Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.Keywords: brain proteome; chronic phase; cynomolgus monkey; ischemia; iTRAQ; stroke biology;

•1st Global phosphoproteomics analyses of the macaque cerebral cortex.•Obtained by applying our recently developed fully automated RP-SA(C)X-RP platform.•14,338 Distinct phosphopeptides in 2705 phosphoproteins at 1% FDR were identified.•784 Novel phosphorylation sites were identified.A fully automated online multidimensional liquid chromatography (MDLC) platform featuring high-/low-pH reversed-phase (RP) dimensions and two other complementary—strong anion exchange (SAX) and strong cation exchange (SCX), respectively—chromatographic separations in tandem, with conventional offline titanium dioxide pre-enrichment, has been applied for the first global phosphopeptide identification from the macaque cerebral cortex in the presence of phosphatase inhibitors. Phosphorylation data interpretation, including site determination, and network construction have been performed: 14,338 distinct phosphopeptides in 7572 non-redundant phosphosites at 1% FDR were identified with 784 novel phosphorylation sites when mapping into the two most-curated public phosphorylation databases, PhosphoSitePlus (PSP) and Phospho.ELM (ELM), using probability-based placements. The net charges of both extremely acidic and basic phosphopeptides depend largely on the pH of the solvent, in turn impacting their retention and subsequent fractionation; the inclusion of the complementary SAX and SCX column chemistries after the high-pH RP dimension allowed effective retention and separation of net-negatively and −positively charged phosphopeptides, thereby leading to extended anionic and cationic phosphopeptide coverage from basophilic and acidophilic kinase substrates. A valuable protein interaction network of known and predicted motifs kinases was constructed from 3064 confident phosphorylation sites in the non-human primate’s brain.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal–ligand–peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen–nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol−1, much lower than the barriers to their dissociation (40.3 kcal mol−1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 − H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.

Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn − H]˙+ ions with [b2 − H − 17]˙+ and [c1 − 17]+ ions, the dominant products in the dissociation of [zn − H]˙+, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [Yπ˙GG]+ establishes that in the formation of [b2 − H − 17]˙+ ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 − 17]+ ion derived from [Yπ˙GG]+ shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Yπ˙GG]+ using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn − H]˙+ ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 − 17]+ ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 − H − 17]˙+ ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 − 17]+ ions.

Herein we report the discovery of a novel lead compound, oxyphylla A [(R)-4-(2-hydroxy-5-methylphenyl)-5-methylhexanoic acid] (from the fruit of Alpinia oxyphylla), which functions as a neuroprotective agent against Parkinson’s disease. To identify a shortlist of candidates from the extract of A. oxyphylla, we employed an integrated strategy combining liquid chromatography/mass spectrometry, bioactivity-guided fractionation, and chemometric analysis. The neuroprotective effects of the shortlisted candidates were validated prior to scaling up the finalized list of potential neuroprotective constituents for more detailed chemical and biological characterization. Oxyphylla A has promising neuroprotective effects: (i) it ameliorates in vitro chemical-induced primary neuronal cell damage and (ii) alleviates chemical-induced dopaminergic neuron loss and behavioral impairment in both zebrafish and mice in vivo. Quantitative proteomics analyses of oxyphylla A-treated primary cerebellar granule neurons that had been intoxicated with 1-methyl-4-phenylpyridinium revealed that oxyphylla A activates nuclear factor-erythroid 2-related factor 2 (NRF2)—a master redox switch—and triggers a cascade of antioxidative responses. These observations were verified independently through western blot analyses. Our integrated metabolomics, chemometrics, and pharmacological strategy led to the efficient discovery of novel bioactive ingredients from A. oxyphylla while avoiding the nontargeting, labor-intensive steps usually required for identification of bioactive compounds. Our successful development of a synthetic route toward oxyphylla A should lead to its availability on a large scale for further functional development and pathological studies.

We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS3 dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%–94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in CuII(2,2′:6′,2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates.

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP–SA(C)X–RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP–SA(C)X–RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.

