of the matrix (Asakawa et al., 2013e). Either 2,5-DHB or 1,5-DAN matrices can be used to cleave disulfide bonds, but such cleavage is suppressed when an ionic liquid and an amorphous structure consisting of both 2,5-DHB and 1,5-DAN is used as a matrix. The efficiency of disulfide bond cleavage (%) estimated from the ratio of signal intensity of the reduced ion [M þ 2H þ H]þ to that of the non-reduced ion [M þ H]þ of calcitonin is summarized in Table 1. These results clearly indicate that the use of a non-crystal matrix suppresses disulfide bond reduction. The low efficiency of disulfide bond cleavage using a non-crystal matrix might be either the consequence of low hydrogen radical production or the low reactivity of the hydrogen radicals produced. To obtain more information, we focused on the matrix signals in MALDI mass spectra, which suggested that, following laser irradiation, hydrogen radical production was induced by an amorphous structure, and an ionic liquid of mixed 2,5-DHB/1,5DAN, as well as crystals (Asakawa et al., 2013e). Therefore, the

low reduction efficiency of a non-crystal matrix can result from low reactivity of the hydrogen radicals produced. In consequence, hydrogen attachment to disulfide bond mainly occurs in matrix crystals and intermolecular hydrogen bonds between the matrix and peptides in the crystal, which are thus essential for efficient disulfide bond reduction (Asakawa et al., 2013e).

B. Hydrogen Attachment to Peptide Backbone and Subsequent N–Ca Bond Cleavage Mechanisms of peptide backbone cleavage are summarized in Scheme 3. In this section, the mechanism of N–Ca bond cleavage, which is initiated by hydrogen attachment to carbonyl oxygen in the peptide backbone during the MALDI-ISD process, is reviewed. The efficiency of N–Ca bond cleavage is dependent on the reducing property and physical state of the matrix, as in the case of disulfide bond reduction. Figure 1 shows the MALDI-ISD mass spectra of substance P with 2,5-DHB,

SCHEME 3. The mechanism of MALDI-ISD via hydrogen attachment.

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FIGURE 2. MALDI-ISD mass spectra of (a) renin substrate (DRVYI HPFHL VIHN) and (b) fibrinopeptide A (ADSGE GDFLA EGGGV R) with 1,5-DAN. Asterisks and asterisk on z fragments indicate metastable ions and 1,5-DAN adducts on z fragments. Reprinted with permission from Asakawa, Smargiasso, and De Pauw (2013a). Copyright 2013, Springer.

FIGURE 1. MALDI-ISD mass spectra of substance P obtained with different matrices, (a) 2,5-DHB, (b) 1,5-DAN, and (c) an ionic liquid mixture of 2,5-DHB/1,5-DAN (2/1). Right inset; picture of the spot. Reprinted with permission from Asakawa et al. (2013e). Copyright 2013, American Chemical Society.

1,5-DAN and an ionic liquid mixture of 2,5-DHB/1,5-DAN. The strongest ISD signal was observed when laser shots were delivered to the 2,5-DHB or 1,5-DAN crystals (Fig. 1a and b), whereas the ionic liquid mixture of 2,5-DHB/1,5-DAN did not produce ISD fragment ions (Fig. 1c). These results indicate that the order of fragment ion abundance correlates with its tendency to reduce the peptide disulfide bond (Asakawa et al., 2013e). In contrast to disulfide bond cleavage, the N–Ca bond cleavage proceeds through a transition state, which results from an endothermic process (Wodrich et al., 2012). Therefore, the existence of a hydrogen-abundant peptide radical containing enough internal energy for decay is necessary for the generation of ISD fragments. The internal energy of ions generated by MALDI can be estimated by the survival yield method (Collette et al., 1998). According to the results obtained with this method, the use of crystal, amorphous and ionic liquid mixtures of 2,5DHB/1,5-DAN produces ions with similar internal energy, indicating that the difference in the internal energies obtained with such matrices does not contribute to ISD fragmentation (Asakawa et al., 2013e). Therefore, it is important to recognize that the peptide backbone cleavage behaviors observed in MALDI-ISD differ markedly according to the physical state of the matrix. Hydrogen attachment to peptides occurs exclusively when favorable intermolecular hydrogen bonds can be formed between peptides and the matrix, which is the case with matrix crystals.

C. Cleavage of N–Ca Bond on Peptide Backbone As described in introduction, MALDI-ISD involves the formation of a hydrogen-abundant radical intermediate, which underMass Spectrometry Reviews DOI 10.1002/mas

goes radical-induced cleavage at the N–Ca bond. Upon forming the hydrogen-abundant peptide, two N–Ca bond cleavage pathways are theoretically possible via a-cleavage, leading, respectively, to c0 /z• and c•/z0 fragment pairs. However, all ISD fragment ions are even-numbered electron-containing species, that is, c0 and z0 ions. Zubarev and coworkers investigated the formation mechanism of c0 and z0 fragments (Kocher, Engstrom, & Zubarev, 2005). As examples, the MALDI-ISD mass spectra of renin substrate and fibrinopeptide A are shown in Figure 2. MALDI-ISD of renin substrate, containing an arginine residue near the N-terminus, preferentially yielded c0 ions (Fig. 2a). The preferential observation of c0 ions but not z0 ions in Figure 2a is attributed to the position of the basic arginine residue. In contrast, the favored site of protonation in fibrinopeptide A is the arginine residue at the C-terminus and, thus, C-terminal side fragment ions are preferentially produced by MALDI-ISD (Fig. 2b). Notably, the matrix adducts on z fragments are observed in Figure 2b, whereas the matrix adducts on c ions are absent in Figure 2a. The matrix adducts of the z fragment are generated by the recombination of z• radical fragments with matrix radicals or by reaction with matrix molecules. Therefore, Zuvarev and coworkers concluded that MALDI-ISD produces the same types of fragment ions as ECD/ETD, that is, a c0 /z• fragment pair, and that the z• ions then gain a hydrogen atom or react with a matrix radical, giving z0 or [z þ matrix] (Kocher, Engstrom, & Zubarev, 2005). The radical z• fragments undergo a variety of radical reactions due to their low stability and the details of these reactions are described in Section II-D. The c0 /z• fragment pair formation through the hydrogenabundant radical intermediate merits a more detailed description. Since MALDI-ISD and ECD/ETD proceed through the aminoketyl radical intermediates, N–Ca bond cleavage similar to that proposed to explain c0 /z• fragment pair formation in ECD/ETD experiments can also be envisaged for MALDI-ISD. One of the most widely accepted mechanisms of N–Ca bond cleavage in ECD/ETD involves the aminoketyl radical undergoing homolytic bond cleavages (a-cleavage) on the C-terminal side of a radical site (Savitski et al., 2007; Syrstad & Turecˇek, 2005). In contrast, according to a recent report, cleavage of the N–Ca bond 539

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SCHEME 4. The c0 /z• fragment pair formation by N–Ca bond cleavage on the (a) right and (b) left sides of the radical site. Reprinted with permission from Asakawa et al. (2014a). Copyright 2014, Springer.

located on the N-terminal side of the radical site is also possible with heterolytic cleavage (Wodrich et al., 2012). Scheme 4 shows proposed mechanisms of homolytic and heterolytic cleavages at the N–Ca bond during MALDI-ISD processes. Notably, the N–Ca bond cleavage at the N-terminal side of a proline (Pro) residue produces the hydrogen-abundant peptide radical containing a radical site on the a-carbon of the proline residue which undergoes no further decomposition (Asakawa et al., 2014a). As a consequence, the aminoketyl radicals containing a radical site at the N- or the C-terminal side of proline residues can only cleave the bonds at Xxx1–Xxx2Pro and Pro–Xxx3, respectively (Scheme 5). This is an important characteristic which sheds light on the N–Ca bond cleavage process. To discuss the homolytic and heterolytic cleavages at the N–Ca bond, we consider the fragmentation of a model hexapeptide containing the proline residue at the 4th position (XXXPXX, where X is any amino acid, except proline), as shown in Scheme 6. If mainly homolytic N–Ca bond cleavages of hydrogen abundant peptide radicals occur in MALDI-ISD (Scheme 4a), the aminoketyl radical containing a radical site at the nth position in the hexapeptide would give a c0 n =zð6n Þ fragment pair, as shown in Scheme 6a. However, the 3rd position peptide radical (blue radical in Scheme 6a) does not produce the c0 3 =z3 fragment pair due to the presence of a proline residue at the 4th position. An alternative heterolytic N–Ca bond cleavage produces a c0 2 =z4 fragment pair. In other words, the peptide radicals containing radical sites at the 2nd and 3rd positions produced a c0 2 =z4 fragment pair via the N–Ca bond cleavage at Xxx1–Xxx2Pro (Scheme 6a). By contrast, if mainly N–Ca bond

