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Proc Natl Acad Sci U S A. Jul 5, 2005; 102(27): 9463–9468.
Published online Jun 27, 2005. doi:  10.1073/pnas.0503189102
PMCID: PMC1172258

Protein identification using sequential ion/ion reactions and tandem mass spectrometry


A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1–2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N—Cα bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15–40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.

Keywords: electron transfer dissociation, fragmentation, ion trap, ion/ion reactions, top down

Perhaps one of the most influential concepts in protein mass spectrometry has been the notion of enzymatic protein digestion to render a collection of peptides of suitable size for conventional tandem mass spectrometry, i.e., bottom-up proteomics. Doubtless this methodology has enabled significant progress for global protein identification (1, 2); however, many investigators now realize this approach has significant limitations (3). First, protein posttranslational modifications (PTMs) on multidomain proteins often work in concert; to determine their biological relevance, these patterns must be detected within the context of one another (across the whole protein). And second, mRNA editing (alternative splicing) has recently gained credit for significantly increasing the protein repertoire of complex organisms, up to three-fourths of all human genes have at least one variant (46). Thus, the use of short peptides as proxy markers for genes is inadequate and often misleading (7).

To detect these biological events, several laboratories are now pursuing mass spectrometry-based methods to analyze whole proteins. Intact proteins are directly dissociated, either by electron capture (ECD) or collision activation (CAD), and the products are measured with the high resolving power of a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) (3, 812). McLuckey and coworkers (1315) have used ion trap-based instrumentation to collisionally activate intact protein ions followed by spectral simplification with ion/ion charge reduction reactions. In either case, the intact protein mass is measured while the product ions are used for sequencing and locating sites of modification. Unfortunately, mainstream implementation of this “top-down”-type analysis is not routine because CAD typically generates only a handful of fragments (cleavage is directed at a few weak linkages, making identification and PTM site location challenging), and the use of ECD requires the most expensive mass spectrometer technology, FT-ICR-MS.

A short time ago, we described modifications to a quadrupole linear ion trap mass spectrometer that make possible a method of peptide ion fragmentation, electron transfer dissociation (ETD) (1618). ETD results from the gas-phase reaction of multiply protonated peptide molecules with radical anions of polyaromatic hydrocarbons such as fluoranthene (Eq. 1)

equation M1

After electron transfer, the charge-reduced peptide ion dissociates through the same mechanisms believed responsible in ECD. These reactions are accomplished rapidly (5–to 65-ms reaction duration) and typically result in extensive peptide backbone dissociation, fragmentation that is indifferent to either peptide sequence or the presence of labile PTMs. Our initial report focused on the analysis of phosphorylated peptides, but we now note the technique is equally well suited for the dissociation of larger peptides (20–75 residues) and even intact proteins.

Direct ETD fragmentation of large peptides and intact proteins, while generally complete and efficient, often leads to spectra too complicated for direct interpretation. For the most part, these larger species are highly charged (z >10) and, thus, the resulting c- and z-type fragments ions are likewise multiply charged. The benchtop ion trap mass spectrometer used in our work has a limited m/z resolution that makes interpretation of these highly charged product ion m/z spectra challenging (the rapid scan speeds used here do not allow determination of z, for z > 2).

Besides the ETD reaction, ion/ion reactions have been harnessed to simplify mixtures of highly charged species, i.e., fragments generated after CAD of intact protein ions, before m/z analysis (1315, 19). That reaction, referred to as proton transfer charge reduction (PTR, Eq. 2) can be

equation M2

effected simply by choosing a different anion, e.g., deprotonated benzoic acid. Because ion/ion reaction rates increase proportionally with the square of the charge (i.e., a +10 ion reacts at a rate 100 times that of a +1 ion), mixtures of highly charged species are readily concentrated at lower charge states (15, 2029).