An automatable, robust, high-performance online multidimensional liquid chromatography (MDLC) platform comprising of pH 10 reversed-phase (RP), strong cation exchange (SCX), and pH 2 RP separation stages has been integrated into a modified commercial off-the-shelf LC instrument with a simple rewiring, enabling accelerated routine qualitative and quantitative proteomics analyses. This system has been redesigned with a dual-trap column configuration to improve the throughput by greatly decreasing the system idle time. The performance of this new design has been benchmarked through analysis of the total lysate of S. cerevisiae, in comparison with that of the former tailor-made system featuring more complicated components; the total run time per “load-and-go” LC/MS analysis was approximately 24 h, with minimal idle time and no labor-intensive steps. This platform features high-resolution fractionations, ease of use and a high degree of user programmability in the first two chromatographic dimensions, allowing flexible and effective sampling with (RP–SCX–RP) or without (RP–RP) the inclusion of SCX sub-fractionation; good proteome coverage and reproducibility was demonstrated through the analyses of bacterial, cell culture, and monkey brain tissue proteomes. The viability of the 3D RP–SCX–RP has been proven in proteome-wide studies of STO fibroblasts and yeast tryptic digests, resulting in extended proteome and protein coverages with high reproducibility—in particular, discovering extra-hydrophilic peptides—at the expense of the acquisition time. The identified inventory of the rat pheochromocytoma PC12 cell proteome—a total of 6345 proteins and 97309 unique peptides is the most comprehensive dataset to date—provides an example of the value of the 3D RP–SCX–RP. The use of orthogonal chromatographic dimensions in the 3D RP–SCX–RP also circumvents the issues of isobaric interference of mass-tagging background contaminations, while significantly improving the accuracy of isobaric tags for relative and absolute quantitation (iTRAQ)-based protein quantitation experiments.

•Online three-dimensional liquid chromatography for simultaneous proteomics and N-glycoproteomics.•First N-glycoproteomics analysis of cynomolgus monkey plasma.In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology—comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension—in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC–RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11 984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

The fragmentation products of the ε-carbon-centered radical cations [Yε˙LG]+ and [Yε˙GL]+, made by 266 nm laser photolysis of protonated 3-iodotyrosine-containing peptides, are substantially different from those of their π-centered isomers [Yπ˙LG]+ and [Yπ˙GL]+, made by dissociative electron transfer from ternary metal–ligand–peptide complexes. For leucine-containing peptides the major pathway for the ε-carbon-centered radical cations is loss of the side chain of the leucine residue forming [YGα˙G]+ and [YGGα˙]+, whereas for the π-radicals it is the side chain of the tyrosine residue that is lost, giving [Gα˙LG]+ and [Gα˙GL]+. The fragmentations of the product ions [YGα˙G]+ and [YGGα˙]+ are compared with those of the isomeric [Yε˙GG]+ and [Yπ˙GG]+ ions. The collision-induced spectra of ions [Yε˙GG]+ and [YGGα˙]+ are identical, showing that interconversion occurs prior to dissociation. For ions [Yε˙GG]+, [Yπ˙GG]+ and [YGα˙G]+ the dissociation products are all distinctly different, indicating that dissociation occurs more readily than isomerization. Density functional theory calculations at B3LYP/6-31++G(d,p) gave the relative enthalpies (in kcal mol−1 at 0 K) of the five isomers to be [Yε˙GG]+ 0, [Yπ˙GG]+ −23.7, [YGGα˙]+ −28.7, [YGα˙G]+ −31.0 and [Yα˙GG]+ −38.5. Migration of an α-C–H atom from the terminal glycine residue to the ε-carbon-centered radical in the tyrosine residue, a 1−11 hydrogen atom shift, has a low barrier, 15.5 kcal mol−1 above [Yε˙GG]+. By comparison, isomerization of [Yε˙GG]+ to [YGα˙G]+ by a 1–8 hydrogen atom migration from the α-C–H atom of the central glycine residue has a much higher barrier (50.6 kcal mol−1); similarly conversion of [Yε˙GG]+ into [Yπ˙GG]+ has a higher energy (24.4 kcal mol−1).

Fascinating N-terminal Cα–C bond cleavages in a series of nonbasic tyrosine-containing peptide radical cations have been observed under low-energy collision-induced dissociation (CID), leading to the generation of rarely observed x-type radical fragments, with significant abundances. CID experiments of the radical cations of the alanyltyrosylglycine tripeptide and its analogues suggested that the N-terminal Cα–C bond cleavage, yielding its [x2 + H]•+ radical cation, does not involve an N-terminal α-carbon-centered radical. Theoretical examination of a prototypical radical cation of the alanyltyrosine dipeptide, using density functional theory calculations, suggested that direct N-terminal Cα–C bond cleavage could produce an ion–molecule complex formed between the incipient a1+ and x1• fragments. Subsequent proton transfer from the iminium nitrogen atom in a1+ to the acyl carbon atom in x1• results in the observable [x1 + H]•+. The barriers against this novel Cα–C bond cleavage and the competitive N–Cα bond cleavage, forming the complementary [c1 + 2H]+/[z1 – H]•+ ion pair, are similar (ca. 16 kcal mol–1). Rice–Ramsperger–Kassel–Marcus modeling revealed that [x1 + H]•+ and [c1 + 2H]+ species are formed with comparable rates, in agreement with energy-resolved CID experiments for [AY]•+.