SCHEME 6. Proposed mechanism for N–Ca bond cleavage of hexapeptide containing proline residue at position 4. (a) Homolytic and (b) heterolytic N–Ca bond cleavage based mechanisms. Red and blue arrows indicate the fragmentation pathways by homolytic and heterolytic N–Ca bond cleavages, respectively. Reprinted with permission from Asakawa et al. (2014a). Copyright 2014, Springer.

cleavage located on the N-terminal side of a radical site are assumed to occur (Scheme 4b), the aminoketyl radical containing a radical site at the nth position in the hexapeptide should yield the c0 ðn1Þ =zð7nÞ fragment pair via heterolytic N–Ca bond cleavages, but this would not occur at the N-terminal side of the proline residue (Scheme 6b). The aminoketyl radical at position 4 (red radical in Scheme 6b) would give c0 4 =z2 fragment pairs via homolytic N–Ca bond cleavage at the Pro–Xxx bond, instead of via heterolytic cleavage of Xxx–Pro bond. According to this hypothesis, MALDI-ISD would be expected to produce strong ISD ions originating from Pro–Xxx bond cleavage. To investigate the influence of proline residues on fragment yield, we used a hexadecapeptide (YYERQ QQQQQ QQQRY Y) as a reference and the same peptide, which includes proline and cysteine residues at positions 8 and 11, respectively (YYERQ QQPQQ CQQRY Y), as a model. To better elucidate cleavage efficiency, the spectra should first be normalized, because the intensity of ISD ions also decreases as their m/z values increase. We used a modified normalization method according to the procedure proposed previously (Takayama, 2012). With this method, the normalized intensity of ISD ions was calculated as the ratio of the intensity of each c0 or z0 ion divided by the sum of the intensities of the previous and the next ISD ions (Equation 1),

SCHEME 5. Radicals located on the (a) N- and (b) C-terminal sides of proline induced N–Ca bond cleavage.

540



allowing us to compensate for the detection efficiency of ISD ions which depends on their m/z values. N I xn ¼

I xn ðx ¼ c0 or z0 Þ I xðn1Þ þ I xðnþ1Þ

ð1Þ

Since the proline has a cyclic structure, the formation of the c0 and z0 fragments could not have originated from the cleavage of Xxx–Pro bond. As a consequence, no c0 and z0 fragment ions originating from the N-terminal side of proline residues are generated. Alternatively, the normalized intensities adjacent to both sides of Xxx–Pro were calculated by Equation (2). N I c0 n ¼

I c0 n ; I c0 ðn1Þ þ I c0 ðnþ2Þ

N I z0 n ¼

I z0 n I z0 ðn2Þ þ I z0 ðnþ1Þ

ðCleavage at Xxx1 Xxx2 ProÞ

N I c0 n ¼

I c0 n ; I c0 ðn2Þ þ I c0 ðnþ1Þ

N I z0 n ¼

ð2Þ

I z0 n I z0 ðn1Þ þ I z0 ðnþ2Þ

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As shown in Figure 3, the cleavage at Gln6–Gln7Pro8 was susceptible to the generation of intense fragments. The strong ISD ion signals originating from Xxx1–Xxx2Pro cleavages are observed in other peptides and proteins (Asakawa et al., 2014a). These results suggest that in general the “Proline effect” holds for Xxx1–Xxx2Pro bonds, where Xxx1 and Xxx2 can be any amino acid except proline. Therefore, experimental observations are in good agreement with the proposed “homolytic N–Ca bond cleavage based mechanism” illustrated in Scheme 6a. In contrast, the presence of the proline residue did not contribute to an increase in the ISD fragment yield originating from Pro–Xxx bond cleavage, indicating that the “heterolytic N–Ca bond cleavage-based mechanism” illustrated in Scheme 6b is not likely to be a dominant process in MALDI-ISD. In summary, our results suggested that the aminoketyl radical generally attacks the N–Ca bond located on the C-terminal side of the radical site and then forms a c0 /z• fragment pair via homolytic cleavage, except for the radical site at the N-terminal side of proline residue thereby inducing heterolytic cleavage at the N–Ca bond located on the N-terminal side of the radical site (Scheme 6a).

D. Reaction of Radical z• Fragment ðCleavage at Pro Xxx3 Þ

ð3Þ

Figure 3 shows the relationship between the normalized intensity of ISD ions and the amino acid sequence. These results clearly indicate the proline and cysteine residues to have promoted the generation of ISD fragments. The proline effect is the focus of this section but that of cysteine residue is described in Section II-F.

FIGURE 3. The influences of amino acid residues on normalized intensities of ISD ions of (a) hexadecapeptide and (b) (Pro8, Cys11)-hexadecapeptide. Reprinted with permission from Asakawa et al. (2014a). Copyright 2014, Springer.


As described in section II-C, MALDI-ISD produces c0 /z• fragment pair by radical-induced N–Ca bond cleavage. Although c0 fragments are stable, radical z• fragments either undergo further decomposition or react with a matrix molecule due to their low stability (Kocher, Engstrom, & Zubarev, 2005). Finally, z, z0 , [z þ matrix], and w fragments are generated from radical z• fragments. As described previously, the observed fragments in the MALDI-ISD mass spectrum are dependent on the positions of basic residues. When peptides and proteins contain a basic residue at the N-terminal side, mainly c0 fragments are observed, facilitating interpretation of MALDIISD mass spectra. However, a peptide with a basic residue at the C-terminal side preferentially yields C-terminal side fragments, that is, z, z0 , [z þ matrix], and w fragments, such that the mass spectra are more complex. To avoid misinterpretation of the spectrum, it is important to determine the predominant factor in the reaction of radical z• fragments. The formation of w fragments can be explained by a-cleavage of z• radicals and thus w fragments would form via unimolecular dissociation of z• radicals. In contrast, the hydrogen and matrix attachments for z• radicals occur during the collision between matrix radicals and molecules in the MALDI plume. Hydrogen detachment from the z• radicals leading to z fragments is less likely to occur and thus the collision between these radicals and matrix molecules probably produces the z fragment, as well as z0 and (z þ matrix) formation. Therefore, the yield of z0 (z þ matrix), z and w fragments is presumably dependent on the collision rate in the MALDI plume. According to the desorption model described by Spengler and Kirsch, the collision rate is proportional to the initial velocities of analytes (Spengler & Kirsch, 2003). The order of these initial velocities is 2,5-DHB > 2-AA > 1,5-DAN 5-ASA 5-FSA CHCA (Table 2), which is the inverse order of that for the w ion yield (Asakawa, Smargiasso, & De Pauw, 2013a; Asakawa, Sakakura, & Takayama, 2013c). A lower initial velocity indicating a lower collision rate in the MALDI plume is expected to suppress the formation of z0 , z and (z þ matrix) fragments. This would allow the formation of w 541

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TABLE 2. Initial velocities of protonated analytes with different matrices normalized to the value obtained with CHCA

2,5-DHBa

2-AAc

5-ASAb

1,5-DANc

1.22

1.03

0.99

1.81 a

b

5-FSAb 0.93 c

Values were taken from Asakawa and Takayama (2012a), Asakawa, Sakakura, and Takayama (2013c), and Asakawa, Smargiasso, and De Pauw (2013a).

fragments due to a longer lifetime of the z• radical fragments. In contrast, the internal energy of the precursor would be an important factor in the fragmentation process, as well as the collision rate. In fact, the collisional activation of z• radical fragments can lead to side-chain loss (Han, Xia, & McLuckey, 2007). The internal energy of analyte ions in the MALDI-ISD process increases as the matrix proton affinity decreases (Demeure, Gabelica, & De Pauw, 2010). However, the proton affinity of the matrix showed no correlation with the abundance of z and w ions and it cannot be explained by the internal energy of analyte ions (Asakawa, Sakakura, & Takayama, 2013c). Therefore, the collision rate between the analyte and the matrix in the MALDI plume is the main factor mediating the reactions of the radical z• fragments. The w ions are very informative fragments and allow discrimination between isobaric residues, leucine and isoleucine (Scheme 7). As described previously, the existence of a charged site is necessary for the observation of ISD fragment ions. In other words, ISD fragments containing basic residues are selectively observed in positive ion MALDI-ISD mass spectra. As in the case of tryptic peptides, observation of z0 and w ions is expected due to the presence of a basic residue at the C-terminus and would allow leucine to be distinguished from isoleucine. In contrast, peptides containing an arginine residue near the N-terminus preferentially yield c0 fragments, which prevents discrimination between leucine and isoleucine. Notably, ISD fragments are also observed in negative-ion mode and the only difference between positive and negative ISD ions is the charge site. In negative-mode MALDI-ISD, the favored sites of deprotonation in the peptides are acidic groups. Typically, peptides containing an acidic carboxyl group at the C-terminus give C-terminal side fragments in negative-ion MALDI-ISD mass spectra, allowing discrimination between leucine and isoleucine. The 1,5-DAN matrix has been found to be good for negative-ion and w fragment formation due to their high proton affinity and low collision rate in this condition. As a consequence, negative-ion MALDI-ISD with 1,5-DAN allows leucine and isoleucine residues to be directly discriminated in peptides and proteins near the C-terminal side, including top-down sequencing of proteins (Asakawa, Smargiasso, & De Pauw, 2013a).

