Chihiro
Nakajima
ab,
Hiroki
Kuyama
*a,
Takashi
Nakazawa
c,
Osamu
Nishimura
a and
Susumu
Tsunasawa
a
aInstitute for Protein Research, Osaka University, Osaka, 565-0871, Japan. E-mail: kuyama@protein.osaka-u.ac.jp
bLife Science Research Center, Shimadzu Corporation, Kyoto, 619-0237, Japan
cDepartment of Chemistry, Nara Women's University, Nara, 630-8506, Japan
First published on 8th October 2010
A method for de novo sequencing of Nα-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of Nα-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two Nα-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.
Mass spectrometry (MS) has had substantial success in the sequencing of terminally blocked peptides, especially when it is employed in combination with a highly efficient scheme for separating or isolating the terminal peptides. The suggested approaches for analyzing N-terminally blocked peptides include those based on enriching the target peptides from peptides generated by proteolytic cleavage.2–9 The most impressive realization of this idea has been reported in connection with ‘positional proteomics’, which exploits the enrichment of Nα-acetylated peptides using an amine-reactive immobilized reagent.2,6–9 However, the absence of a free α-amino group in a peptide thus enriched often impedes the access of the chemical reagents generally used in the sequencing strategies of MS to enhance the sensitivity or simplify the spectra. In principle, every Nα-blocked peptide still retains a C-terminal carboxyl group free to be modified, albeit to little avail because of the difficulty in activating it specifically and effectively for chemical derivatization.
In earlier studies, we developed a method for isolating the N-terminal peptides of proteins using p-phenylenediisothiocyanate (DITC) glass from proteolytic digest.8,9 We have also devised a scheme for selectively modifying C-terminal α-carboxyl groups based on oxazolone chemistry.10,11 An oxazolone forms specifically at the C-termini of peptides, so that the C-terminal carboxyl group can be strictly distinguished from those in the side-chains of glutamic acid and aspartic acid. However, the reaction yields were not high enough to envisage applying this to the sequence analysis of isolated terminal peptides at sub-nanomolar quantities, until the successful optimization of the reaction conditions has been achieved.12 On the technical grounds elaborated so far, we suggest here a method for sequencing Nα-blocked peptides (Fig. 1). The procedures include: (1) proteolytic digestion of Nα-blocked proteins with peptidyl-Lys metalloendopeptidase (LysN),13–15 (2) isolation of terminal Nα-blocked peptides from the enzyme digest by removing all of the peptides having free amino groups with the DITC glass,16 and (3) selective activation of the C-terminal α-carboxyl group for amidation with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine17,18), which might have the effect of preferentially giving a defined set of fragment peaks in MALDI-TOF/TOF-MS, thus facilitating the amino acid sequencing. We chose to tag the peptides with the TMPP group, which has already proved useful for de novo sequencing as demonstrated in the previous studies, where it was utilized as a tag for the α-amino group.8,19–21
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Fig. 1 Schematic depiction of this protocol. |
In this paper we will also present an extension of the method, adapted to the case where the use of LysN digestion to obtain the N-blocked peptide is unsuccessful. A discussion about the wider applicability of modifying α-carboxyl groups with TMPP-propylamine in proteomics studies based on mass spectrometry is included.
The matrix used in this experiment was α-cyano-4-hydroxycinnamic acid (CHCA), which was dissolved to saturation in 50% aqueous ACN containing 0.05% TFA. We used MDPNA, which has been proven useful for MALDI analysis of salt-containing samples, as a matrix additive.23 MDPNA was used as 1% aqueous solution. An aliquot (0.4 μL) of sample solution was mixed with an equivalent volume of matrix and matrix-additive solutions on the MALDI target plate and analyzed after drying.
The m/z values in the spectra were externally calibrated with angiotensin II (human) and ACTH fragment 18–39 (human) using CHCA as a matrix.
Hence, we focused on employing the α-carboxyl group, another characteristic group of a protein or peptide, for derivatizing N-terminal peptide isolated from Nα-blocked protein.
In this study, we used specific activation of an α-carboxyl group through oxazolone formation and TMPP-propylamine as a derivatizing agent (Fig. 1) because TMPP-acetylation of the α-amino group of a peptide is known to provide better fragmentation for de novo sequence analysis by MS.
