2-Aminobenzamide and 2-Aminobenzoic Acid as New MALDI Matrices Inducing Radical Mediated In-Source Decay of Peptides and Proteins
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One of the mechanisms leading to MALDI in-source decay (MALDI ISD) is the transfer of hydrogen radicals to analytes upon laser irradiation. Analytes such as peptides or proteins may undergo ISD and this method can therefore be exploited for top-down sequencing. When performed on peptides, radical-induced ISD results in production of c- and z-ions, as also found in ETD and ECD activation. Here, we describe two new compounds which, when used as MALDI matrices, are able to efficiently induce ISD of peptides and proteins: 2-aminobenzamide and 2-aminobenzoic acid. In-source reduction of the disulfide bridge containing peptide Calcitonin further confirmed the radicalar mechanism of the ISD process. ISD of peptides led, in addition to c- and z-ions, to the generation of a-, x-, and y-ions both in positive and in negative ion modes. Finally, good sequence coverage was obtained for the sequencing of myoglobin (17 kDa protein), confirming the effectiveness of both 2-aminobenzamide and 2-aminobenzoic acid as MALDI ISD matrices.
Key wordsMALDI In-Source decay Fragmentation Peptides Proteins
Name, Sequence and Masses of Peptides Used in this Study
As thermal activation allows a redistribution of the internal energy received by the analyte, it is not suitable for top-down sequencing of large proteins that have a high number of degrees of freedom. The radical-mediated pathway overcomes this limitation, as the redistribution of internal energy of the analyte is not the driving force of the reaction. This pathway would not be theoretically limited by the mass of the analyte, thus allowing ISD to be used to sequence large proteins. Moreover, it was demonstrated that during radical-induced ISD and just like ETD or ECD, post-translational modifications are preserved, including labile ones such as phosphorylation [19, 20, 21]. Nevertheless, since radical-mediated ISD is a reductive reaction, disulfide bonds could be altered , and information concerning their position could be lost. On the other hand, this reductive property was used to identify disulfide-linked peptides [23, 24] or to classify cyclical peptides, such as microcystins, which contain modified amino acids (such as dehydroalanine) that are also susceptible to being reduced upon ISD .
The MALDI matrices 2,5-dihydroxybenzoic acid (2,5-DHB) , 5-aminosalycilic acid (5-ASA) , and 1,5-diaminonaphthalene (1,5-DAN) [4, 26] were previously described to efficiently induce radical mediated ISD. Among them, 1,5-DAN is the most efficient due to its high radical transfer ability, but it promotes the formation of intense matrix cluster ion peaks, potentially interfering with the detection of analyte fragment peaks . More recently, two additional matrices, 5-nitrosalicylic acid (5-NSA) and 5-formylsalicylic acid (5-FSA) that lead to the formation of a- and x-ions were described by Asakawa and Takayama . In this case, a transfer of hydrogen from a NH group of a peptide bond to the matrix would occur, leading to the formation of a nitrogen-centered radical. This radical would subsequently induce the fragmentation of the Cα–C bond.
Here, we report the ability of two new MALDI matrices to produce radical-induced ISD: 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA). These matrices are chemically stable under normal laboratory conditions, very soluble, and nontoxic compounds. As 1,5-DAN is an unstable carcinogenic compound , these new easy-to-use matrices could become useful alternatives to previously described ISD matrices.
2-Aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) and 1,5-diaminonaphthalene (1,5-DAN) were purchased from Sigma-Aldrich (Steinheim, Germany). The peptides calcitonin (salmon I), (Tyr0)-C-peptide (dog), (Glu1)-fibrinopeptide B, acetyl-amylin were purchased from Bachem (Weil am Rhein, Germany). All the solvents used were HPLC grade quality. The proteins myoglobin and human serum albumin (HSA) were purchased from Sigma-Aldrich (Steinheim, Germany).
2.2 MALDI Spots Preparation
All peptides (Table 1) were prepared at 20 μM in water. Stock solutions of myoglobin and HSA were prepared at 1 mg/mL and were further diluted to spot 200 pmol on the MALDI plate.