SCHEME 7. Side-chain loss from d• and z• radicals at (a) leucine and (b) isoleucine residues.

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E. Formation Mechanism of a•/y 0 Fragment Pair MALDI-ISD with reducing matrix also produces a and y0 ions, which cannot be explained by the N–Ca bond cleavage model alone. To investigate the matrix effect on a and y0 ion yield, two model peptides were analyzed by MALDI-ISD with different matrices, 1,5-DAN (Fig. 2) and 2,5-DHB (Fig. 4). By comparing Figures 2 and 4, it can be seen that the use of 2,5-DHB promotes to generate the a and y0 ions by MALDI-ISD. The order of a and y0 ion yields is 2,5-DHB 5-ASA > 1,5-DAN, which is inverse proportion to the order of the matrix proton affinity (Demeure, Gabelica, & De Pauw, 2010). Since protonated analyte molecule was formed by proton transfer from protonated matrix to analyte, matrix proton affinity correlates with the internal energy of the protonated analyte produced by MALDI. Importantly, CHCA does not efficiently work as a hydrogen radical donor during the MALDI process and MALDI-ISD with CHCA mainly leads peptide bond cleavage, giving b and y0 ions (Bae, Moon, & Kim, 2011). Recent studies have shown that the addition of ammonium persulfate in CHCA solution enhanced the production of b and y0 fragments by MALDI-ISD (Horvatic et al., 2013). Consequently, b and y0 were probably generated via thermal activation processes in the MALDI plume. However, interestingly, y0 fragment ions are often observed in MALDIISD mass spectra with reducing matrix such as 2,5-DHB and 1,5-DAN, whereas their counterpart b fragment ions are usually absent (Figs. 2 and 4). Alternatively, a ions are usually observed in MALDI-ISD mass spectra, instead of b ions. Moreover, radical a• ions are occasionally present in MALDI-ISD mass spectra (Smargiasso, Quinton, & De Pauw, 2012). The radical fragments are less likely to originate from closed shelled intact molecules via thermal activation. Therefore, peptide bond

FIGURE 4. Positive-ion MALDI-ISD mass spectra of (a) renin substrate (DRVYI HPFHL VIHN) and (b) fibrinopeptide A (ADSGE GDFLA EGGGV R) using 2,5-DHB matrix. Asterisks and asterisk on z fragments indicate metastable ions and 2,5-DHB adducts on z fragments. Reprinted with permission from Asakawa, Smargiasso, and De Pauw (2013a). Copyright 2013, Springer.



cleavage by thermal activation, leading to the b/y0 fragment pair, is less likely to occur in the MALDI-ISD process with reducing matrix, the exception being proline-rich peptides without a basic chemical group. These peptides are very labile and leads to peptide bond cleavage at the N-terminus nearest the first proline of the XPP motif by MALDI-ISD (Sachon et al., 2009). Notably, the a and y0 fragments are barely produced using a non-crystal mixture of 2,5-DHB/1,5-DAN which suppresses the formation of hydrogen-abundant peptide radicals, as described in Sections II-A and II-B (Asakawa et al., 2013e). This provides evidence supporting the involvement of hydrogen-abundant peptide radicals in the formation of a•/y0 fragments during the MALDI-ISD process. Since MALDI-ISD shares some mechanistic similarities with ECD (Kocher, Engstrom, & Zubarev, 2005), a mechanism proposed to explain the occurrence of a•/y0 ion pairs in ECD experiments can be envisaged for MALDI-ISD (Scheme 8a). According to the proposed process, dissociation of hydrogen-abundant peptide radicals resulted in the formation of a b•/y0 fragment pair and then fragmentation of the b• fragments, giving only a• fragments via loss of a carbon monoxide molecule (Zubarev et al., 1999). As to MALDI-ISD processes, intermediate b• fragments are formed by cleavage at the Xxx–Pro bonds by using an oxidizing matrix, and these b• fragments can yield both a and b fragments, indicating that b• fragments have sufficient stability to form a band b0 fragments via radical recombination (Asakawa & Takayama, 2011b). The details of these fragmentation processes are described in Section III-C. As a consequence, the b• fragments would produce b0 and b fragments, whereas these fragments are absent in MALDI-ISD mass spectra obtained with a reducing matrix. This suggests that

SCHEME 8. Proposed formation pathways of the a•/y0 fragment pair by hydrogen attachment via (a) b•/y0 and (b) a•/x0 fragments pair intermediates. Reprinted with permission from Asakawa et al. (2013e). Copyright 2013, American Chemical Society.


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formation of a•/y0 fragment pair likely involves an alternative intermediate. It should be noted that an a•/x0 fragment pair is observed in MALDI-ISD mass spectra with 2-AA and 2-AB (Smargiasso, Quinton, & De Pauw, 2012), suggesting the x0 fragment to be an intermediate of the y0 fragment. Because the transfer of a hydrogen atom from a matrix molecule to the carbonyl group of a peptide backbone initiates MALDI-ISD, the existence of an oxygen-centered radical is possible. If this were the case, an a•/x0 fragment pair would be generated by the acleavage of the oxygen-centered radical. A proposed pathway for the formation of an a•/y0 fragment pair is shown in Scheme 8b. However, x0 ions, which are intermediate species in the proposed mechanism, were only rarely observed in MALDIISD mass spectra, probably due to their low stability. According to H/D exchange experiments, MALDI-ISD produced c0 , z0 , and y0 fragments with low hydrogen scrambling levels (Bache et al., 2008). These results indicated that the formation of y0 fragments is less likely to occur via thermal activation, which usually involves extensive H/D scrambling. The proposed formation mechanism of a•/y0 fragment pair via hydrogen-abundant peptide radical is supported by the results of experiments using MALDI-ISD with H/D exchange. Although the mechanistic origins of a and y0 fragments are uncertain, the formation of these fragment pairs likely involves alternative fast dissociation pathways of the hydrogen-abundant peptide radicals.

F. Hydrogen Attachment to Cysteine Side Chain and Subsequent Reactions An interesting fragmentation behavior in MALDI-ISD was found to occur at residues adjacent to free cysteine residues (Kocher, Engstrom, & Zubarev, 2005) The MALDI-ISD mass spectrum of a cysteine-containing peptide shows strong intensities of ISD fragments, which are generated from Xxx–Cys bond cleavage. By comparing Figure 3a and b, it can be seen that the presence of cysteine residues in the (Pro8, Cys11)-hexadecapeptide contributes to increases in the ISD ion yields originating from Gln10–Cys11 cleavage. This clearly indicates the cysteine residue to have promoted the generation of ISD fragments. Notably, w fragments are generated by cleavage of the Nterminal side of cysteine residues, instead of z0 . As described in Section II-C, MALDI-ISD with a reducing matrix yields a c0 /z• fragment pair, and radical z• fragments then undergo either gain of a hydrogen atom or side-chain loss, leading to z0 or w fragments, respectively. These reactions occur competitively and the yield of z0 fragments is usually higher than that of w fragments. In contrast, the cleavage of Xxx–Cys yields only c0 and w fragments, not z0 fragments, suggesting the c0 and w fragments originating from Xxx–Cys bond cleavage to be generated without the formation of c0 /z• fragment pair. To explain this phenomenon, we proposed the fragmentation mechanism shown in Scheme 9 (Asakawa et al., 2013d). As sulfur atom has a high affinity for hydrogen radicals, the formation of c•/w fragment pairs would be initiated by hydrogen attachment of a thiol functional group via the b-carbon-centered radical. The high intensity of w ions and the lack of z0 ions can be better explained by this pathway. Interestingly, the MALDI spectrum of (Pro8, Cys11)hexadecapeptide (YYERQ QQPQQ CQQRY Y) shows [M þ H32]þ and [M þ H þ 124]þ, both of which were absent from 543

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attachment to a disulfide bond produces free cysteine residues. As expected, the presence of disulfide bonds in peptides and proteins suppresses the formation of b-carbon-centered radicals, whereas [M 32 þ H]þ and [M þ 124 þ H]þ were still observed. Formation of these fragments could be explained by a three-step process in which, first, a hydrogen attachment on a disulfide bond leads to the analytes containing free cysteine residues that further undergo a second hydrogen attachment, forming a b-carbon-centered radical. Subsequently, radicalinduced cleavage and a hydrogen/matrix attachment result in the formation of c•/w fragment pair and [M 32 þ H]þ/[M þ 124 þ H]þ, respectively (Scheme 9). For sequencing of peptides and proteins, the disulfide bonds are usually subjected to reduction followed by alkylation such as S-carbamidomethylation before mass spectrometric analysis. However, MALDI-ISD of peptides containing S-carbamidomethylated cysteine residues resulted in the loss of 57 Da which might be assigned as a CHCONH2 (Asakawa et al., 2013d). The loss of 57 Da is probably initiated by hydrogen attachment to the side-chain of an S-carbamidomethylated cysteine residue with subsequent •CH2CONH2 radical loss, as shown in Scheme 9. The formation of a b-carboncentered radical with CH2(SH)CONH2 loss is also possible via hydrogen attachment to a sulfur atom in the S-carbamidomethylated cysteine residue. However, the loss of CH2(SH) CONH2 was barely observed, indicating the b-carbon-centered radical to be not a dominant process in MALDI-ISD of peptides containing S-carbamidomethylated cysteine residues. Thus, MALDI-ISD highly favors the formation of free cysteine residues with •CH2CONH2 radical loss rather than a bcarbon-centered radical with CH2(SH)CONH2 loss. The S-carbamidomethylation therefore suppress the formation of b-carbon-centered radicals at cysteine residues, as in the case of the presence of a disulfide bond.

