Abstract

Numerous studies of cluster formation and dissociation have been conducted to determine properties of matter in the transition from the condensed phase to the gas phase using materials as diverse as atomic nuclei, noble gasses, metal clusters, and amino acids. Here, electrospray ionization is used to extend the study of cluster dissociation to peptides including leucine enkephalin with 7–19 monomer units and 2–5 protons, and somatostatin with 5 monomer units and 4 protons under conditions where its intramolecular disulfide bond is either oxidized or reduced. Evaporation of neutral monomers and charge separation by cluster fission are the competing dissociation pathways of both peptides. The dominant fission product for all leucine enkephalin clusters studied is a proton-bound dimer, presumably due to the high gas-phase stability of this species. The branching ratio of the fission and evaporation processes for leucine enkephalin clusters appears to be determined by the value of z²/n for the cluster where z is the charge and n the number of monomer units in the cluster. Clusters with low and high values of z²/n dissociate primarily by evaporation and cluster fission respectively, with a sharp transition between dissociation primarily by evaporation and primarily by fission measured at a z²/n value of 0.5. The dependence of the dissociation pathway of a cluster on z²/n is similar to the dissociation of atomic nuclei and multiply charged metal clusters indicating that leucine enkephalin peptide clusters exist in a state that is more disordered, and possibly fluid, rather than highly structured in the dissociative transition state. The branching ratio, but not the dissociation pathway of [somatostatin₅ + 4H]⁴⁺ is altered by the reduction of its internal disulfide bond indicating that monomer conformational flexibility plays a role in peptide cluster dissociation.

Article Outline

• Experimental

• Mass spectrometry

• Chemicals

• Disulfide bond reduction

• Cluster formation

• Double resonance and collisional activation

• Results

• Leucine enkephalin 7-mer²⁺

• Leucine enkephalin 9-mer²⁺

• Leucine enkephalin 11-mer³⁺

• Leucine enkephalin 15-mer⁴⁺

• Leucine enkephalin 19-mer⁵⁺

• Somatostatin 5-mer⁴⁺ clusters

• Discussion

• Structure of peptide clusters

• Cluster dissociation and the liquid-drop model

• The use of double resonance in dissociation experiments

• Conclusions

• Acknowledgements

• References

Bulk properties of matter and the properties of isolated gas-phase atoms and molecules can have radically different characteristics in terms of structure, work function, ion solvation, etc. (for a review see reference [1]). One motivation behind cluster research is that the transitional characteristics between bulk and molecular properties can be discovered by studying intermediate states of matter. Investigators have explored a wide variety of materials, including charged droplets of various liquids [2 and 3], clusters of noble metals [4, 5, 6, 7 and 8], metal-ligand clusters [1, 9, 10 and 11], highly charged clusters of noble gasses [12 and 13], small molecules [14 and 15], and ultra-cold clusters of transition metals [16]. Recently, investigators have formed noncovalently bound clusters of biologically important molecules, including clusters of amino acids and small peptides [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28], and have studied their dissociation processes [18, 20, 26, 27 and 28]. Multiply charged “clusters” of proteins consisting of multimeric, noncovalently associated protein complexes with a high degree of solution-phase structural specificity have been liberated into the gas phase [29, 30, 31, 32, 33, 34 and 35]. From a simple mass measurement, the stoichiometry of the complex can be determined. There has also been much interest in determining if structural information can be inferred from gas-phase dissociation of the complexes [30, 33, 35 and 36].

Gas-phase dissociation of a wide variety of clusters have been investigated [1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 24, 26, 27, 30, 35, 36, 37, 38, 39 and 40]. In the case of multiply charged clusters, the two competing processes for dissociation are the evaporation of a neutral atom or molecule and the ejection of charged subunits. The latter occurs through a fission process in which the cluster dissociates into two or three charged fragments that then separate due to coulombic repulsion [41]. The competing processes of evaporation and fission have been described by various liquid-drop models for clusters as diverse as atomic nuclei [42], multiply charged metal clusters [5 and 6], and highly charged solvent droplets [2 and 3]. The recent development of femtosecond lasers capable of rapidly ionizing many molecules in atomic or molecular clusters has permitted access to another dissociation process whereby a cluster undergoes a coulombic explosion, resulting in the isotropic ejection of many charged fragments [12, 13, 14 and 15].