Here, we describe an approach for large peptide and whole protein characterization using sequential ion/ion reactions and online chromatography with a benchtop linear ion trap mass spectrometer. Eluting, multiply protonated peptides and proteins are first isolated and then reacted with the radical anion of fluoranthene for a relatively short duration (≈10 ms, ETD). After this reaction, and the expulsion of excess fluoranthene anions, the resulting product ions are reacted with even electron anions of benzoic acid (≈100–200 ms). This second reaction (PTR) serves to both simplify the spectrum, making spectral interpretation much easier, and concentrate the various c- and z-type product ion signals into predominantly one charge state.

Materials and Methods

Instrument Modification and Operation. All experiments were performed with a commercial RF linear quadrupole ion trap (LTQ), the Finnigan LTQ mass spectrometer (Thermo Electron) equipped with either a modified factory nano-flow electrospray ionization source (chromatography experiments) or a nanospray robot (Advion Biosciences, Ithaca, NY; infusion). The LTQ was modified to accept a Finnigan 4500 chemical ionization source (Thermo Electron) placed at the rear of the instrument (16, 17). A batch inlet was used to volatilize molecules of both fluoranthene and benzoic acid into the chemical ionization source, where an electron beam generated anions of both species. The instrument control software (itcl) was modified to accommodate the following sequence after precursor ion selection (isolation width 4 m/z units) and storage: (i) anion injection (≈2 ms); (ii) fluoranthene anion isolation (m/z 202, 10 ms); (iii) ion/ion reaction of anion and precursor cation (≈10–15 ms); (iv) removal of excess fluoranthene anions and storage of ETD products; (ν) injection of anions (≈2 ms); (vi) application of selective waveform to remove m/z 202 and other background anion species (≈5 ms); (vii) ion/ion reaction of purified benzoic acid anions (m/z 121) with ETD product ions (≈100–150 ms); and (viii) removal of excess benzoic acid anions and mass analysis of product ions.

Sample Preparation. Histone H3.1 was isolated and separated from asynchronously growing HeLa cells as described (30, 31). An aliquot containing 5 μg of histone H3.1 was digested with Glu-C (Roche, Palo Alto, CA) in 100 mM ammonium acetate (pH 4.0) at an enzyme-to-protein ratio of 1:15 for 4 h at 37°C. The resulting peptides were fractionated by HPLC; fractions containing the 1–50 residue were concentrated, resuspended in 100 mM ammonium bicarbonate (pH 8.5), and treated with propionylation reagent as described (32). The reaction mixture was lyophilized to dryness and resuspended in 0.1% aqueous acetic acid. Histone H2A.Z was isolated from chicken erythrocytes as described (33).

Chromatography. An Agilent Technologies (Palo Alto, CA) 1100 Series binary HPLC system was interfaced with the linear quadrupole ion trap mass spectrometer for online protein/peptide separations. Approximately 100 fmol each (Sigma unless noted) of vasoactive intestinal peptide fragment 1–12, angiotensin I, bovine ubiquitin, bovine cytochrome c, recombinant histone H2B, and bovine albumin (Upstate Biotechnology, Waltham, MA) and ≈10 pmol of H2A.Z mixture were pressure-loaded onto a monolithic capillary column (360 × 100 μm i.d., 5-cm column length, LC Packings, Sunnyvale, CA) equipped with a 30-μm SilicaTip electrospray ionization emitter (New Objective, Woburn, MA) and gradient-eluted with a linear gradient of 0–60% B for 12 min and 60–100% B for 2 min (A = 0.1 M formic acid, B = 70% acetonitrile in 0.1 M formic acid, flow rate = 1 μl/min).

Propionylated histone H3.1 (1–50 residue, 10 pmol) was pressure-loaded onto a self-prepared nano-HPLC column [360 × 50 μm i.d. fused silica packed with 7 cm of C18 reversed-phase material (ODS-AQ, YMC, Milford, MA)] equipped with an integrated, laser-pulled, electrospray ionization emitter (34). Peptides were eluted with a flow rate of 60 nl/min, using a linear gradient of 0–5% B for 15 min and 5–100% B for 15 min (A = 0.1 M acetic acid, B = 70% acetonitrile in 0.1 M acetic acid).