G. Influence of Ion Source Pressure SCHEME 9. Reactions of Cys residue with hydrogen attachment in MALDI-ISD. Reprinted with permission from Asakawa et al. (2013d). Copyright 2013, John Wiley & Sons, Ltd.

the MALDI spectrum of the hexadecapeptide of the reference (YYERQ QQQQQ QQQRY Y) (Asakawa et al., 2014a). These ions presumably formed due to side-chain loss from the cysteine residue with subsequent radical recombination, as shown in Scheme 9. In order to confirm the structures of [M þ H 32]þ and [M þ H þ 124]þ, we performed a PSD experiment, unambiguously showing the sites of 32 Da loss and 124 Da gain to be at a cysteine residue (Asakawa et al., 2014a). Thus, [M þ H 32]þ and [M þ H þ 124]þ must have been formed by sulfur loss at a cysteine residue and subsequent hydrogen/matrix attachment, indicating the b-carbon-centered radicals at the radical site on the cysteine residue to be intermediates of the c0 / w fragments, [M þ H 32]þ and [M þ H þ 124]þ (Scheme 9). The presence of disulfide bonds in peptides contributes to a decrease in the ion yields of c0 /w fragments originating from cleavage at Xxx–Cys bonds, [M–32 þ H]þ and [M þ 124 þ H]þ (Asakawa et al., 2013d). As described in Section II-A, hydrogen 544

The MALDI ion source is well-suited for use with axial time-offlight (TOF) mass spectrometers fitted with ion sources working at a pressure below 105 mbar. In order to obtain high mass resolution and accuracy, MALDI-ISD has been combined with Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer (Calligaris et al., 2013). In the FTICR configuration, the MALDI ion source is operated with an intermediate pressure ion source which contributes to cooling the generated intact peptide ions and ISD ions. The types of ISD ions observed in a MALDI source differ according to the ion source pressure and the mass analyzer used (Soltwisch & Dreisewerd, 2010). In this section, the mechanism leading to the final ISD ions for FTICR mass analyzer with an intermediate pressure ion source are reviewed. Figure 5 shows MALDI-ISD FTICR mass spectra of renin substrate and fibrinopeptide A (Asakawa et al., 2013b). The pressure in the MALDI ion source was adjusted to 4.2 mbar. Renin substrate and fibrinopeptide A contain an arginine residue near the N- and the C-terminus, respectively, and the observed ISD fragments are dependent on the position of this arginine residue in the MALDI-ISD TOF mass spectrum (Asakawa, Smargiasso, & De Pauw, 2013a). This phenomenon strongly supports the assumption that the proton is localized on the arginine residue in the protonated peptides formed by MALDI. Mass Spectrometry Reviews DOI 10.1002/mas


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FIGURE 5. Positive-ion MALDI-ISD mass spectra of (a) renin substrate (DRVYI HPFHL VIHN) and (b) fibrinopeptide A (ADSGE GDFLA EGGGV R) obtained with FTICR MS. 1,5-DAN was used as the matrix. The fragments annotated with an asterisk correspond to 1,5-DAN adducts on the fragments. Reprinted with permission from Asakawa et al. (2013b). Copyright 2013, American Chemical Society.

However, results of MALDI-ISD performed with FTICR mass spectrometer favors the formation of z0 ions from renin substrate (Fig. 5a) and c0 ions from fibrinopeptide A (Fig. 5b), as compared with that in MALDI TOF mass spectrometer (Fig. 2). The major difference between the MALDI ion sources in TOF and FTICR mass spectrometers is pressure. During MALDITOF measurements, resulting ions are transferred to the fieldfree region in TOF mass spectrometer without collision with background gases. In contrast, the intermediate pressure ion source in FTICR mass spectrometer causes desorbed ions to collide with background gases. The collisional process during desorption contributes to increase the lifetimes of ions by internal energy transfer from peptide ions to background gases (Soltwisch et al., 2009). In contrast, MALDI-ISD FTICR results indicate that the proton is mobilized from the arginine residue to less favored protonation sites during the ISD process (Asakawa et al., 2013b). It is well known that ions are not only cooled but can also be activated by collision and low-energy collisional activation of protonated peptides leads to various isomers containing different protonation sites (Dongre et al., 1996). After the formation of c0 and z• fragments, proton transfers between c0 and z• fragments are no longer possible. Therefore, proton mobilization occurs before or at the same time as the formation of c0 /z• fragment pairs which are generated rapidly after desorption. During the early desorption step, the presence of buffer gases probably contributes to activation of the generated ions (Asakawa et al., 2013b). MALDI-ISD FTICR with 1,5-DAN produced not only c0 and z0 ions, but also a and y0 ions. As described in Section II-E, the yield of a and y0 ions as the internal energy of peptide ions, which depends on matrix proton affinity, increases (Demeure, Gabelica, & De Pauw, 2010). Notably, these fragments only rarely appeared in the high-vacuum MALDI-ISD experiment with 1,5-DAN (Asakawa, Smargiasso, & De Pauw, 2013a). This Mass Spectrometry Reviews DOI 10.1002/mas

indicates that 1,5-DAN did not provide enough internal energy to produce a•/y0 fragment pair. In contrast, when intermediate pressure MALDI-ISD was used, a•/y0 fragment pair formed via hydrogen attachment followed by collisional activation. Furthermore, intermediate pressure MALDI-ISD (FTICR mass spectrometer) generates d and w fragments, which formed via radical-induced cleavages at the Cb–Cg bonds of a• and z• radical fragments, respectively (Scheme 7). The presence of d ions is evidence for the existence of a• radicals which are considered to be intermediates of a and d fragments. The proposed a•/y0 fragment pair formation mechanism, as shown in Scheme 8, is supported by this observation. The d and w ions are informative fragments for discriminating between the isobaric amino acid residues, leucine and isoleucine. As described in Section II-D, the use of 1,5-DAN in vacuum MALDI-ISD highly favors the formation of w ions, via a-cleavage of z• fragments due to the prolonged lifetimes of the intermediates under these conditions. In contrast, d fragments which are generated by side-chain loss from a• fragments, are rarely observed in high-vacuum MALDIISD (TOF mass spectrometer) (Demeure, Gabelica, & De Pauw, 2010). Therefore, only the C-terminal side ISD fragments gave side chain information, allowing discrimination between leucine and isoleucine (Asakawa, Smargiasso, & De Pauw, 2013a). For renin substrate, which contains three isobaric residues, positive ion high-vacuum MALDI-ISD producing mainly N-terminal side fragments provided no information for discriminating between isobaric residues (Fig. 2a). By contrast, all isobaric amino acid residues in renin substrate and fibrinopeptide A could be discriminated by MALDI-ISD combined with FTICR mass spectrometer (inset of Fig. 5). Therefore, intermediate pressure MALDI-ISD (FTICR mass spectrometer) is a better method than high-vacuum MALDI-ISD (TOF mass spectrometer) for discriminating between leucine and isoleucine. 545