General characteristics of cluster dissociation are well illustrated by multiply charged metal clusters. Evidence for their dissociation was first observed by Sattler et al. who, by measuring the mass and charge of Pb, Xe, and NaI clusters, observed half-integer cluster numbers that were interpreted to be doubly charged clusters [4]. It was found that the cluster size necessary to support two charges is much higher for Xe than for Pb or NaI due to the much weaker intermolecular attraction in Xe. Doubly charged Au clusters containing 9–17 atoms were isolated and collisionally activated by Saunders [5 and 6]. These clusters were found to dissociate by competing mechanisms of neutral Au atom evaporation, and fission by the ejection of Au₃⁺ and other products. Following collisional activation, larger Au clusters dissociate primarily by successive evaporation of neutral Au atoms while the smaller clusters, such as Au₁₀²⁺ and Au₉²⁺, dissociate primarily through fission. Interestingly, the log of the ratio of the rates of fission to evaporation was found to vary linearly with z²/n, where z is the cluster charge and n is the number of gold atoms in the cluster. This behavior corresponds closely to nuclear fission [43] and led Saunders to suggest that a similar liquid-drop model used to describe nuclear fission would also describe metal cluster dissociation [5 and 6]. Recent work on the dissociation of noble metal clusters has shown that neutral evaporation and trimer fission are also the primary dissociation pathways of larger Au clusters and other metals [7 and 8].

In contrast to the many cluster dissociation experiments that have been done with atoms and small molecules, only a few studies have been conducted with clusters of large, biologically relevant molecules. These have included studies of the dissociation and charge partitioning among the fragments of both homogeneous protein complexes that are biologically active in solution [29, 30, 31, 32, 33, 34, 35, 44 and 45] and proteins that are nonspecifically aggregated [36 and 44]. Studies have also been conducted on clusters of smaller, biologically relevant molecules, and these have tended to focus on amino acids and small peptides. Amino acid and peptide clusters can be readily formed by electrospray ionization (ESI) and initial reports were concerned with clusters of monomers (M) of the form [M_n + nH]ⁿ⁺ that appear at the same nominal m/z as the singly protonated monomer. These ions are indistinguishable in low resolution mass spectra from the monomer and larger clusters and could potentially complicate the interpretation of dissociation spectra [17, 19, 21, 23 and 46]. While the formation of such highly charged molecular clusters by ESI is interesting in its own right and indicative of the gentle nature of the ESI process, the elucidation of dissociation pathways for clusters of the type [M_n + nH]ⁿ⁺ is more complicated as the fragments are likely to be at the same m/z as the parent ion.

Some observations have been made of amino acid and peptide clusters formed by ESI of the type [M_m + nH]ⁿ⁺ where m > n [18, 20, 22, 23, 24, 25, 26, 27, 28, 40 and 47]. Much of this work has focused on octamers of the amino acid serine that have been found to form with a preference for homochirality [20, 22, 25, 26, 27 and 48]). Clusters of serine as large as [44Ser + 4H]⁴⁺ were isolated by Beauchamp and coworkers and collisionally activated in an ion trap mass spectrometer [20]. The cluster dissociation revealed that the large serine clusters dissociate by a combination of fission and evaporation, but the specific dissociation pathways are difficult to determine because the primary fragment ions of noncovalently bound complexes are often sufficiently activated to undergo subsequent dissociation. For example, although the activation of [30Ser + 3H]³⁺ was shown to produce a dissociation spectrum with fragments ranging in size from [23Ser + 3H]³⁺ to [29Ser + 3H]³⁺ [20], it is not known whether the fragments are a result of sequential evaporation of single amino acids or if several dissociation pathways exist whereby different sizes of neutral serine clusters evaporate from the original cluster. Because the primary activation products are more likely to provide the most information about the structure of the initial cluster, it is important to distinguish between primary and secondary fragments. A useful technique for distinguishing between primary and secondary dissociation products is double resonance (DR).

DR was initially developed to identify chemically coupled processes in equilibrium experiments conducted in ion cyclotron resonance mass spectrometers [49] and was later adapted for Fourier-transform mass spectrometry [50] and other kinds of mass spectrometry [51]. DR experiments are done by applying an on-resonance ejection waveform simultaneous with the dissociation event, with the aim of ejecting primary fragment ions before they undergo subsequent dissociation. If the ejection of an ion perturbs the abundance of another ion, it indicates that the second ion is formed by subsequent dissociation of the ejected ion and not directly from the activated precursor ion.

In this work, the dissociation pathways of multiply charged peptide clusters of leucine enkephalin and both oxidized and reduced somatostatin are elucidated using collisionally activated dissociation and DR. The effects of cluster size and charge on the dissociation pathways of leucine enkephalin clusters are compared with the dissociation of multiply charged metal clusters. To the authors' knowledge, this is the first application of DR to multiply charged cluster dissociation.