Sequential Ion/Ion Reactions. A 15-ms reaction of the +13 charge state of ubiquitin (8.5 kDa, m/z 659, 76 residues) with radical anions of fluoranthene generates the tandem mass spectrum displayed in Fig. 1A. Several hundred highly charged, unresolved fragment ions are observed after this relatively short reaction. Theoretically, these product ions possess charges (z), ranging from 1 to 12, recall the benchtop ion trap system used here can resolve z for z ≤ 2. And, with ≈146 possible unique c- and z-type fragments spread among numerous charge states, more or less confined within a 1,000-m/z range, spectral interpretation, at this point, is simply not possible.

Fig. 1.
Tandem mass spectrum of ubiquitin generated by sequential ion/ion reactions. (A) Whole protein dissociation (ubiquitin +13, m/z 659) after a 15-ms reaction with the radical anion of fluoranthene. Note production of several hundred highly charged unresolved ...

That mass spectrum (Fig. 1 A), resulting from the initial ETD ion/ion reaction, can be simplified by sequestering the entire mixture of highly charged product ions and reacting them with a second anion, deprotonated benzoic acid. This second reaction (PTR) removes excess charge from the diverse population of multiply charged fragment ions. Recall ion/ion reaction rates increase proportionally with charge squared; therefore, adjustment of the PTR reaction duration allows one to control the charge state of the resulting products. In this experiment, multiple PTR reaction times were used (50, 100, and 150 ms; Fig. 1 B, C, and D, respectively). As the reaction period is extended, the higher-charged fragments are preferentially concentrated to lower charge states, predominately singly charged products in the case of the 150-ms reaction (Fig. 1D). This effect can be observed by following the small expanded region of each spectrum plotted in Fig. 1. Mass analysis after the brief 15-ms ETD reaction produces several isobaric, highly charged fragments within the 60-m/z expanded range. Gradually these multiply protonated products are removed from the spectrum and after 150 ms only the three significant product ions remain: the doubly protonated z17 and c17 and singly charged c8. Note, whereas the doubly protonated signal of c17 and z17 is progressively degraded with increased reaction time, the singly protonated form (m/z 1919) increases proportionally.

From the spectrum displayed in Fig. 1D, the entire amino and carboxyl terminus of the protein can be sequenced by subtracting consecutive c- and z-type product ion mass-to-charges within each respective series (17 residues deep from either end). The mass spectrometer used in these studies has an m/z range limited to 2,000, which, of course, constrains the depth of observed coverage. Obviously higher mass c- and z-type fragment ions are produced after the ETD reaction (Fig. 1 A); however, the simplifying proton transfer reaction increases their m/z values beyond our mass range. Even with this limited m/z range, coverage can be extended by identifying doubly protonated fragment ions, the upper limit of our resolving power (with the 100-ms PTR conditions we observe a population of doubly protonated fragment series that increases coverage to ≈35 residues from either end).

Whole Protein Sequencing with Online Chromatography. To demonstrate the viability of intact protein sequencing with online chromatography, 100 fmol (each) of three proteins (ubiquitin, cytochrome c, and human histone H2B) was loaded onto a monolithic capillary column and gradient-eluted into the mass spectrometer (Fig. 2A). After a full m/z scan, the two most abundant m/z ratios were selected for interrogation by using sequential ion/ion reactions (15-ms ETD followed by 150-ms PTR, each spectrum the average of four single-scan spectra), this process was repetitively cycled throughout the course of the experiment. Fig. 2 BD displays the tandem mass spectra generated after automated selection and interrogation of each eluting protein. Each spectrum, acquired in ≈2 s, defines the amino and carboxyl terminus of the precursor protein (up to ≈20 residues). Because of the heme group located on the amino terminus of cytochrome c, the c-type fragment series ceases at the ninth residue (likewise observed with ECD fragmentation) (35).

