Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Fragmentation Reactions of Methionine-Containing Protonated Octapeptides and Fragment Ions Therefrom: An Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions as well as the b₇, a₇, and a₇* ions derived therefrom have been studied in detail for the octapeptides MAAAAAAA, AAMAAAAA, AAAAMAAA, and AAAAAAMA. Ionization was by electrospray using a QqToF mass spectrometer, which allowed a study of the evolution of the fragmentation channels as a function of the collision energy. Not surprisingly, the product ion mass spectra for the b₇ ions are independent of the original precursor sequence, indicating macrocyclization and reopening to the same mixture of protonated oxazolones prior to fragmentation. The results show that this sequence scrambling results in a distinct preference to place the Met residue in the C-terminal position of the protonated oxazolones. The a₇ and a₇* ions also produce product ion mass spectra independent of the original peptide sequence. The results for the a₇ ions indicate that fragmentation occurs primarily from an amide structure analogous to that observed for a₄ ions (Bythell et al. in J Am Chem Soc 132:14766–14779, 2010). Clearly, the rearrangement reaction they have proposed applies equally well to a_n ions as large as a₇. The major fragmentation modes of the MH⁺ ions at low collision energies produce b₇, b_6, and b₅ ions. As the collision energy is increased further fragmentation of these primary products produces, in part, non-direct sequence ions, which become prominent at lower m/z values, particularly for the peptides with the Met residue near the N-terminus.

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a₁ ions. The advantageous generation of a₁ ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported. Figure

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Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Characterization of Electron Transfer Dissociation in the Orbitrap Velos HCD Cell

Electron transfer dissociation (ETD) is commonly employed in ion traps utilizing rf fields that facilitate efficient electron transfer reactions. Here, we explore performing ETD in the HCD collision cell on an Orbitrap Velos instrument by applying a static DC gradient axially to the rods. This gradient enables simultaneous three dimensional, charge sign independent, trapping of cations and anions, initiating electron transfer reactions in the center of the HCD cell where oppositely charged ions clouds overlap. Here, we evaluate this mode of operation for a number of tryptic peptide populations and the top-down sequence analysis of ubiquitin. Our preliminary data show that performing ETD in the HCD cell provides similar fragmentation as ion trap-ETD but requires further optimization to match performance of ion trap-ETD.      

Differentiation of Diastereoisomers of Protected 1,2-Diaminoalkylphosphonic Acids by EI Mass Spectrometry and Density Functional Theory

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions.

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Sulfation is a common post-translational modification of tyrosine residues in eukaryotes; however, detection using traditional liquid chromatography-mass spectrometry (LC-MS) methods is challenging based on poor ionization efficiency in the positive ion mode and facile neutral loss upon collisional activation. In the present study, 193 nm ultraviolet photodissociation (UVPD) is applied to sulfopeptide anions to generate diagnostic sequence ions, which do not undergo appreciable neutral loss of sulfate even using higher energy photoirradiation parameters. At the same time, neutral loss of SO₃ is observed from the precursor and charge-reduced precursor ions, a spectral feature that is useful for differentiating tyrosine sulfation from the nominally isobaric tyrosine phosphorylation. LC-MS detection limits for UVPD analysis in the negative mode were determined to be around 100 fmol for three sulfated peptides, caerulein, cionin, and leu-enkephalin. The LC-UVPD-MS method was applied for analysis of bovine fibrinogen, and its key sulfated peptide was confidently identified.

Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M?+?H?+?O]⁺), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M?+?H?+?O]⁺ product is observed at a much greater abundance than the proton transfer product (viz., [M?+?H]⁺). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to ‘label’ methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

N-Terminal Peptide Sequence Repetition Influences the Kinetics of Backbone Fragmentation: A Manifestation of the Jahn-Teller Effect?

Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance (P?<?1?10^–3) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b₂ ⁺ ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems.

Activation Energies of Fragmentations of Disaccharides by Tandem Mass Spectrometry

A simple multiple collision model for collision induced dissociation (CID) in quadrupole was applied for the estimation of the activation energy (E_o) of the fragmentation processes for lithiated and trifluoroacetated disaccharides, such as maltose, cellobiose, isomaltose, gentiobiose, and trehalose. The internal energy-dependent rate constants k(E_int) were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) or the Rice-Ramsperger-Kassel (RRK) theory. The E_o values were estimated by fitting the calculated survival yield (SY) curves to the experimental ones. The calculated E_o values of the fragmentation processes for lithiated disaccharides were in the range of 1.4–1.7 eV, and were found to increase in the order trehalose < maltose < isomaltose < cellobiose < gentiobiose.