Experimental

Mass spectrometry

The experiments were conducted on a 110 mm bore 9.4 T Fourier-transform mass spectrometer that was constructed in collaboration with Bruker Daltonics (Billerica, MA) (Figure 1). This instrument has been described elsewhere [36]. The ion optics and introduction system are a standard Bruker design. Ions are generated by nanoelectrospray using borosilicate capillaries that are pulled to a 4 μm tip with a model P-87 capillary puller (Sutter Instruments, Novato, CA). A small volume of sample solution (4-10 μL) is loaded into the borosilicate capillary and a platinum wire is inserted into the solution at the end of the capillary. The capillary is positioned 2 mm from the source inlet and a potential of 900 V is applied to the platinum wire.

Chemicals

Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), somatostatin (Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys [Disulfide bridge: 3–14]) and dithiolthreitol (DTT) were purchased from Sigma-Aldrich C. (St. Louis, MO) and were used without further treatment except for the disulfide reduction described below. Methanol and acetic acid (99.9%) were purchased from EM Science (Gibbstown, NJ) and from Fisher Scientific (Pittsburgh, PA) respectively.

Disulfide bond reduction

For each day of experiments, a fresh solution with a concentration of 5 mM somatostatin (ST) and 50 mM DTT was made in Millipore 18.2 MΩ H₂O. The solution was placed in a sealed eppendorf tube covered with parafilm, and heated in a water bath at 80–95 °C for 50 min. Disulfide reduction was nearly complete as verified by an average increase of 2 Da in the peptide mass following the reduction procedure. ST remained reduced for several hours following the reduction procedure. Fresh, reduced solutions of ST were prepared for each day of experiments.

Cluster formation

Solutions of leucine enkephalin (LE) and oxidized ST for ESI were prepared at concentrations of 2.5 and 1.3 mM, respectively, in 1:1 water:methanol + 2 % acetic acid by volume. The relatively high peptide concentrations were found to enhance cluster formation. Solutions of reduced ST for ESI were prepared by diluting stock solutions of 5 mM somatostatin and 50 mM DTT to final concentrations of 1.3 mM and 13 mM, respectively, in 1:1 water:methanol + 2 % acetic acid. In order to stabilize the nanoelectrospray emission, ammonium acetate was added to the reduced ST solution containing 13 mM DTT until the final concentration was 100 mM. This technique is often useful for obtaining nanoelectrospray signal from solutions containing a high concentration of small molecules, such as DTT, which often result in unsteady nanoelectrospray currents.

The signal intensity of peptide clusters is enhanced by tuning for selective accumulation in the external hexapole of the instrument. This is done by accumulating the ions in the hexapole for 3 s (versus ≤ 1.0 s typically), increasing the hexapole dc offset from 2.7 to 3.5–4.5 V and injecting multiple hexapole accumulations into the ion cell prior to detection. The particular clusters measured in the mass spectra using a given set of tuning conditions are also sensitive to the voltage applied to the nanoelectrospray needle. In general, slowly decreasing the applied nanoelectrospray voltage after initiating ion current favors the formation of larger clusters. By tuning the hexapole offset and the second skimmer potentials and adjusting the electrospray voltage, it is possible to dramatically change the appearance of the mass spectra so that the abundance of a cluster of interest is greatly enhanced.

Double resonance and collisional activation

Modifications to the electronics of the APEX II mass spectrometer were implemented in order to do DR experiments. An additional transmitter board and a HI-222 analog switch from Bruker Daltonics were installed in the high frequency unit of the APEX II mass spectrometer. Modifications to the multiplexer board of the mass spectrometer were made in-house according to specifications provided by Scott Daniels (Bruker Daltonics, Billerica MA) that permit two excite frequencies to be applied simultaneously to ions in the cell of the mass spectrometer. The software to implement DR experiments is a modification of code written by Dr. Christian Berg (Bruker Daltonics, Billerica MA).

Peptide clusters of interest were isolated using two identical correlated sweeps with a pulse of nitrogen gas introduced into the ion cell between the sweeps. Following the double isolation, the clusters were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) [52] in which an excitation waveform was applied +600 Hz off-resonance for two seconds with an applied peak-to-peak potential of 6–10 V. A pulse of nitrogen gas was introduced for 90 ms, briefly raising the pressure to 2 × 10⁻⁶ torr. During DR experiments, a second excitation waveform is applied on-resonance to the fragment ion under investigation for the duration of the SORI excitation and for an additional 1.0 s to eject fragment ions that continue to dissociate after the SORI waveform is turned off. The amplitude of the DR waveform was adjusted to eject the fragment ions as rapidly as possible while avoiding additional excitation of the precursor ion or any of the other fragment ions. The maximum voltage that could be applied without additionally exciting the precursor or other fragment ions varied from 4–32 V pk–pk depending on the m/z and the proximity of other fragment ions of the DR waveform.