Fig. 2.
Tandem mass spectra of proteins in a mixture generated by a combination of on-line chromatography and sequential ion/ion reactions. (A) Chromatogram of the peptide/protein separation. (BD) Tandem mass spectra for ubiquitin (B), cytochrome c ( ...

Shown in Fig. 3A is the tandem mass spectrum of the (M+49H)+49 ion of albumin (66 kDa, m/z 1,381) generated by using a combination of ETD (10 ms) and PTR (150 ms). This spectrum defines the first 31 aa at the N terminus of the protein. These data were obtained by averaging 100 single scans acquired over 60 s from an infused sample. No sequence ions, however, from the carboxyl terminus could be identified in the spectrum. Previous works have noted gas-phase protein conformation can affect the production, or at least, the observation of fragmentation after ECD (36). Such an explanation is likely the case here. Even so, this result provides direct evidence that whole proteins of ordinary size (≈66 kDa) are readily identified, without prior processing, on a benchtop mass spectrometer. To further assess the capabilities of the method, 100 fmol of the protein was loaded on a column and gradient-eluted into the mass spectrometer under the same conditions described above. As the protein eluted, abundant charge states were automatically isolated for direct analysis by using the same sequential ion/ion reactions used in the infusion experiment. Fig. 3B displays one of the resulting mass spectra, the average of five single-scan spectra (≈3-s acquisition). Here, every singly charged c-type ion that was previously observed (during the infusion experiment) is readily distinguishable with sufficient signal/noise. Some of the higher-m/z doubly protonated c-type ions are no longer discernable from the background; nonetheless, the spectrum clearly defines the first 23 residues of the intact protein.

Fig. 3.
Tandem mass spectrum of albumin generated by sequential ion/ion reactions. (A) The tandem mass spectrum generated after direct analysis of the +49 charge state of albumin (≈66 kDa) by using sequential ion/ion reactions (≈100 single-scan ...

Sequencing Highly Modified, Large Peptides. The N-terminal half of histone H3.1, a highly posttranslationally modified region of the protein (residues 1–50, harvested from asynchronous human cells), was isolated and analyzed with a chromatographic separation coupled online to sequential ion/ion reactions and mass spectrometric analysis. The first scan (Fig. 4A, the average of four single-scan spectra, ≈2-s acquisition) results from the automated interrogation, 15-ms ETD followed by 150-ms PTR, of an early eluting peptide. A near-complete series of c-type ions (10 of 11) at the N terminus demonstrates that K4 and K9 are modified with monomethyl and dimethyl groups, respectively. Analysis of the z-type ion series indicates the C terminus is not modified until K36, which contains a dimethylated lysine. At this point, given the limited m/z range, the “center” portion of the peptide will remain uncharacterized, although technical advances are expected to relieve this restriction (see below). Still, the present system can uncover global modification patterns that would otherwise remain obscured. For example, Fig. 4B displays a later eluting peptide (≈6 s) containing a similar, but different, modification pattern. Inspection of the c-type ion series reveals the N terminus of this peptide is modified identically to the previous species; however, an m/z shift in the higher-mass z-type ions confirms an unmodified K36 residue followed by a monomethylated K37. Note, this later spectrum comprises fragment ions from both species (coelution), e.g., the presence of two z14 ions, one unmodified at K37 (the earlier species, ≈60%) and the K37 monomethylated form (the later peptide, ≈40%).

Fig. 4.
Online chromatographic separation of large peptides (residues 1–50, ≈½ the protein) from histone H3.1 followed by automated sequential ion/ion reactions (ETD/PTR) and mass spectrometry. (A) The tandem mass spectrum generated from ...

