Label Scrambling During CID of Covalently Labeled Peptide Ions

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification. Graphical Abstract

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Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H₃PO₄), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H₃PO₄ elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H₃PO₄ upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence

Glycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., ^2,4A_R, ^2,4A_R-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + ^0,2X₀–H]^– and [peptide–NH₃–H]^–). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn–36]^–, was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides.

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.

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.

Evidence for Sequence Scrambling and Divergent H/D Exchange Reactions of Doubly-Charged Isobaric b-Type Fragment Ions

To date, only a limited number of reports are available on structural variants of multiply-charged b-fragment ions. We report on observed bimodal gas-phase hydrogen/deuterium exchange (HDX) reaction kinetics and patterns for substance P b₁₀ ²⁺ that point to presence of isomeric structures. We also compare HDX reactions, post-ion mobility/collision-induced dissociation (post-IM/CID), and sustained off-resonance irradiation-collision induced dissociation (SORI-CID) of substance P b₁₀ ²⁺ and a cyclic peptide with an identical amino acid (AA) sequence order to substance P b₁₀. The observed HDX patterns and reaction kinetics and SORI-CID pattern for the doubly charged head-to-tail cyclized peptide were different from either of the presumed isomers of substance P b₁₀ ²⁺, suggesting that b₁₀ ²⁺ may not exist exclusively as a head-to-tail cyclized structure. Ultra-high mass measurement accuracy was used to assign identities of the observed SORI-CID fragment ions of substance P b₁₀ ²⁺; over 30 % of the observed SORI-CID fragment ions from substance P b₁₀ ²⁺ had rearranged (scrambled) AA sequences. Moreover, post-IM/CID experiments revealed the presence of two conformer types for substance P b₁₀ ²⁺, whereas only one conformer type was observed for the head-to-tail cyclized peptide. We also show that AA sequence scrambling from CID of doubly-charged b-fragment ions is not unique to substance P b₁₀ ²⁺.

Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSⁿ, n >2) and collision-induced dissociation (CID) of Z₁ ions in the negative ion mode. Under low energy CID conditions, disaccharides ¹⁸O-labeled on the reducing carbonyl group gave rise to Z₁ product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS³ CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z₁ ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSⁿ CID (n = 3 – 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z₁ ions. CID data of these Z₁ ions were collected and compared with the standard database of Z₁ ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling

An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic b_n-44 ions instead of b_n-28 (a_n) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH₃CHO (44.0262) from a Thr side chain; (2) loss of CO₂ (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel b_n-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway. Graphical Abstract

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Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S–S bonds is often used in “bottom up” sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the “hidden” C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S–S bond rupture does not occur in this case.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

Loss of Internal Backbone Carbonyls: Additional Evidence for Sequence-Scrambling in Collision-Induced Dissociation of y-Type Ions

It is shown that y-type ions, after losing C-terminal H₂O or NH₃, can lose an internal backbone carbonyl (CO) from different peptide positions and yield structurally different product fragment ions upon collision-induced dissociation (CID). Such CO losses from internal peptide backbones of y-fragment ions are not unique to a single peptide and were observed in four of five model peptides studied herein. Experimental details on examples of CO losses from y-type fragment ions for an isotopically labeled AAAAHAA-NH₂ heptapeptide and des-acetylated-α-melanocyte-stimulating hormone (dα-MSH) (SYSMEHFRWGKPV-NH₂) are reported. Results from isotope labeling, tandem mass spectrometry (MSⁿ), and ion mobility-mass spectrometry (IM-MS) confirm that CO losses from different amino acids of m/z-isolated y-type ions yield structurally different ions. It is shown that losses of internal backbone carbonyls (as CID products of m/z-isolated y-type ions) are among intermediate steps towards formation of rearranged or permutated product fragment ions. Possible mechanisms for generation of the observed sequence-scrambled a-“like” ions, as intermediates in sequence-scrambling pathways of y-type ions, are proposed and discussed. ?      