Constant-Momentum Acceleration Time-of-Flight Mass Spectrometry with Energy Focusing

Fundamental aspects of constant-momentum acceleration time-of-flight mass spectrometry (CMA-TOFMS) are explored as a means to improve mass resolution. By accelerating all ions to the same momentum rather than to the same energy, the effects of the initial ion spatial and energy distributions upon the total ion flight time are decoupled. This decoupling permits the initial spatial distribution of ions in the acceleration region to be optimized independently, and energy focus, including ion turn-around-time error, to be accomplished with a linear-field reflectron. Constant-momentum acceleration also linearly disperses ions across time according to mass-to-charge (m/z) ratio, instead of the quadratic relationship between flight time and m/z found in conventional TOFMS. Here, CMA-TOFMS is shown to achieve simultaneous spatial and energy focusing over a selected portion of the mass spectrum. An orthogonal-acceleration time-of-flight system outfitted with a reduced-pressure DC glow discharge (GD) ionization source is used to demonstrate CMA-TOFMS with atomic ions. The influence of experimental parameters such as the amplitude and width of the time-dependent CMA pulse on mass resolution is investigated, and a useful CMA-TOFMS focusing window of 2 to 18 Da is found for GD-CMA-TOFMS.

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

High Energy Collisions on Tandem Time-of-Flight Mass Spectrometers

Long before the introduction of matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), Orbitraps, and any of the other tools that are now used ubiquitously for proteomics and metabolomics, the highest performance mass spectrometers were sector instruments, providing high resolution mass measurements by combining an electrostatic energy analyzer (E) with a high field magnet (B). In its heyday, the four sector mass spectrometer (or EBEB) was the crown jewel, providing the highest performance tandem mass spectrometry using single, high energy collisions to induce fragmentation. During a time in which quadrupole and tandem triple quadrupole instruments were also enjoying increased usage and popularity, there were, nonetheless, some clear advantages for sectors over their low collision energy counterparts. Time-of-flight (TOF) mass spectrometers are high voltage, high vacuum instruments that have much in common with sectors and have inspired the development of tandem instruments exploiting single high energy collisions. In this retrospective, we recount our own journey to produce high performance TOFs and tandem TOFs, describing the basic theory, problems, and the advantages for such instruments. An experiment testing impulse collision theory (ICT) underscores the similarities with sector mass spectrometers where this concept was first developed. Applications provide examples of more extensive fragmentation, side chain cleavages, and charge-remote fragmentation, also characteristic of high energy sector mass spectrometers. Moreover, the so-called curved-field reflectron has enabled the design of instruments that are simpler, collect and focus all of the ions, and may provide the future technology for the clinic, for tissue imaging, and the characterization of microorganisms.      

Effects of Column and Gradient Lengths on Peak Capacity and Peptide Identification in Nanoflow LC-MS/MS of Complex Proteomic Samples

Reversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing ion suppression. Here we examined the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatographic conditions for a proteomics experiment.

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

Estimation of Activation Energy from the Survival Yields: Fragmentation Study of Leucine Enkephalin and Polyethers by Tandem Mass Spectrometry

A simple collision model for multiple collisions occurring in quadrupole type mass spectrometers was derived and tested with leucine enkaphalin a common mass spectrometric standard with well-characterized properties. Implementation of the collision model and Rice-Ramsperger-Kassel-Marcus (RRKM) algorithm into a spreadsheet software allowed a good fitting of the calculated data to the experimental survival yield (SY) versus collision energy curve. In addition, fitting also ensured to estimate the efficiencies of the kinetic to internal energy conversion for Leucine enkephalin in quadrupole-time-of-flight and triple quadrupole instruments. It was observed that the experimental SY versus collision energy curves for the leucine enkephalin can be described by the Rice-Ramsperger-Kassel (RRK) formalism by reducing the total degrees of freedom (DOF) to about one-fifth. Furthermore, this collision model with the RRK formalism was used to estimate the critical energy (E _o ) of lithiated polyethers, including polyethylene glycol (PEG), polypropylene glycol (PPG), and polytetrahydrofurane (PTHF) with degrees of freedom similar to that of leucine enkephalin. Applying polyethers with similar DOF provided the elimination of the effect of DOF on the unimolecular reaction rate constant. The estimated value of E _o for PEG showed a relatively good agreement with the value calculated by high-level quantum chemical calculations reported in the literature. Interestingly, it was also found that the E _o values for the studied polyethers were similar.

Activated Ion ETD Performed in a Modified Collision Cell on a Hybrid QLT-Oribtrap Mass Spectrometer

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO₂ laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety’s high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m₂ and m₂+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.