In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [GnW]•+ (n = 2–4) and [GXW]•+ (X = C, S, L, F, Y, Q) species. The Cβ–Cγ bond cleavage of a C-terminal decarboxylated tryptophan residue ([M – CO2]•+) can generate [M – CO2 – 116]+, [M – CO2 – 117]•+, and [1H-indole]•+ (m/z 117) species as possible product ions. Competition between the formation of [M – CO2 – 116]+ and [1H-indole]•+ systems implies the existence of a proton-bound dimer formed between the indole ring and peptide backbone. Formation of such a proton-bound dimer is facile via a protonation of the tryptophan γ-carbon atom as suggested by density functional theory (DFT) calculations. DFT calculations also suggested the initially formed ion 2, the decarboxylated species that is active against Cβ–Cγ bond cleavage, can efficiently isomerize to form a more stable π-radical isomer (ion 9) as supported by Rice–Ramsperger–Kassel–Marcus (RRKM) modeling. The Cβ–Cγ bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WGnR]•+ (n = 1–3) radical cations consistently resulted in predominant formation of [M – 116]+ product ions. It appears that the basic arginine residue tightly sequesters the proton and allows the charge-remote Cβ–Cγ bond cleavage to prevail over the charge-directed one. DFT calculations predicted that the barrier for the former is 6.2 kcal mol–1 lower than that of the latter. Furthermore, the pathway involving a salt-bridge intermediate also was accessible during such a bond cleavage event.

Under the conditions of low-energy collision-induced dissociation (CID), the canonical glycylphosphoserinyltryptophan radical cation having its radical located on the side chain of the tryptophan residue ([GpSW]•+) fragments differently from its tautomer with the radical initially generated on the α-carbon atom of the glycine residue ([G•pSW]+). The dissociation of [G•pSW]+ is dominated by the neutral loss of H3PO4 (98 Da), with backbone cleavage forming the [b2 – H]•+/y1+ pair as the minor products. In contrast, for [GpSW]•+, competitive cleavages along the peptide backbone, such as the formation of [GpSW – CO2]•+ and the [c2 + 2H]+/[z1 – H]•+ pair, significantly suppress the loss of neutral H3PO4. In this study, we used density functional theory (DFT) to examine the mechanisms for the tautomerizations of [G•pSW]+ and [GpSW]•+ and their dissociation pathways. Our results suggest that the dissociation reactions of these two peptide radical cations are more efficient than their tautomerizations, as supported by Rice–Ramsperger–Kassel–Marcus (RRKM) modeling. We also propose that the loss of H3PO4 from both of these two radical cationic tautomers is preferentially charge-driven, similar to the analogous dissociations of even-electron protonated peptides. The distonic radical cationic character of [G•pSW]+ results in its charge being more mobile, thereby favoring charge-driven loss of H3PO4; in contrast, radical-driven pathways are more competitive during the CID of [GpSW]•+.

In this study, we observed unprecedented cleavages of the Cβ–Cγ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M – 116]+ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent Cβ–Cγ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH – 43]+ and [WGGGH – 116]+, obtained from the CID of [LGGGH]•+ and [WGGGH]•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]•-2), in agreement with the CID data for [WGGGH]•+ and [W1-CH3GGGH]•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from Cβ–Cγ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the Cβ–Cγ bond and, therefore, decreases the dissociation energy barrier dramatically.