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III. PRINCIPLE OF MALDI-ISD WITH OXIDATION A. Cleavage of Ca–C Bond in the Peptide Backbone With Hydrogen Abstraction MALDI-ISD fragmentation behaviors differ markedly according to the chemical properties of the matrix used. Our research group recently reported an alternative Ca–C bond cleavage in MALDI-ISD which can be observed when employing an oxidizing matrix (Asakawa & Takayama, 2011a, 2012a). The use of an oxidizing matrix, 5-NSA or 2,5-bis(2-hydroxyethoxy)7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ) generated a ions with strong signal intensities and no c0 ions were produced (Fig. 6). Additionally, the x fragments were observed as Cterminal side fragments in MALDI-ISD performed with an oxidizing matrix (Asakawa & Takayama, 2011a), indicating Ca–C bonds to be cleaved under these conditions. The inset of Figure 6 shows the oxidized product [M–2H þ H]þ which was formed by hydrogen transfer from a peptide to the oxidizing matrix. It suggests that the use of oxidizing matrix results in MALDI-ISD involving “hydrogen-deficient” peptides as radical precursors, instead of “hydrogen-abundant” peptide radicals. The abstraction site of the hydrogen involved in the MALDIISD with oxidizing matrix was investigated using peptides containing deuterium at the a-carbon (Asakawa & Takayama, 2011a). No a ions originating from deuterium abstraction from the a-carbon were detected. According to these results, the a/x fragment pair is formed via abstraction of the amide hydrogen on the peptide backbone with subsequent Ca–C bond cleavage. The MALDI-ISD processes performed with oxidizing matrices are summarized in Scheme 10. Upon forming this hydrogen-deficient peptide, two Ca–C bond cleavage pathways, giving either a•/x or a/x• fragment pairs, are theoretically possible. However, all ISD fragment ions contain even numbers of electrons as in the case of MALDI-ISD performed with a reducing matrix. To identify the most plausible pathway for the formation of a and x fragments, we focused on

FIGURE 6. MALDI-ISD of ACTH18-35 with (a) 5-NSA and (b) bisHETCNQ. Reprinted with permission from Asakawa and Takayama (2012a). Copyright 2012, American Chemical Society.

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SCHEME 10. Mechanism of MALDI-ISD via hydrogen abstraction.

the Ca–C bond cleavages adjacent to proline residues. Since a nitrogen-centered radical site is absent from the proline residue, Ca–C bond cleavage at Pro–Xxx and Xxx–Pro bonds would lead to a•/x and a/x• fragment pairs, respectively (Scheme 11). The x fragments originating from Ca–C bond cleavage at the Nterminal side of a proline residue are absent in MALDI-ISD mass spectra obtained with an oxidizing matrix (Asakawa & Takayama, 2011b). This indicates that fragmentation leading to an a/x• fragment pair does not occur, which further suggests that the mechanism proposed in Scheme 11a generally holds for Ca– C bonds that do not involve a Xxx–Pro bonds. Although x ions arising from the Ca–C cleavage at Xxx–Pro are absent, their counterpart a ions are observed. The observed a fragments

SCHEME 11. Formation of (a) a•/x and (b) a/x• fragment pairs originating from the Ca–C bond cleavage adjacent to the proline residue. Reprinted with permission from Asakawa and Takayama (2011b). Copyright 2011, John Wiley & Sons, Ltd.



originating from Xxx–Pro bond cleavage would thus be formed via alternative peptide bond cleavage (Asakawa & Takayama, 2011b). The details of this formation mechanism are described in Section III-C. An ab initio calculation showed that formation of an a•/x fragment pair is more likely than that of an a/x• fragment pair (Anusiewicz et al., 2005). Therefore, the proposed Ca–C bond cleavage mechanism leading to a•/x fragment pair is supported by the ab initio calculation. However, radical a• ions are not observed, and instead a ions are detected. It is likely that the amounts of excited oxidizing matrix molecules leaving the MALDI plume are sufficient to form a ions via further hydrogen abstraction after the Ca–C bond cleavage. Notably, the Ca–C bond cleavage at C-terminal side of glycine residue, Gly–Xxx is less susceptible to fragment formation in MALDI-ISD with an oxidizing matrix than are those of other residues (Asakawa & Takayama, 2011b). If thermochemistry were the driving force for Ca–C bond cleavage, cleavage efficiency would be expected to depend on Ca–C bond strength which correlates with the stability of a•/x fragment pair. The stability of the radical a• fragment would be lower than that of the closed-shell x fragment. Thus, the required energy for Ca–C bond cleavage increases as the stability of the a• fragment decreases, which would be regarded as the stability of an individual amino acid radical that contained a radical site on the a-carbon atom. According to the ab initio calculation, glycine radicals have the lowest stability of the 12 calculated amino acids (Savitski et al., 2007). When the a• fragment contained a valine side-chain at the radical site, the formation of an a•/x fragment pair from a hydrogen-deficient peptide radical is nearly thermoneutral (Anusiewicz et al., 2005). The formation enthalpy of a glycine radical is 20–25 kJ/mol higher than that of a valine radical (Savitski et al., 2007). In consequence, formation of an a•/ x fragment pair from Gly–Xxx bond cleavage would be regarded as an endothermic process. The low abundance of a ions originating from Gly–Xxx cleavage can be understood from the low stability of the radical a• fragment. The formation of peptide radical cation, Mþ• via CID of Cu2þligand-peptide complexes has been reported (Hopkinson, 2009). Peptides having an aromatic residue formed abundant hydrogendeficient peptide radical by this method. Aromatic residues are easily oxidized due to their low ionization energy and produce stable radical cation containing the charge and radical sites at aromatic side chain. In the other case, peptide radical cation took a protonated hydrogen-deficient radical form in which the a-carbon was radical center and that a proton was present elsewhere in the peptide. Although CID of Cu2þ-ligand-peptide complexes and MALDI-ISD performed with an oxidizing matrix produced same composition of peptide radicals, their chemical property are different each other. In contrast, MALDI-ISD performed with an oxidizing matrix shares some mechanistic similarities with fragmentation techniques involving electron ejection from analyte ions, such as electron detachment dissociation (EDD) (Budnik, Haselmann, & Zubarev, 2001), negative electron transfer dissociation (NECD) (Coon et al., 2005) and femtosecond laser-induced ionization/dissociation (fs-LID) (Kalcic et al., 2009). EDD and NECD have been developed for the identification of multiple deprotonated peptides which cause electron detachment/abstraction by using a fast electron and a radical cation, respectively. Electron detachment/abstraction from multiple deprotonated peptides results in the formation of a charge-reduced peptide anion that contains a radical site on the carboxyl group of the side chain or C-terminal Mass Spectrometry Reviews DOI 10.1002/mas

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carboxyl group. Subsequently, a nitrogen-centered radical product is formed via hydrogen transfer from the backbone amide nitrogen to the radical site on the carboxyl group. The radical on the amide nitrogen induces dissociation of the Ca–C bond, leading to the formation of a•/x fragment pair (Kjeldsen et al., 2005). In contrast, fs-LID is a fragmentation method operating in positive-ion mode. Fs-LID causes electron ejection from protonated peptides via femtosecond laser irradiation, forming a charge-increased peptide cation. Subsequently, the radical induces dissociation of the Ca–C bond, as in the case of EDD and NECD. Although EDD/NECD, fsLID and MALDI-ISD performed with oxidizing matrices involve different precursors, backbone cleavages by these techniques are mechanistically similar, leading to the formation of hydrogendeficient peptide radicals and subsequent radical-induced Ca–C bond cleavage.

B. Further Hydrogen Abstraction From HydrogenDeficient Peptide Radicals The focus of this section is the formation mechanism of [M 2H þ H]þ. Hydrogen abstraction from peptides results in the formation of a hydrogen-deficient peptide radical in which the radical site is on the amide nitrogen. Subsequently, further hydrogen abstraction from the peptide radical to the matrix leads to the formation of [M 2H þ H]þ. The hydrogen-deficient peptide radical undergoes either loss of a hydrogen atom or radical-induced Ca–C bond cleavage and these processes competitively occur during MALDI-ISD via hydrogen abstraction. The 5-NSA matrix highly favors the formation of the oxidized product [M 2H þ H]þ as compared with bisHE-TCNQ, whereas the use of bisHE-TCNQ produces higher yields of a fragments (Fig. 6). An ab initio calculation showed that the formation of an a•/x fragment pair from the hydrogen-deficient peptide radical is nearly thermoneutral (Anusiewicz et al., 2005), which is expected when formed via a unimolecular dissociation process. In contrast, the hydrogen detachment from the hydrogen-deficient peptide radical is an endothermic process and is relatively unlikely to occur in the MALDI plume. Therefore, the hydrogen abstraction reactions from the hydrogen-deficient peptide radical occur via the collisions between hydrogen-deficient peptide radicals and matrix molecules in the MALDI plume. The yield of these products is expected to be dependent on the collision rate in the MALDI plume, as in the case of reaction of z• radical generated by MALDI-ISD with reducing matrix. As described in Section IID, the collision rate could be estimated based on the initial velocities of the analytes (Spengler & Kirsch, 2003). The collision rate order in the MALDI plume is 5-NSA > 4-NSA >3NSA > 5-FSA >TCNQ > bisHE-TCNQ (Table 3), which is proportional to the order of the yields of [M 2H þ H]þ (Asakawa & Takayama, 2012a; Asakawa, Sakakura, & Takayama, 2013c). The high abundance of [M 2H þ H]þ with higher initial velocities of analyte ions can be explained by the high collision rate in the MALDI plume allowing further hydrogen abstraction from hydrogen-deficient peptide radicals.