Sequencing Protein Mixtures. Fig. 5 displays results, after online chromatographic elution and sequential ion/ion reactions, for the intact analysis in a wild-type protein mixture (chicken histone H2A.Z). The full m/z spectrum, obtained for major eluting species, indicates the presence of two distinct protein forms: Mr ≈13,380 and 13,456 Da, after deconvolution. The lighter protein, with a measured molecular mass of 13,380 Da, is within 2 Da of the unmodified, previously described chicken histone H2A.Z isoform. We surmised the heavier form, ≈76 Da, was probably caused by PTM. Precursor m/z peaks were selected for further interrogation by sequential ion/ion reactions (15-ms ETD/150-ms PTR). The product ion spectrum of the lower molecular mass species identifies the protein as unmodified histone H2A.Z (consecutive c- and z-type ions are observed to define the approximately the first 30 residues from either end of the protein, Fig. 5C). Inspection of the product ion spectrum for the heavier species, however, rules out the possibility of PTM; instead an m/z shift of 30 units at the 12th and 14th residues of the amino terminus (c12 and c14), compared with the unmodified H2A.Z sequence, is observed. This mass shift indicates that Ala at positions 12 and 14 of H2A.Z both are replaced with Thr in the heavier protein. Another difference was found in the z-type ion series of the heavier protein; it is increased by 28 m/z units (Fig. 5D, although this discrepancy can be located only to the first three amino acids). Because the c-type ion series indicated two amino acid changes, the first 30 N-terminal amino acids, as we interpreted them from the dissociation spectrum (de novo), were subjected to a blast (37) search to identify any H2A.Z isoforms containing the detected amino acid changes. Surprisingly, that search returned a protein having the exact N-terminal amino acid sequence, as identified by ETD/PTR, with threonine residues at positions 12 and 14. This protein was termed “hypothetical protein” from chicken and is not presently described as an H2A.Z variant. Comparison of the entire H2A.Z sequence with the hypothetical protein sequence revealed four amino acid changes: A12T,A14T,T38S,A128V. The change of valine to alanine corresponds to a mass addition of 28 Da and explains the 28-Da increase of the z-type ion series of the heavier protein. The total net change of 74 Da, caused by those four changed amino acids, is in agreement with the molecular mass determined by the full m/z scan (≈76 Da). Further confirmation that the isolated protein contained the above amino acid changes was obtained by sequencing peptides in a Glu-C digest by using tandem mass spectrometry (ETD/PTR).

Fig. 5.
Analysis of a mixture of histone H2A.Z isoforms by on-line chromatography and sequential ion/ion reactions. (A) Chromatogram of the protein separation. (B) The protein charge envelope of two coeluting proteins and the corresponding m/z values that were ...

In conjunction with PTM, histone variants add to the structural repertoire that imparts these chromosomal proteins with their ability to epigenetically control gene expression; histone H2A contains the largest number of variants described to date (38). The molecular mechanisms by which these variants participate in the regulation of gene expression are not clear. Evidence suggests that H2A.Z is functionally involved in transcriptional activation (39) and gene silencing (40, 41). This controversial role is mirrored in structural studies (33). The existence of the two H2A.Z variants observed here provides a possible explanation for the above observations. Furthermore, it has not escaped our notice that the two alanine-to-threonine substitutions, both of which follow lysine residues, create a variant with a potential “binary switch” that could control protein function. It has been suggested that modules that recognize trimethyl lysine (Lys-9 in human H3.1) can be inhibited by phosphorylation of the adjacent serine residue (Ser-10) (42).


Ion/Ion Chemistry. Ion/ion reaction duration is an important parameter that remains, as yet, unoptimized. For example, in these experiments the ETD reaction period was kept low to minimize multiple electron transfer events; consecutive electron transfer can result in the production of internal fragments (unpublished data). For example, a c50 fragment, produced after a single electron transfer to the whole protein cation, could subsequently receive an electron, cleave, and form two product ions, e.g., c25 and z25. Of course, the N-terminal fragment, c25, is still recognizable within the context of the original precursor protein; however, the z25 product contains neither the amino nor carboxyl terminus of the original precursor and thus appears, with a variety of other similar products, as elevated noise. Besides increasing chemical noise, multiple electron transfer events can also serve to generate a disproportionate amount of low m/z c- and z-type fragment ions.

