A comparison of negative and positive ion time-of-flight post-source decay mass spectrometry for peptides containing basic residues

Dedicated to professor J.L. Beauchamp on the occasion of his 60th birthday for his many seminal contributions to gas-phase ion chemistry and mass spectrometry.
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Abstract

Nine peptides containing highly basic residues were studied by post-source decay (PSD) in a reflectron time-of-flight (TOF) mass spectrometer. Although these compounds produced more abundant yields of protonated ions, [M+H]+, by matrix-assisted laser desorption ionization (MALDI), deprotonated ions, [MH], were formed in sufficient intensities for study by tandem mass spectrometry (MS/MS). PSD was conducted in both the positive and negative ion modes. Peptide backbone cleavage involving the y-ion series is generally seen in both modes and the two Dalton difference in mass between yn+ and yn can be useful in identifying these ions. For negative ions, PSD also generated cn and (to a lesser extent) an, while bn+ are produced from positive ions. When a peptide contains a mixture of acidic and basic residues, the negative PSD spectra are more complex and the locations of acidic residues dictate some fragmentations. The most extensive and abundant production of cn occurs in peptides with no acidic residues. This suggests that the mechanism for c-ion formation does not involve a deprotonated side chain, but may invoke a mobile deprotonation site along the peptide amide backbone or may possibly involve a charge remote cleavage. Negative ion PSD of basic peptides yields structurally informative spectra that complement the positive data. Even highly basic peptides, such as ACTH (11–24), can be studied in the negative ion mode.

Introduction

Peptides and proteins containing basic amino acid residues (i.e., arginine, lysine, histidine) are known to play important roles in enzyme catalysis mechanisms [1], [2]. They are major sources of protons for proton transfer reactions such as the bovine pancreatic RNAse hydrolysis of RNA, which involves a histidine residue [3], [4]. Furthermore, in the case of tryptic digests cleavage of the peptide chain occurs at basic residues [5].

Historically, mass spectrometric studies of peptides have involved positive ions [6]; these reports dwarf the relatively few negative studies. However, it has recently been shown that the analysis of phosphopeptides can benefit from examination in both positive and negative ion modes [7], [8]. In addition, Harrison [9] has used electrospray ionization (ESI) to produce deprotonated peptide ions, [MH], while Beauchamp [10], Bowie [11], [12], [13], [14] and their co-workers have produced peptide [MH] by fast atom bombardment (FAB). In these studies, collision-induced dissociation (CID) was used to fragment [MH] and the dissociation mechanisms were explored. Negative ion CID spectra were generally found to be as informative as the positive ion spectra [12]. In addition, our group has shown that negative mode post-source decay (PSD) time-of-flight (TOF) analysis of peptides can provide complementary structural information to positive ion studies [15], [16]. Negative mode PSD has been effective on peptides with neutral amino acid chains, as well as peptides containing acidic residues [16].

The protonated and deprotonated ions from a peptide differ by both polarity and charge location. Charge location is especially important because it can dramatically impact the structural information that is obtained by tandem mass spectrometry (MS/MS). Charge-directed fragmentation is a major mode of peptide dissociation. For example, Harrison and Yalcin [17] and Wysocki and co-workers [18], [19] reported that protonation of an amido nitrogen along the peptide backbone causes the (OC)NHR bond to weaken, leading to cleavage at the stressed site. In the positive ion mode, sequence-determining fragments are believed to result predominantly when the peptide backbone is protonated. The mobile proton model suggests that initial ionization may occur on the side chain of basic residues [20] and that the proton may migrate to other sites upon ion activation [17]; the location of these specific sites drives the fragmentation patterns. The probability of a proton being attracted to a basic residue is high, thus causing greater fragmentation adjacent to basic residues [21], [22], [23]. The locations of basic sites can complicate peptide dissociation; for example, if a basic residue is at or near the C-terminal, then more C-terminal ions may be observed than N-terminal ions and vice versa [23]. Arginine residues are particularly problematic because their highly basic side chains can sequester the proton and limit its mobility across the peptide backbone. Consequently, in positive mode analysis, the presence of arginine in a peptide may lead to spectra dominated by arginine-containing fragments and to a significant reduction in the amount of sequencing information obtained [23], [24], [25], [26], [27].

Basic residues have other implications upon positive ion mass spectrometry. The gas-phase basicities of the major basic residues decrease in the order of arginine (Arg,R)>lysine(Lys,K)≈histidine (His, H) [28], [29], [30], [31]. In a study of synthetic tryptic peptides by positive mode matrix-assisted laser desorption ionization (MALDI), this basicity ordering explains the preferred protonation and enhanced detection of peptides containing a C-terminal arginine residue relative to peptides with a lysine residue [24]. In addition, arginine-containing peptides are known to undergo preferential cleavage at the C-terminal side of aspartic acid residues [32], [33], [34], while peptides containing C-terminal lysine and at least one arginine yield a rearrangement that results in the loss of the terminal lysine [35]. Also, arginine residues may participate in gas-phase zwitterion formation, which can complicate dissociation patterns. It is believed that bradykinin ions (which have N- and C-terminal arginine residues) can exist as stable zwitterions in the gas phase [36], [37], [38], [39], as can arginine itself [40].

As the studies discussed before indicate, basic residues play a major role in the dissociations of protonated peptide ions and their effects can at times be detrimental to obtaining sequence information. In contrast, for the negative ion mode, the side chains of basic residues should not be charge sites. Thus, major differences in fragmentation may be observed in comparing the two modes. The present study will explore this using PSD of [M+H]+ and [MH] produced by MALDI. All three common basic amino acids are represented in the peptides examined:

Section snippets

Experimental

All experiments were performed on a Bruker Daltonics (Billerica, MA) Reflex III TOF mass spectrometer, which is equipped with a two-stage reflectron. The instrument has an effective flight path of 2.9 m and sample ionization by MALDI involves a Laser Science (Franklin, MA) model VSL-337ND-S nitrogen laser emitting at 337 nm.

Ions were moved from the source to the flight tube via delayed extraction [41] with an extraction delay of 250 ns. For PSD experiments, a gated pulse was used to selectively

Ion formation by MALDI

For the basic peptides under study, MALDI yields weaker [MH] signals compared to [M+H]+. However, the [MH] intensities from all peptides were still sufficient for study, which is undoubtedly due to the carboxylic acid group of the C-terminus offering a ready site for deprotonation. To illustrate this, Fig. 1 shows the MALDI/TOF mass spectra of ACTH (11–24) in both the positive and negative ion modes. This is the most basic peptide studied here, with 6 of its 13 residues being highly basic

Effects of charge location on fragmentation

The dominant backbone fragmentations in the positive PSD spectra are b- and y′′-ions. In contrast, the negative PSD spectra showed primarily c- and y-ions, although some a- and b-ions also form. These fragmentations generally occur throughout the peptide backbone. This is consistent with previous studies [21], [43], which have found that for singly protonated peptides, PSD yields fragments at almost every residue. In comparison to PSD, both high [43] and low [21] energy CID generally have more

Conclusions

Basic peptides readily form negative ions, [MH], by MALDI. Although not as abundant as the corresponding positive ions, [M+H]+, these negative ions are of sufficient intensity for PSD studies. The y-ion series is generally seen in both positive and negative modes and the two Dalton difference in mass between yn+ and yn can be useful in identifying these ions. Production of cn and, to a lesser extent, an are also notable in negative mode PSD, while bn+ are produced from positive ions. When

Acknowledgements

Financial support from the National Institutes of Health (R01-GM51384) is gratefully acknowledged.

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