Oxazolone formation is a well-known characteristic reaction with an α-carboxyl group. Oxazolone is a five-membered ring selectively formed between an α-carboxyl group and a carbonyl group of a penultimate amino-acid residue from the C-terminus (Fig. 2), and serves as a reactive electrophile for reactions with various types of nucleophiles. In a previous report,12 we described the methyl amidation of an α-carboxyl group with high selectivity. In this paper, we applied the protocol to the selective derivatization of an α-carboxyl group using TMPP-propylamine (Fig. 2).
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Fig. 2 Outline of α-carboxyl group derivatization using TMPP-propylamine. |
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Fig. 3 (a) Derivatization of Ac-SQNY with TMPP-propylamine. (b) MS/MS spectrum of the derivatized peptide. |
It has been estimated from the peak intensities in MALDI-MS that 60–90% of the C-terminal carboxyl group underwent amidation with methylamine through the oxazolone.12 Although we have not determined the yield of the reaction with TMPP-propylamine, it would be somewhat lower because the concentration of the amine used was about 2.5 mM, much lower than the 0.5–1 M for methylamine. Nevertheless, the possible low yield of the derivatization with TMPP-propylamine could have been successfully compensated for by a sensitivity-enhancing effect of the TMPP group, thus apparently suppressing the detection of undesirable peaks including those of unmodified N-terminal peptides. This is consistent with the tendency to detect a-type (y-type) ions when the TMPP group is attached to an N-terminus (C-terminus) in MS/MS experiments.
As judged from the peaks at m/z 973.4 for phosphorylase b and m/z 1156.6 for orexin A, and in agreement with the mass values expected from their amino-acid sequences and N-terminal structures, N-terminal peptides of these proteins were isolated by the reported protocol7 using metalloendoprotease LysN digestion and DITC glass (Fig. 4a and 5a). The simplicity of the spectra suggests that the isolation procedure of these N-terminal peptides using DITC glass has effectively removed other internal and C-terminal peptides. Due to the derivatization of C-termini with TMPP-propylamine, the mass of each peptide increased by 572 Da, measured by the shift in the corresponding peaks to m/z 1544.9 for phosphorylase b and m/z 1727.7 for orexin A (Fig. 4b and 5b).
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Fig. 4 (a) Isolated N-terminal peptide of phosphorylase b (rabbit). (b) Derivatization of the N-terminal peptide with TMPP-propylamine. (c) MS/MS spectrum of the derivatized N-terminal peptide. |
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Fig. 5 (a) Isolated N-terminal peptide of orexin A (human). (b) Derivatization of the N-terminal peptide with TMPP-propylamine. (c) MS/MS spectrum of the derivatized N-terminal peptide. C*: S-carbamidomethyl cysteine residue; Pyr: pyroglutamic acid residue. |
We noted that the terminal Nα-acetylated peptide of calmodulin was not obtained by this protocol, probably because of a mis-cleavage along peptide bond, say Phe12–Lys13 in the N-terminal peptide during digestion with LysN, leaving an ε-amino group of lysine residue (Lys13) that could react with an isothiocyanate group in the DITC glass. In fact, the N-terminal peptide, Ac-ADQLTEEQIAEFJ (J indicates homoarginine) was isolated from calmodulin after digestion with trypsin used in place of LysN, guanidination of the ε-amino group of Lys residues, and treatment with DITC glass. The peptide thus isolated gave a peak at m/z 1605.8 (Fig. 6a), which shifted to m/z 2177.2 with an increase in the mass of 572 Da, characteristic of derivatization with TMPP-propylamine (Fig. 6b). The PSD spectrum of the peak at m/z 2177.2 (Fig. 6c) clearly indicates that the derivatization occurred exclusively at the C-terminal carboxyl group in the presence of four side-chain carboxyl groups of aspartic acid (Asp2) and glutamic acid (Glu6, Glu7, and Glu11), in agreement with the results of preceding experiments conducted on the basis of oxazolone chemistry.10–12 Furthermore, a series of y-type ions appeared predominantly in the PSD spectrum, making it very easy to clarify the amino acid sequence. Note that the preference for y-type ions was observed commonly in the MS/MS spectra for peptides attached with the TMPP group at the C-termini (Fig. 4c, 5c, and 6c). This is in sharp contrast to the tendency of observed a-type ions in the fragmentation of peptides modified with the TMPP-acetyl group at the N-termini.8,19–21 Unlike Nα-TMPP-acetylated peptides in which the N-terminal arginine residue causes deterioration in the appearance of the MS/MS spectra,24 the modification of the C-terminal arginine residue with TMPP-propylamine had no such damaging effect on the fragmentation pattern of the peptide (see, for example, Fig. 4c and 5c). If need be, however, derivatization of arginine with acetylacetone to N5-(4,6-dimethyl-2-pyrimidinyl)ornithine (Pyo)24,25 (Fig. 7a) could be useful for further simplifying the MS/MS spectrum, as we demonstrated by showing that the peak of the x7-ion, potentially confusing the MS/MS spectrum (Fig. 5c) was converted to that of the desired y7-ion to complete a set of y-type ions (Fig. 7b).