For matrices, 2-AA and 2-AB solutions were both prepared in H2O/ACN (1/1), 0.1% formic acid at 20 mg/mL. 1,5-DAN solutions were prepared at saturation in H2O/ACN (1/1), 0.1% formic acid shortly before use due to its instability.
All spots were prepared by mixing equivolumes of matrix and analyte following the “dried droplet” method. Spots prepared with both 2-AA and 2-AB as the MALDI matrix are not homogeneous and analytes are located in needle-shaped crystals and in the rim of the spots (Figure S-1). 2-AB spots were found to be stable under vacuum while 2-AA spots degraded slowly under these conditions, therefore limiting the available time for spot analysis. Both spots were stable at atmospheric pressure and room temperature and analytes were not degraded by these conditions, thus allowing preservation of spots for further analysis.
2.3 Mass Spectrometry
Spectra were recorded on a Bruker Ultraflex II TOF/TOF, controlled by the FlexControl 3.0 software (Bruker Daltonics, Bremen, Germany). Desorption/Ionization was performed using a frequency tripled Nd:YAG laser emitting at 355 nm with a 100 Hz shot frequency. Variable laser power was used according to the MALDI matrix: roughly, 2-AB needs a higher laser power than 2-AA while the laser power used for 2-AA is similar to that used for 2,5-DHB. Concerning spectra of peptides, 1000 laser shots were acquired for experiments on calcitonin and (Glu1)-fibrinopeptide B and 5000 shots for experiments on acetyl-amylin and (Tyr0)-C-peptide. For proteins, 10,000 shots were summed. The spectrometer was tuned with the following parameters: 25 kV acceleration voltage, 21.85 kV pulse voltage, 30 ns ion extraction delay, and 1.4 kV detector gain. FlexAnalysis 3.0 and Biotools 3.2 softwares were used to analyze and annotate spectra (Bruker Daltonics, Bremen, Germany).
3 Results and Discussion
3.1 Evaluation of the Hydrogen Radical Transfer Ability
3.2 ISD of Peptides in Positive Ion Mode
It was shown previously that, according to MALDI matrix properties, different type of ions could be produced upon ISD of peptides. In order to determine which fragments are produced during ISD using 2-AA and 2-AB, the peptides (Glu1)-fibrinopeptide B, (Tyr0)-C-peptide (dog), and acetyl-amylin were chosen as test models. Indeed, (Glu1)-fibrinopeptide B contains an arginine (easily protonated) in the C-terminal part, which leads to the preferential observation of ions containing this part (i.e., x-, y- and z-type ions) in positive ion mode. Inversely, (Tyr0)-C-peptide and acetyl-amylin contain an arginine in the N-terminal part, leading to the preferential formation of a-, b-, and c-type ions [17, 28]. Ions are named here according to the Zubarev et al. nomenclature .
Results obtained for (Glu1)-fibrinopeptide B (Figure S-2) show that the most abundant fragments are y- and z′-ions. The presence of z-type ions confirms the activation by the radical-mediated fragmentation pathway. Besides intense y- and z-ions, x-ions were also observed using these two matrices (Figure S-2B). As described by Takayama et al., these ions would result from the fragmentation of an N-centered radical produced after the transfer of a hydrogen radical from the peptide NH group onto the matrix. This would indicate that 2-AA and 2-AB are both able to accept and transfer H radicals. This behavior was also previously described for 5-formylsalicylic acid (5-FSA) . Moreover, x′-ions are present, indicating either a reduction of x-ions, or the capture of a hydrogen radical by x• ions that would be formed after the fragmentation of the Cα–C bond. Nevertheless, the pathway leading to the formation of the a/x• pair was showed to be unfavored when 5-NSA or 5-FSA are used, indicating possible differences compared with fragmentation pathways induced by 2-AB and 2-AA.
The presence of w-ions was previously described as produced by ISD by fragmentation of z′-ions using the DAN matrix . Interestingly, 2-AA and 2-AB promote the formation of w-ions, but also reduced w-ions (Figure S-2C) and v-ions, possibly resulting from the degradation of y- or x•-ions  (Figure S-2D). However, the ratios between w- and v-ions are different between 2-AA and in 2-AB, indicating possible matrix related differences in the activation pathways of the first generation of ions during the ISD process.