Results

Multiply charged, gas-phase noncovalent clusters of leucine enkephalin (LE) and somatostatin (ST) can be readily formed from concentrated solutions using nanoelectrospray ionization. The gas-phase dissociation pathways of these clusters were investigated using collisional activation and double resonance (DR) ejection. Many of the primary fragment ions produced from the dissociation of these clusters have sufficient internal energies to undergo further fragmentation before ion detection making DR experiments useful for determining dissociation pathways. In DR experiments, one of the fragment ions resulting from dissociation of a precursor cluster ion is continuously ejected from the ion cell while the precursor ion is activated. If subsequent dissociation of the fragment ion requires more time than ion ejection (3–24 ms for these experiments depending on the amplitude of the DR ejection pulse), the products of this ion will not appear in the DR spectrum. Thus, secondary dissociation products are identified by their decrease in abundance when a DR waveform is applied to eject their parent ion. Dissociation pathways for several different clusters of LE and one cluster of ST with its individual peptides containing either oxidized or reduced disulfide bonds are described in the following sections.

It should be noted that each cluster ion is isolated by two correlated sweeps with a pulse of gas between the sweeps. After an initial correlated sweep, the isolated cluster undergoes some dissociation after a pulse of nitrogen collision gas is introduced. When the cluster is re-isolated by a second, identical correlated sweep, the remaining ions do not dissociate as readily. This result suggests that either the nonspecific peptide clusters examined in this study exist in more than one gas-phase configuration that have different stabilities or that the ions are kinetically excited after trapping. The clusters investigated in this study are those that survive the double isolation.

Leucine enkephalin 7-mer²⁺

Doubly protonated LE clusters consisting of seven monomers (7LE²⁺) are readily formed by nanoelectrospray (Figure 2a). Dissociation of 7LE²⁺ (Figure 3a) results in abundant LE⁺ and 2LE⁺ ions along with lower abundance 3LE⁺, 4LE⁺, and 5LE⁺ ions. Interestingly, the abundance of 2LE⁺ greatly exceeds that of its complimentary ion, 5LE⁺, and the complimentary ion to LE⁺, 6LE⁺, is absent from the dissociation spectrum. Although mass discrimination may account for a very small fraction of the asymmetry observed, secondary fragmentation accounts for the majority. Application of a DR ejection pulse to the m/z corresponding to 6LE⁺ causes no change in the appearance of the mass spectrum indicating that 6LE⁺ is not a significant dissociation product of 7LE²⁺. When a DR ejection pulse is applied to the 5LE⁺ peak (Figure 3b), the abundance of the other fragmentation peaks change considerably. The abundance of 4LE⁺ and 3LE⁺ are attenuated, suggesting that a substantial fraction of these ions are formed by subsequent dissociation of the primary 5LE⁺ fragment. Application of a DR waveform at the frequency of the 4LE⁺ ion (Figure 3c) results in a further attenuation of 3LE⁺ and 2LE⁺ ions. The isotopic distribution of the 3LE⁺ peak shows that 6LE²⁺ is present in the dissociation spectra with an intensity as high as 10% of the 3LE⁺ peak indicating that the 7LE²⁺ → 6LE²⁺ + LE⁰ process does occur. The abundance of 6LE²⁺ varies considerably from scan to scan and is often not detectable within the signal to noise of the experiment. Based on the observed abundance of the 6LE²⁺ peak, the 7LE²⁺ → 6LE²⁺ + LE⁰ process does not constitute more than 0.005% of the total dissociation.

Leucine enkephalin 9-mer²⁺

A slight adjustment of the hexapole accumulation parameters and the voltage applied to the nanoelectrospray needle causes a dramatic shift in the relative peak intensities of the LE cluster ions, such that the abundance of 9LE²⁺ greatly exceeds that of 7LE²⁺ (Figure 2b). The isotopic distribution of 9LE²⁺ indicates the presence of 18LE⁴⁺ (Figure 2b inset). Both 9LE²⁺ and 18LE⁴⁺ are activated during SORI excitation and some products from dissociation of 18LE⁴⁺ are apparent as indicated by the appearance of 16LE³⁺ formed by the pathway 18LE⁴⁺ → 16LE³⁺ + 2LE⁺. Unlike 9LE²⁺, which dissociates into many fragments by multiple pathways, 18LE⁴⁺ appears to dissociate only by the ejection of 2LE⁺. The 16LE³⁺ does not appear to undergo further dissociation as DR ejection changes the appearance of other peaks in the spectrum very little (data not shown). It should be noted that the ratio of 9LE²⁺ and 18LE⁴⁺ varied during the DR experiment, resulting in a slight variation in the relative abundance of 16LE³⁺ to the other fragment clusters.