C. Cleavage of Peptide Bond at N-Terminal Side of the Proline Residue As described in Section III-A, there are no x ions arising from cleavage at the N-terminal side of proline, with b and y ions 547

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TABLE 3. Initial velocities of protonated ACTH18-35 with different matrices normalized to the value obtained with CHCA

5-NSAa

4-NSAb

3-NSAb

5-FSAb

TCNQa

bisHE-TCNQa

1.30

1.13

0.93

0.70

0.57

1.74 a

b

Values are taken from Values were taken from Asakawa and Takayama (2012a), Asakawa, Sakakura, and Takayama (2013c).

resulting from the peptide bond cleavage at the same position instead being observed. Thus, alternative hydrogen abstraction from the Ca–H bond at a proline residue generates the a-carboncentered radical due to the lack of amide hydrogen. Subsequently, the peptide bond at the N-terminal side of the proline residue would be cleaved by the a-cleavage of the a-carbon-centered radical. A proposed pathway for the formation of b•/y fragment pair is shown in Scheme 12. To ascertain the most probable pathway for the formation of b and y fragments originating from the cleavage of Xxx–Pro bonds, this section focuses on the ISD fragments arising from a cleavage adjacent to the sarcosine residue (N-methyl glycine residue, Sar), which does not contain a hydrogen radical at the amide portion of the peptide backbone, as in the case of proline residues. The cleavage at the N-terminal side of the sarcosine residue gave both a and b ions, supporting the proposed fragmentation pathway shown in Scheme 12 (Asakawa & Takayama, 2011b). The abstraction of hydrogen atom from the Ca–H in proline and sarcosine residues results in the formation of a carbon-centered radical with subsequent radical-induced peptide bond cleavage, leading to the generation of b and y ions. As described in Section III-A, no evidence was

found for the production of x fragments originating from a cleavage at Xxx–Pro, whereas the counterpart a ions were still observed. The cleavage of the N-terminal side the sarcosine residue also gave a fragments. These results suggest that a ions originating from the cleavage of Xxx–Pro and Xxx–Sar bonds are formed by the further degradation of b• ions (Scheme 12), indicating the competitive formation of b and a fragments from b• to be occurred during MALDI-ISD via hydrogen abstraction. Then, the b• radical fragment undergoes either the loss of a formyl radical (•CHO) or of a hydrogen radical. As shown in Figure 6, the use of 5-NSA favors the formation of b6 ions, whereas MALDI-ISD with bisHE-TCNQ gave an intense a6 ion signal. The intensity ratio of b/a ions would be expected to be affected by the initial velocities of analyte ions, reflecting the collision rate in the MALDI plume. Indeed, the order of the intensity ratios of b6/a6 in MALDI-ISD of ACTH18-35 was 5NSA > 4-NSA 3-NSA > 5-FSA bisHE-TCNQ, which is proportional to the order of the initial velocities of the analyte ions (Asakawa & Takayama, 2012a; Asakawa, Sakakura, & Takayama, 2013c). This suggests that the formyl radical loss from b• fragments leading to the formation of a fragments occurs via a unimolecular dissociation process, whereas the b fragments formed via collisions with b• ions and matrix molecules in the MALDI plume. It is important to recognize that the initial velocities of analyte ions area prominent factor in the fragmentation processes of MALDI-ISD employing both reducing and oxidizing matrices.

IV. DEVELOPMENT OF THE MALDI-ISD APPLICATIONS IN THE FIELD OF PROTEOMICS A. Combination of Redox Reactions

SCHEME 12. The mechanism of peptide bond cleavage at the N-terminal side of the proline residue. Reprinted with permission from Asakawa and Takayama (2011b). Copyright 2011, John Wiley & Sons, Ltd.

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As explained before, MALDI-ISD is initiated by a hydrogen radical transfer reaction between an analyte and the matrix, which leads to the peptide radical. Two radical formation pathways in MALDI-ISD have been described to date, that is, hydrogen attachment onto the peptide backbone and hydrogen abstraction from the peptide backbone. Hydrogen attachment to peptides in MALDI-ISD occurs when using a reducing matrix, whereas MALDI-ISD with hydrogen abstraction can be achieved by employing an oxidizing matrix. The resulting hydrogen-abundant and -deficient peptide radicals undergo radical-induced cleavages at N–Ca and Ca–C bonds, respectively. The mechanism of MALDI-ISD is briefly summarized in Scheme 13. The use of reducing and oxidizing matrices preferentially yields c0 /z0 and a/x fragment pairs, respectively. An oxidizing matrix provides useful information complementary to that obtained by MALDI-ISD, in contrast to a reducing matrix, for the identification of sequence ions in MALDI-ISD mass spectra. A phosphopeptide was used as an example to show the applicability of MALDI-ISD employing both reducing and oxidizing matrices (Asakawa & Takayama, 2012c). To Mass Spectrometry Reviews DOI 10.1002/mas


SCHEME 13. Mechanisms of peptide fragmentation by MALDI-ISD with redox reactions.

determine the peptide sequence, MALDI-ISD with reducing and oxidizing matrices were recorded and compared to each other. In MALDI-ISD with 1,5-DAN, the peptides principally cleaved at the N–Ca bond on the peptide backbone without degradation of the phosphate group, thereby allowing sequence identification including the location of the phosphorylation sites (Fig. 7b). However, consecutive series of c0 ions were difficult to identify, due to interference by a ions and ions from contaminants. To avoid misinterpreting ISD fragment ions, the MALDI-ISD mass spectrum with 1,5-DAN was compared to that obtained with 5-

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NSA, which is known to induce specific Ca–C bond cleavages (Fig. 7b). Notably, the multiple peaks shown in Figure 7a are 45 Da heavier than that in Figure 7b. Therefore, the c0 ions in the MALDI-ISD mass spectrum obtained with 1,5-DAN can be identified by comparing the peaks with those for a ions. MALDI-ISD with both reducing and oxidizing matrices used in conjunction provides information allowing unambiguous sequencing of peptides. However, MALDI-ISD performed with an oxidizing matrix did not provide enough useful information for top-down sequencing of proteins, due to the low fragmentation efficiency under these conditions. Since the choice of matrix can dramatically affect the quality of the MALDI mass spectrum, it will be necessary to find better oxidizing matrices that allow identifying proteins directly.

B. Use of High Resolution Mass Spectrometer The focus of this section is the advantages of high mass resolution/mass accuracy measurement for MALDI-ISD experiments. Recent studies have shown that the use of FTICR mass spectrometer for MALDI-ISD experiments provides high mass resolution and accuracy measurements and thereby facilitates the interpretation of MALDI-ISD mass spectra (Asakawa et al., 2013b; Calligaris et al., 2013). For MALDI-ISD experiments using the FTICR mass spectrometer, the MALDI ion source is operated with an intermediate pressure ion source

FIGURE 7. Positive-ion MALDI-ISD mass spectra of phosphorylated peptide (RELEELNVPGEIVEpSLpSpSpSEESITR) obtained with (a) 1,5-DAN and (b) 5-NSA. Asterisks indicate the signals originated from 1,5-DAN matrix. Reprinted with permission from Asakawa and Takayama (2012c). Copyright 2012, John Wiley & Sons, Ltd.


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ISD ions. Moreover, MALDI-ISD FTICR MS imaging approach is potentially applicable to the analysis of tissue sections and allows the simultaneous identification and localization of proteins present in tissue sections (Calligaris et al., 2013). Therefore, MALDI-ISD FTICR can be used for rapid detection and identification of proteins present in mixtures or tissue sections by comparison with spots of pure target proteins.