PTR reaction duration is also a critical parameter and is ideally adjusted to coincide with the charge and size of the precursor protein. We envision that future implementations of this methodology will doubtless contain the ability to automatically prescreen precursor ion charge state (obtain charge and molecular weight) by using PTR. With this information the optimal ETD and PTR reaction period will be calculated and used in the subsequent ion/ion reaction series. Depending on the determined m/z, the PTR time could be adjusted so as to reduce the entire c- and z-type ion series to the single charge state (large protein) or to shorten the reaction period to leave both singly and doubly charged species and increase sequence coverage (large peptide/small protein).

Instrumentation. Future enhancements of this ion/ion technology will almost certainly come in the form of instrumentation. For example, multisegmented ion traps that allow fully independent anion and cation isolations will reduce acquisition time and enhance anion purity. Higher-capacity devices will allow increased ion storage and, thus, decrease the need for spectral averaging (at present, we start with approximately two to five times the number of precursor ions used for a conventional CAD experiment). Finally, hybridization of the device with other mass analyzers (e.g., FT-ICR-MS, TOF-MS, etc.) will be of obvious utility for increasing mass accuracy, mass resolution, and/or m/z range.

Data Analysis. Translation of tandem mass spectra to peptide/protein sequence is usually accomplished with a protein database-searching algorithm, e.g., sequest (43). These search algorithms were designed especially for the type of fragmentation achieved with CAD, fragmentation that highly depends on which amino acids are present, their order, and the presence of PTMs. Neutral losses of amino acid side chains or PTMs are common. With all of these caveats, direct interpretation (by computer, de novo sequencing) of CAD tandem mass spectra (peptide or whole protein) remains challenging. In contrast, ETD does not suffer from these limitations; rather, peptide backbone fragmentation occurs randomly to generate a homologous series of c- and z-type fragment ions. For example, note the consecutive c-type ion series in Fig. 4B that allows direct “reading” of the protein's amino terminus. We propose this predictability will make possible automated de novo sequencing, which, in turn, may eliminate the reliance on protein database searching. We envision ETD/PTR-derived tandem mass spectra, from whole proteins, could be analyzed in the following manner: (i) preprocessing via a de novo algorithm to generate sequence tags (44) from the present c- and z-type ion series, (ii) calculated amino acid sequences searched via a blast alignment of a genomic database, and (iii) all possible sequence-containing proteins (identified in step ii) are fragmented in silico with subsequent spectral alignment and comparison with the measured protein intact molecular weight.


The sequential ion/ion reactions described above allow rapid sequence analysis of intact proteins on low-cost, benchtop ion trap mass spectrometers. As demonstrated, each spectrum contains a series of c-type ions, characteristic of the amino acid sequence of the amino terminus of the protein, and a series of z-type ions to define the carboxyl terminus. Beyond that, the protein charge envelope (obtained in the full m/z spectrum) allows determination of the intact molecular weight of each protein from which the N/C-terminal amino acids have been characterized. This information could be used to either confirm the protein identity or suggest the presence of PTMs or mutations in the molecule. Alternatively, discrepancies, from the predicted sequence, in either the intact molecular weight or the N/C-terminal amino acid sequence can identify mRNA alternative splicing. Finally, besides proteomics applications, this technology should be particularly valuable for characterization of recombinant proteins, including truncated isoforms, used as drugs or diagnostics in the biotechnology/pharmaceutical industry.


We thank Jim Stephenson for helpful discussions and Thermo Electron for its support (specifically George Stafford, Scott Quarmby, and Jae Schwartz). This work was supported by National Institutes of Health Grants GM37537 and AI33993 (to D.F.H.), National Institutes of Health Grant RR018688 (to J.J.C.), National Science Foundation Grant MCB-0209793 (to D.F.H.), and Canadian Institutes of Health Research Grant MOP-57718 (to J.A.).


This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PTM, posttranslational modification; ECD, electron capture; CAD, collision activation; ETD, electron transfer dissociation; PTR, proton transfer charge reduction.


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