Unlike the 7LE²⁺, which dissociates almost entirely by fission, 9LE²⁺ dissociates by a combination of evaporation and fission processes. The dissociation spectrum of 9LE²⁺ (Figure 4a) shows that a large number of cluster fragments are formed. The most abundant of these is the 8LE²⁺/4LE⁺ peak. Although 8LE²⁺ and 4LE⁺ have the same nominal m/z, the isotopic distribution indicates that while both clusters are present, approximately 70% of the peak is 8LE²⁺, produced directly from 9LE²⁺ by the evaporation of neutral LE. A comparison of Figure 4a and e shows that double resonance ejection of 8LE²⁺ results in the elimination of 7LE²⁺ and 3LE⁺ and a decrease in the relative abundance, compared to the parent ion, of all other fragment clusters except for 16LE³⁺ and 7LE⁺. The abundance of 7LE²⁺ and 3LE⁺ during DR ejection of 8LE²⁺ indicates that 7LE²⁺ is formed by two consecutive losses of neutral LE from 9LE²⁺ and that 3LE⁺ is formed by neutral loss from 4LE⁺. The decrease in abundance of 5LE⁺ and 6LE⁺ during DR ejection of 8LE²⁺ indicates that after dissociation by the 8LE²⁺→ 2LE⁺ + 6LE⁺ pathway, 6LE⁺ can undergo neutral loss to form 5LE⁺. Note that the abundance of 6LE⁺ normalized to 9LE²⁺ is significantly attenuated by a DR pulse on 8LE²⁺ (Figure 4e) from the abundance of 6LE⁺ normalized to 9LE²⁺ in the regular activation spectrum (Figure 4a). It is evident that very little LE⁺ is formed directly from the 8LE²⁺ as the ejection of 2LE⁺ virtually eliminates LE⁺ (Figure 4h). DR ejection of 7LE⁺, 6LE⁺ and 5LE⁺ all cause decrease in signal and a change in the isotopic distribution for 8LE²⁺/4LE⁺ indicating that the 4LE⁺ in the 8LE²⁺/4LE⁺ peak is primarily produced by evaporation of neutral LE from larger singly charged clusters. DR ejection of 7LE²⁺ (Figure 4f) alters the relative abundance of the peaks in the spectra very little except for a small decrease in 5LE⁺ and 2LE⁺ suggesting that the 9LE²⁺ → 8LE²⁺ → 7LE²⁺ → (fragments) pathway is not a dominant process under the dissociation conditions used.

Leucine enkephalin 11-mer³⁺

The hexapole accumulation and nanoelectrospray voltage can be tuned to produce a high abundance of 11LE³⁺ and 15LE⁴⁺ (Figure 2c). The activation spectrum of 11LE³⁺ is much simpler than that for 9LE²⁺ (Figure 5a). As in the case of 7LE²⁺, the primary dissociation pathway is asymmetric fission, specifically the ejection of a proton-bound dimer, 2LE⁺, and complimentary 9LE²⁺. The subsequent dissociation of 9LE²⁺ by the evaporation of a neutral to form the 8LE²⁺ and by fission and subsequent evaporation to form 5LE⁺ and 4LE⁺ is consistent with the dissociation pathways of the 9LE²⁺ cluster discussed above. The peak labeled 10LE²⁺/5LE⁺ is identified as a combination of the two clusters by the isotopic distribution. The formation of 10LE²⁺ from 11LE³⁺ indicates that the 11LE³⁺ cluster also dissociates by a highly asymmetric fission process ejecting LE⁺. This is clearly a minor pathway with a branching ratio of no more than 2%, and is difficult to measure in spectra with poor signal to noise (as in Figure 5c, d). It should be noted that ideally, the relative peak intensity for Fourier-transform mass spectrometry is proportional to the charge of the ions under investigation. For example, an equal number of 9LE²⁺ and 2LE⁺ ions would produce a relative peak intensity ratio of 2:1 for 9LE²⁺ and 2LE⁺. Even considering the effect of charge, the sum of the intensities for the 2LE⁺ and LE⁺ is more than half of the sum of the intensities of the 9LE²⁺, 5LE⁺/10LE²⁺, and 8LE²⁺ peaks (Figure 5a). This indicates that there is a slight mass bias towards lower m/z ions.