C. Development of Software

FIGURE 8. MALDI-ISD mass spectra of amyloid b protein obtained by (a) TOF mass spectrometer in reflectron mode and (b) FTICR mass spectrometer. 1,5-DAN was used as the matrix. Reprinted with permission from Asakawa et al. (2013b). Copyright 2013, American Chemical Society.

which contributes to cooling the generated intact peptide ions and ISD ions. The influence of the buffer gas in the ion source on the ISD fragmentation processes is described in Section II-G. The high mass resolution of the FTICR mass spectrometer helps to resolve isobaric matrix–analyte and analyte–analyte doublet peaks. The amyloid b protein 1–28 was used as an example to show the applicability of MALDI-ISD FTICR mass spectrometer (Asakawa et al., 2013b). The monoisotopic masses of protonated amyloid b protein 1–28 fragments, c0 17 and z0 18 are 2,066.98 and 2,069.04, respectively. Figure 8 shows enlarged MALDI-ISD mass spectra for these fragment regions. Although a monoisotopic signal of z0 18 (m/z 2,069.04) overlapped with that of a c0 17 fragment, it contained two 13C atoms (m/z 2,068.99) in the TOF mass spectrum (Fig. 8a), while FTICR with a narrow band measurement allows the detection of both ISD ions (Fig. 8b). Thus, well-resolved peaks with high mass accuracy in MALDI-ISD FTICR mass spectra allow the construction of reliable tags for peptide and protein sequencing. The high mass resolution/mass accuracy measurement provided by FTICR mass spectrometer gives useful information for the identification of proteins in mixture. Figure 9 shows a MALDI-ISD FTICR MS imaging experiment performed on five spots, pure solution of ubiquitin, a-crystalline, bovine serum albumin (BSA), myelin basic protein (MBP), and b-casein, as well as an additional spot corresponding to an equimolar mixture of these proteins (Calligaris et al., 2013). According to the signals observed on the ion images in Figure 9, it is possible to identify all proteins by comparing series of analog ISD ion peaks between from the protein mixture and the spots of pure protein solutions. Additionally, the sequence tag based searching using Mascot confirmed protein identification from the spot of protein mixture except for a-crystallin, which has a Mascot score below the identity threshold due to the low intensity of its 550

As described previously, the application of MALDI-ISD employing both reducing and oxidizing matrices and high mass resolution/accuracy measurement can enable highly accurate identifications of proteins. However, if more than one protein is present in a sample, MALDI-ISD can lead to a complicated mass spectrum. As a consequence, specialized software for automated identification of proteins from a mixture decreases the time needed for interpretation of the MALDI-ISD mass spectrum. Software for MALDI-ISD imaging has been developed that automates sequencing and constructs ion images (Zimmerman et al., 2011). For MALDI-ISD imaging of protein mixtures, the localizations of proteins differ among matrix crystals as shown in Figure 9, and each protein must be identified from mass spectra selected from pixels of high intensity in the imaging dataset (Calligaris et al., 2013). First, the imaging dataset is searched, where the software finds groups of highly correlated peaks that occur together and that are likely to correspond to a single protein. Second, the software then identifies the highest quality spectra from the imaging dataset for automated de novo sequencing using mass differences calculated from the spectral peak list. After the first protein has been automatically sequenced or identified, another search is run in the imaging dataset to find spectral patterns of peaks that are different from that of the first sequenced protein, which are likely to correspond either to a second protein or to the first protein with post-translational modification. This semi-automated process is iterated for an exhaustive characterization of all of the proteins in the MALDI-ISD imaging dataset (Zimmerman et al., 2011). Since the software uses mass differences calculated from spectral peak lists to perform protein sequencing, we found that the sequencing ability of MALDI-ISD could be improved by high-resolution mass spectrometer, which provides accurate mass differences between a series of consecutive c0 and z0 ions. The ISD fragments in the low m/z region are often hidden by the interference of isobaric matrix peaks. It hampers the identification of sequence ions required for the full sequence coverage of peptides and proteins. To identify these ions, we used our new software for in silico elimination of MALDI matrix peaks, which can be applied to any high mass resolution MALDI mass spectra (Asakawa et al., 2013b). The high mass resolution of FTICR MS facilitates the mass resolution of isobaric matrix–analyte doublet peaks, and matrix elimination further improves peak annotation. The removal of interfering matrix peaks aids the identification of ISD signals in the low m/z region and contributes to increased protein sequence coverage.

D. Pseudo-MSn Analysis Fragmentations during MALDI process are categorized as ISD and PSD, occurring in the MALDI ion source and in the analyzer between the ion source and the detector, respectively. In this Mass Spectrometry Reviews DOI 10.1002/mas


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FIGURE 9. MALDI-ISD FTICR MS imaging of an equimolar mixture of five proteins and of pure protein solutions. Ion images correspond to the signals acquired, for the equimolar mixture of five proteins and the pure protein solutions, for the ISD ions of ubiquitin (c0 11 and z0 15 ), a-crystallin (c0 14 and c0 16 ), BSA (c0 12 and c0 24 ), MBP (c0 10 and c0 12 ), and b-casein (y0 17 and y0 19 ). Numbers indicate the positions of each pure protein solution (1, ubiquitin; 2, a-crystallin; 3, BSA; 4, MBP; 5, b-casein). Reprinted with permission from Calligaris et al. (2013). Copyright 2013, American Chemical Society.

section, the usefulness of the pseudo-MS3, that is, the combination of ISD and PSD, for protein sequencing is reviewed. PSD involves the fragmentation of metastable ions occurring in the mass spectrometer after the ion extraction step and allows for


precursor ion selection (Spengler et al., 1992). Briefly, PSD involves excess energy being deposited on peptide ions during desorption and ionization processes, followed by conversion into vibrational energy that is distributed over the entire ion,

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leading to fragmentation (Chaurand, Luetzenkirchen, & Spengler, 1999). When the number of vibrational modes increases, fragmentation efficiency decreases due to less energy received per mode. Therefore, PSD is not directly applicable to top-down protein sequencing. On the other hand, the pseudoMS3, that is, mass selection of ISD ions followed by PSD, provides information on the identity of the C- and N-terminus of the protein, which are usually hidden by the interference of intense matrix peaks in MALDI-ISD mass spectrum (Suckau & Resemann, 2003). Moreover, pseudo-MS3 can facilitate confirming the protein sequences determined by MALDI-ISD and thereby contributes to increased protein sequence coverage. Additionally, pseudo-MS3 is a potentially useful method for discrimination of N- and C-terminal side fragments in a complex MALDI-ISD mass spectrum (Asakawa, Smargiasso, & De Pauw, 2014b). Since peptide and protein sequencing by MALDI-ISD is generally conducted by interpreting mass differences between series of consecutive c0 and z0 ions, discrimination of c0 and z0 ions is important to perform unambiguous sequencing of peptides and proteins. As described previously, the observed fragments in the MALDI-ISD mass spectrum are dependent on the positions of basic residues. In the case of topdown sequencing of proteins by MALDI-ISD, both N- and Cterminal side fragments are often present, because basic residues are located at various positions within proteins. This can make the attribution of m/z values to specific series of consecutive c0 and z0 ions difficult and thereby discrimination of N- and Cterminal side fragments by pseudo-MS3 analysis is important to identify the protein sequence. The pseudo-MS3 mass spectra were usually recorded via c0 or z0 fragments which are abundant ions in MALDI-ISD mass spectra. However, c0 and z0 ions could not be distinguished by their pseudo-MS3 mass spectra, because PSD of such fragments generates the same type of ions, mostly b and y0 ions. In contrast, our research group performed pseudoMS3 analysis via 1,5-DAN adduct on zn ions, [zn þ 1,5-DAN þ H]þ, which are formed by radical reaction between z• and 1,5DAN (Asakawa, Smargiasso, & De Pauw, 2014b). Herein, a small protein, myoglobin was used as an example to show the applicability of pseudo-MS3 analysis in top-down sequencing approach. As shown in Figure 10a, both N- and C-terminal side fragments of myoglobin are presented in the MALDI-ISD mass spectrum. PSD of the 1,5-DAN adduct on zn ions resulted in the dominant loss of an amino acid with 1,5-DAN from [zn þ 1,5DAN þ H]þ, giving [bxnþ1/zn þ 1,5-DAN], and y0 n1 fragments, where x represents the number of amino acid residues in the protein (Fig. 10b–e). Notably, [z17 þ 1,5-DAN þ H]þ at m/z 2,111.0 overlaps with ½c0 19 þ Hþ at m/z 2,114.1 and the pseudoMS3 mass spectrum shows the fragment ions originated from both [z17 þ 1,5-DAN þ H]þ and ½c0 19 þ Hþ . Although the signal abundance of [z17 þ 1,5-DAN þ H]þ is lower than that of ½c0 19 þ Hþ (right inset of Fig. 10d), the ½y0 16 þ Hþ that originated from [z17 þ 1,5-DAN þ H]þ is observed as an intense signal as compared with PSD fragments generated from ½c0 19 þ Hþ . Therefore, this pseudo-MS3 method can use to identify the C-terminal side fragments, even when 1,5-DAN adduct on z fragments overlapped with other ISD fragments. To understand the formation mechanism of [bxnþ1/zn þ 1,5-DAN] and y0 n1 fragments from [zn þ 1,5-DAN þ H]þ in detail, we performed PSD analysis of [zn þ 1,5-DAN H]. According to the result, [zn þ 1,5-DAN H] does not preferentially generate [bxnþ1/zn þ 1,5-DAN] and y0 n1 fragments by 552