Comparison of high- and low-energy collision-induced dissociation tandem mass spectrometry in the analysis of glycoalkaloids and their aglycons

Four aglycons (tomatidine, demissidine, solanidine, and solasodine) and three glycoalkaloids (α-tomatine, α-chaconine, and α-solanine) have been analyzed by positive ion liquid secondary ion high-energy and low-energy collision-induced dissociation (CID) tandem mass Spectrometry, performed on a four-sector (EBEB) and a hybrid (EBQQ) instrument, respectively. Both high- and low-energy collision-induced dissociation mass spectra of [M+H]⁺ ions of these compounds provided structural information that aided the characterization of the different aglycons and of the carbohydrate sequence and linkage sites in the glycoalkaloids. Low-energy CID favors charge-driven fragmentation of the aglycon rings, whilst high-energy CID spectra are more complex and contain additional ions that appear to result from charge-remote fragmentations, multiple cleavages, or complex charge-driven rearrangements. With respect to the structural characterization of the carbohydrate part, low-energy CID fragmentations of sugar residues in the glycoalkaloids generate Y _n ⁺ ions and some low intensity Z _n ⁺ ions; the high-energy spectra also exhibit strong ^1,5X _n ⁺ ions, formed by multiple cleavage of the sugar ring, and significant Z _n ⁺ ions.     

Resonance Activation and Collision-Induced-Dissociation of Ions Using Rectangular Wave Dipolar Potentials in a Digital Ion Trap Mass Spectrometer

Collision-induced dissociation (CID) of ions by resonance activation in a quadrupole ion trap is usually accomplished by resonance exciting the ions to higher kinetic energy, whereby the high kinetic energy ions collide with a bath gas, such as helium or argon, inside the trap and dissociate to fragments. A new ion activation method using a well-defined rectangular wave dipolar potential formed by dividing down the trapping rectangular waveform is developed and examined herein. The mass-selected parent ions are resonance excited to high kinetic energies by simply changing the frequency of the rectangular wave dipolar potential and dissociation proceeds. A relationship between the ion mass and the activation waveform frequency is also identified and described. This highly efficient (CID) procedure can be realized by simply changing the waveform frequency of the dipolar potential, which could certainly simplify tandem mass spectrometry analysis methods.

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.

ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell. Figure

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A Distonic Radical-Ion for Detection of Traces of Adventitious Molecular Oxygen (O2) in Collision Gases Used in Tandem Mass Spectrometers

We describe a diagnostic ion that enables rapid semiquantitative evaluation of the degree of oxygen contamination in the collision gases used in tandem mass spectrometers. Upon collision-induced dissociation (CID), the m/z 359 positive ion generated from the analgesic etoricoxib undergoes a facile loss of a methyl sulfone radical [^?SO₂(CH₃); 79-Da] to produce a distonic radical cation of m/z 280. The product-ion spectrum of this m/z 280 ion, recorded under low-energy activation on tandem-in-space QqQ or QqTof mass spectrometers using nitrogen from a generator as the collision gas, or tandem-in-time ion-trap (LCQ, LTQ) mass spectrometers using purified helium as the buffer gas, showed two unexpected peaks at m/z 312 and 295. This enigmatic m/z 312 ion, which bears a mass-to-charge ratio higher than that of the precursor ion, represented an addition of molecular oxygen (O₂) to the precursor ion. The exceptional affinity of the m/z 280 radical cation towards oxygen was deployed to develop a method to determine the oxygen content in collision gases. Figure

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Elimination of Benzene from Protonated N-Benzylindoline: Benzyl Cation/Proton Transfer or Direct Proton Transfer?

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp²-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn

A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MSⁿ, n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MSⁿ, with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions (m/z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MSⁿ. High confidence stereo-structural determination was achieved by matching MSⁿ CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MSⁿ (n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.