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Fig. 6 (a) Isolated N-terminal peptide of calmodulin (bovine). (b) Derivatization of the N-terminal peptide with TMPP-propylamine. (c) MS/MS spectrum of the derivatized N-terminal peptide J: homoarginine residue. |
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Fig. 7 Simplifying fragmentation by converting Arg to Pyo residue. (a) Arg derivatization using acetylacetone to Pyo residue. (b) MS/MS spectrum of the arginine-derivatized N-terminal peptide. Pyo: N5-(4,6-dimethyl-2-pyrimidinyl)ornithine residue; C*: S-carbamidomethyl cysteine residue. |
Other fragmentation patterns characteristic of the peptides modified with TMPP-propylamine at the C-termini include the observation of z-type ions coupled with y-type ions, both of which occur due to fragmentation on the N-terminal side of aspartic acid. In this pair, the peaks of z-type ions are often more intense than those of y-type ions (Fig. 4c, 5c, and 6c). Another pair of peaks representing y-type and w-type (y-SCH2CONH2) ions was observed at a comparable intensity, suggesting that the y-type ion produced by the cleavage of the bond at the N-terminal side of (S-carboxamidomethyl)cysteine is likely to undergo β-elimination (Fig. 5c). Although an x-type ion corresponding to fragmentation at the bond of Pro1–Leu2 was detected in the MS/MS of the N-terminal peptide from orexin A (Fig. 5c), this might not be a common feature of bond breaking where proline or leucine residue is involved because no peak of an x-type ion appeared in the PSD spectrum of the N-terminal peptide of phosphorylase b. Instead, a peak of a y5-ion, signifying fragmentation in Pro4–Leu5, was clearly observed. For all of these observations of unpredicted peaks, the C-terminal TMPP-propylamide group could effectively simplify MS/MS spectra by providing an almost complete set of peaks of y-type ions, thus making it easier to determine the amino-acid sequence.
One of the distinguishing features of C-terminal TMPP-propylamidation is the simplicity of the fragmentation pattern in MS/MS that exhibits a complete or near complete y-type ion series. This greatly facilitates the de novo sequencing of peptides. The cleanliness of the reaction is such that no peak was observed at a mass exceeding the expected value by 28 Da, corresponding to formylation of the hydroxyl group of a serine or threonine residue.
It is well known that in using a TMPP-Ac charge tag, sample clean up to remove excess reagent is necessary prior to MALDI-MS analysis.26,27 As described in the previous report,8 we used MDPNA23 as a co-matrix for the detection of TMPP-acetylated peptide. Just an addition of the reagent as a co-matrix improved the S/N ratio of the derivatized peptide peaks. This is an advantage where conventional ZipTip pretreatment is often not effective. And if need be, the excess reagent in the C-terminal TMPP-propylamidation can be removed by an appropriate amine-reactive medium such as DITC glass, which does not capture the peptide(s) of interest because of the lack of the free amine group.
Concerning C-terminal carboxyl derivatization in connection with de novo sequencing of peptides, Lindh et al.28 employed a condensing reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride: EDC) for coupling with 4-aminonaphthalenesulfonic acid as a charge tag. With this approach, however, it is inherently impossible to distinguish between the C-terminal and side-chain carboxyl groups. Our method, requiring a strict distinction between these two types of carboxyl groups, is incompatible with the non-specific nature of this simple condensation scheme. Note that satisfactory performance of the present method has been achieved through restricting the position of charge tagging to the C-termini of peptides, so that an almost complete set of peaks due to y-type ions could be observed in the MS/MS spectra.
The oxazolone reaction used for the introduction of the TMPP-propylamine is site-specific for the C-terminus, and the side chain carboxyl group is not derivatized. The TMPP group is a good tag for MALDI, because it increases the S/N ratio and gives a high quality PSD spectrum in MS analysis, often with a complete or near complete y-type ion series when attached to the C-terminus, and the simplified ion series can be used directly to interpret the amino-acid sequence. The fragmentation pattern differs from that of the peptide incorporating the TMPP-acetyl group at its α-amino group, which includes mainly a-type ions.
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