3.3 ISD of Peptides in Negative Ion Mode
As it is difficult to observe acidic peptides in positive ion mode due to the lack of proton acceptor sites, they can be analyzed in negative ion mode. To ensure that 2-AA and 2-AB are able to induce ISD in negative ion mode, we decided to use (Glu1)-fibrinopeptide B as model peptide because this peptide possess four acidic residues. Obtained spectra (Figure S-4) show that both 2-AA and 2-AB are efficient matrices in negative ion mode. Moreover, ISD was also observed in this mode, emphasizing the versatility of these new matrices. The fragments that were observed in negative ion modes are similar to those that were found in positive ion mode. Of course, the negative ion mode allowed observing both N-terminal (a- and c-ions) and C-terminal (x-, y- and z-ions) of (Glu1)-fibrinopeptide B, due to the presence of several potential negatively charged sites.
3.4 ISD of Proteins with 2-AA or 2-AB as the Matrix
To evaluate the ability of 2-AA and 2-AB to be used for top-down sequencing of proteins, ISD was tested on myoglobin (Mw ≈ 17 kDa) and oxidized HSA (Human serum albumin, Mw ≈ 66 kDa). For both proteins, the limit at which ISD was no longer observed was around 10–20 pmol per spot for 2-AA, and 50–60 pmol per spot for 2-AB (data not shown). For myoglobin (Figure S-5), sequence coverages of more than 60% were obtained for both 2-AA and 2-AB. These coverage values are similar to those obtained with the 1,5-DAN matrix. However, the accumulation of more spectra was needed with 2-AA and 2-AB as matrices than for 1,5-DAN to obtain workable spectra for sequencing purpose. This is probably due to the lower efficiency of these two compounds in inducing hydrogen radical transfer. As expected, numerous a- and x-type ions were produced, allowing the sequencing to overcome the presence of proline gaps. Concerning HSA (Figure S-6), its oxidized form was used in order to evaluate the ability of the matrices to sequence proteins containing several disulfide bonds . In Figure S-6, it can be seen that practically only c-ions are present in the spectra, whichever the matrix (2-AA or 2-AB) is used. This may be rationalized by the presence of several cysteines located on the C-terminal part of the sequence, implying that the disulfide bonds in which these cysteines are involved are preferentially reduced during ISD, thus impeding the sequencing. The obtained sequence covers the 30 first amino acids in the case of 2-AA, which is similar to the results obtained with 1,5-DAN on the same protein (data not shown), while a smaller sequence coverage was obtained for 2-AB. Interestingly, c′12 is the most intense fragment with both matrices and the c′22 ion is more intense compared with its neighbors, with 2-AA as matrix, indicating preferential fragmentation sites. In addition, with both 2-AA and 2-AB, the intensity of the c′33 ion is relatively important, as this residue is located before the first cysteine of the protein. After that residue, ISD peaks are not detected anymore, probably due to the preferential reduction of disulfide bonds, potentially impeding sequencing.
2-AA and 2-AB, two molecules that had never been described as potential MALDI matrices for analysis of peptides and proteins, were described here as suitable matrices for MALDI-ISD. Besides production of c- and z-ions, it was shown that 2-AA and 2-AB are able to produce different other types of ions, including a-, x-, and v-ions. These observations raise questions about the exact mechanisms governing the formation of these ion types and the potential differences existing between ISD fragmentation induced by well-established matrices like 1,5-DAN or 2,5-DHB and these new matrices. As an important difference in the hydrogen radical transfer ability was observed between these two molecules, this implies that others factors (crystallization, ability to ionize, and/or desorb efficiently analytes) are essential for the ISD efficiency of the matrix. Finally, due to their lower toxicity and their good stability (2-AA and 2-AB could be kept for several days in the dark without alteration of their properties) compared with the highly unstable 1,5-DAN matrix, these two compounds represent good alternatives for MALDI-ISD.
N.S. is an F.R.S.–F.N.R.S. logistic collaborator. The F.N.R.S. and Walloon Region contributed the mass spectrometry facility funding. Dr. Tyler A. Zimmerman is acknowledged for useful comments and additional editing of the manuscript.
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