PSD, indicating that the specific peptide bond cleavage adjacent to the binding site of 1,5-DAN occurring in PSD of [zn þ 1,5DAN þ H]þ was mediated by a mobile proton (Asakawa, Smargiasso, & De Pauw, 2014b). In MALDI, a proton would be localized at the arginine residue, which is the most efficient residue for protonation. Vibrational activation, including CID and PSD of protonated peptides, leads to various species containing different protonation sites by relocation of the excess protons (Paizs & Suhai, 2005) The proton affinity of 1,5-DAN is 942 kJ/mol (Demeure, Gabelica, & De Pauw, 2010), which is higher than those of amino acids, except for the basic residues, that is, arginine, lysine, and histidine. Consequently, the other favored site of protonation in [zn þ 1,5-DAN] is that at which 1,5-DAN binds to the N-terminus in the zn fragment. Therefore, the proton can be mobilized from an arginine residue to the 1,5DAN moiety in [zn þ 1,5-DAN þ H]þ by vibrational activation. The proton would then be transferred to the amide nitrogen located adjacent to 1,5-DAN. In such a case, the ½bxnþ1 =zn þ 1; 5-DAN=y0 n1 fragment pair would be formed by peptide bond cleavage of [zn þ 1,5-DAN þ H]þ, as shown in Scheme 14. Although the use of 2,5-DHB and 2-AA produces matrix adducts on z fragments, the preferential formation of y0 n1 is not likely to constitute a dominant process in PSD of [z þ 2,5DHB þ H]þ and [z þ 2-AA þ H]þ probably due to the low proton affinities of 2,5-DHB and 2-AA. Consequently, 1,5-DAN was found to be a better matrix than either 2,5-DHB or 2-AA for the discrimination of C-terminal side fragments employing a MALDI-ISD mass spectrum by pseudo-MS3 analysis via the matrix adduct on z fragments. This method can be helpful to identify the C-terminal side fragments from a complex MALDIISD mass spectrum, allowing unambiguous sequencing of peptides and proteins. Pseudo-MS3 can provide useful information about the direct identification of a target protein in a mixture. A recent study has shown the direct identification of a- and b-tubulin variants by MALDI-ISD (Calligaris et al., 2010). MALDI-ISD of intact HeLa cell tubulin preferentially generated y0 ions characteristic of each tubulin isotype contained in the solution. Identification of each tubulin isotypes has been confirmed using a pseudo-MS3 method. By this way, the major tubulin isotypes contained in the solution, that is, a1B, a1C, bI, bIVb, have been identified but not bIII. An additional step consisting to the guanidination of the C-terminal lysine of bIII isotype allowed performing pseudo-MS3 on one ISD fragment that confirmed the presence of the minor tubulin isotypes, which is a marker of drug resistance and tumor progression, in the solution. Moreover, pseudo-MS3 can be used for the direct identification of proteins in tissue sections during MALDI imaging analysis (Debois et al., 2010). MALDI-ISD mass spectra have been recorded with a hybrid quadrupole ion trap time-of-flight (TOF) mass spectrometer, which opened the possibility to perform pseudo-MSn analysis with CID (Sellami et al., 2012). CID is the most widely available fragmentation method and effectively cleaves the peptide backbone. Therefore, this method improves the quality of pseudo-MS3 results as compared with PSD spectra and also allows preudo-MS5 spectra to be recorded (Sellami et al., 2012). MALDI-ISD followed pseudo-MSn analysis opened new research avenues for unambiguous characterization of protein modification when multiple sites are concerned or when the modification type is complex. Mass Spectrometry Reviews DOI 10.1002/mas


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FIGURE 10. (a) MALDI-ISD mass spectrum of myoglobin with 1,5-DAN. PSD of (b) [z15 þ 1,5-DAN þ H]þ, (c) [z16 þ 1,5-DAN þ H]þ, (d) [z15 þ 1,5-DAN þ H]þ and ½c0 19 þ Hþ , and (e) [z18 þ 1,5-DAN þ H]þ. The fragments annotated with an asterisk correspond to 1,5-DAN adducts on the fragments. Reprinted with permission from Asakawa, Smargiasso, and De Pauw (2014b). Copyright 2014, American Chemical Society.

V. CONCLUSION MALDI-ISD appears to have a promising future and will likely have increasing importance in MS-based applications to the field of proteomics. MALDI-ISD allows rapid characterization of Nterminal and C-terminal sequences of a protein and requires only several picomoles of compounds. The aim of this review of the MALDI-ISD mechanism of identifying and studying peptides and proteins is to promote wider application of this method. Mass Spectrometry Reviews DOI 10.1002/mas

Since MALDI-ISD is initiated by a hydrogen radical transfer reaction between an analyte and the matrix, the choice of matrix is essential and dramatically impacts MALDI-ISD fragmentation. To date, two radical fragmentation pathways in MALDI-ISD have been described, that is, hydrogen attachment onto the peptide backbone and hydrogen abstraction from the peptide backbone. Briefly, reducing and oxidizing matrices cleave N–Ca bonds and Ca–C bonds, respectively. For MALDI-ISD with a reducing matrix, the attachment of hydrogen from the matrix to the peptide, which is the initial step of

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SCHEME 14. Proposed mechanism of the formation of [bxnþ1/zn þ 1,5-DAN] and y0 n1 from 1,5-DAN adduct on zn, where x represents the number of amino acid residues in the peptide. Reprinted with permission from Asakawa, Smargiasso, and De Pauw (2014b). Copyright 2014, American Chemical Society.

MALDI-ISD, is suggested to occur via hydrogen bonding between peptides and the matrix before the desorption process (Asakawa et al., 2013e). The resulting aminoketyl radical generally attacks the N–Ca bond located on the C-terminal side of the radical site and then forms a c0 /z• fragment pair via homolytic cleavage (Asakawa et al., 2014a). Subsequently, the z• radical either reacts with a matrix molecule or loses its sidechain, leading to the z, z0 [z þ matrix] or w fragments (Asakawa, Smargiasso, & De Pauw, 2013a). The use of a reducing matrix for MALDI-ISD also results in a and y0 ions, which is enhanced by decreasing matrix proton affinity (Demeure, Gabelica, & De Pauw, 2010) and increasing the ion source pressure (Asakawa et al., 2013b). However, the mechanism by which these fragments are formed is not yet fully understood. In contrast to a reducing matrix, the use of an oxidizing matrix allows the formation of ISD fragments initiated by hydrogen abstraction from the peptide backbone (Asakawa & Takayama, 2011a). The resulting hydrogen-deficient nitrogen-centered radical can lead to Ca–C bond cleavage, forming a•/x fragment pair (Asakawa & Takayama, 2011b). MALDI-ISD with hydrogen abstraction provides information complementary to that obtained by ISD with hydrogen attachment and facilitates the identification of ISD fragment ions (Asakawa & Takayama, 2012c). Therefore, MALDI-ISD is a potentially useful method for de novo peptide sequencing. In addition, the use of high resolution mass spectrometer and the software for automated identification of proteins contributed to decreasing the time needed for interpretation of a MALDI-ISD mass spectrum. Pseudo-MS3 analysis provides additional information for ISD ions. By gaining a full understanding of MALDI-ISD processes, employing high resolution mass spectrometer, pseudo-MS3 analysis and developing software, it is anticipated that the applications of MALDI-ISD MS will be further expanded. The recent developed methods are applied in unison toward a MALDI-ISD experiment on proteomics research to achieve better results.

VI. ABBREVIATIONS 2-AA 2-AB 5,1-ANL

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2-aminobenzoic acid 2-aminobenzamide 5,1-aminonaphthol

5-ASA bisHE-TCNQ CHCA CID Cys 1,5-DAN 2,5-DHB 1,5-DHN ECD EDD ESI ETD 5-FSA Fs-LID FTICR H/D ISD MALDI MS NECD NSA PSD PTM Pro Sar TCNQ TOF

5-aminosalicylic acid 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ) a-cyano-4-hydroxycinnamic acid collision-induced dissociation cysteine 1,5-diaminonaphthalene 2,5-dihydroxybenzoic acid 1,5-dihydroxylnaphthalene electron capture dissociation electron detachment dissociation electrospray ionization electron transfer dissociation 5-formylsalicylic acid femtosecond laser-induced ionization/ dissociation Fourier transform-ion cyclotron resonance hydrogen/deuterium in-source decay matrix-assisted laser desorption mass spectrometry negative electron transfer dissociation nitrosalicylic acid post-source decay post-translational modification proline sarcosine 7,7,8,8-tetracyanoquinodimethane time-of-flight

ACKNOWLEDGMENTS The author gratefully acknowledges Dr. Nicolas Smargiasso, Prof. Loı¨c Quinton and Prof. Edwin De Pauw (Mass Spectrometry Laboratory, University of Lie`ge, Belgium), Dr. David Calligaris (Harvard Medical School, USA) and Dr. Tyler A. Zimmerman (Northwestern University, USA) for helpful discussions. My research was supported by a fellowship from the Japan Society for the Promotion of Science for Young Scientists (PD: 23-10272). Mass Spectrometry Reviews DOI 10.1002/mas


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