The gas phase fragmentations of aliphatic radical cationic glycylglycyl(iso)leucine tripeptides ([G•G(L/I)]+), with well-defined initial locations of the radical centers at their N-terminal α-carbon atoms, are significantly different from those of their basic glycylarginyl(iso)leucine ([G•R(L/I)]+) counterparts; the former lead predominantly to [b2 – H]•+ fragment ions, whereas the latter result in the formation of characteristic product ions via the losses of •CH(CH3)2 from [G•RL]+ and •CH2CH3 from [G•RI]+ through Cβ–Cγ side-chain cleavages of the (iso)leucine residues, making these two peptides distinguishable. The α-carbon-centered radical at the leucine residue is the key intermediate that triggers the subsequent Cβ–Cγ bond cleavage, as supported by the absence of •CH(CH3)2 loss from the collision-induced dissociation of [G•RLα-Me]+, a radical cation for which the α-hydrogen atom of the leucine residue had been substituted by a methyl group. Density functional theory calculations at the B3LYP 6-31++G(d,p) level of theory supported the notion that the highly basic arginine residue could not only increase the energy barriers against charge-induced dissociation pathways but also decrease the energy barriers against hydrogen atom transfers in the GR(L/I) radical cations by ∼10 kcal mol–1, thereby allowing the intermediate precursors containing α- and γ-carbon-centered radicals at the (iso)leucine residues to be formed more readily prior to promoting subsequent Cβ–Cγ and Cα–Cβ bond cleavages. The hydrogen atom transfer barriers for the α- and γ-carbon-centered GR(L/I) radical cations (roughly in the range 29–34 kcal mol–1) are comparable with those of the competitive side-chain cleavage processes. The transition structures for the elimination of •CH(CH3)2 and •CH2CH3 from the (iso)leucine side chains possess similar structures, but slightly different dissociation barriers of 31.9 and 34.0 kcal mol–1, respectively; the energy barriers for the elimination of the alkenes CH2═CH(CH3)2 and CH3CH═CHCH3 through Cα–Cβ bond cleavages of γ-carbon-centered radicals at the (iso)leucine side chains are 29.1 and 32.8 kcal mol–1, respectively.

In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal–ligand phosphorylated peptide complexes [CuII(terpy)pM]·2+ and [CoIII(salen)pM]·+ [pM: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations (pM·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M – H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the pM·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model—N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

Fragmentation of radical cationic peptides [R(G)n−2X(G)7−n]˙+ and [R(G)m−2XG]˙+ (X = Phe or Tyr; m = 2–5; n = 2–7) leads selectively to an+ product ions through in situ Cα–C peptide backbone cleavage at the aromatic amino acid residues. In contrast, substituting the arginine residue with a less-basic lysine residue, forming [K(G)n−2X(G)7−n]˙+ (X = Phe or Tyr; n = 2–7) analogs, generates abundant b–y product ions; no site-selective Cα–C peptide bond cleavage was observed. Studying the prototypical radical cationic tripeptides [RFG]˙+ and [KFG]˙+ using low-energy collision-induced dissociation and density functional theory, we have examined the influence of the basicity of the N-terminal amino acid residue on the competition between the isomerization and dissociation channels, particularly the selective Cα–C bond cleavage via β-hydrogen atom migration. The dissociation barriers for the formation of a2+ ions from [RFG]˙+ and [KFG]˙+via their β-radical isomers are comparable (33.1 and 35.0 kcal mol−1, respectively); the dissociation barrier for the charge-induced formation of the [b2 − H]˙+ radical cation from [RFG]˙+via its α-radical isomer (39.8 kcal mol−1) was considerably higher than that from [KFG]˙+ (27.2 kcal mol−1). Thus, the basic arginine residue sequesters the mobile proton to promote the charge-remote selective Cα–C bond cleavage by energetically hindering the competing charge-induced pathways.

Previously, we described an online high-/low-pH RP–RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP–RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation. Relative to the RP–RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.

Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP–RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP–RP can be harnessed to complement SCX–RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.

Gas phase fragmentations of two isomeric radical cationic tripeptides of glycylglycyltryptophan—[G•GW]+ and [GGW]•+—with well-defined initial radical sites at the α-carbon atom and the 3-methylindole ring, respectively, have been studied using collision-induced dissociation (CID), density functional theory (DFT), and Rice−Ramsperger−Kassel−Marcus (RRKM) theory. Substantially different low-energy CID spectra were obtained for these two isomeric GGW structures, suggesting that they did not interconvert on the time scale of these experiments. DFT and RRKM calculations were used to investigate the influence of the kinetics, stabilities, and locations of the radicals on the competition between the isomerization and dissociation channels. The calculated isomerization barrier between the GGW radical cations (>35.4 kcal/mol) was slightly higher than the barrier for competitive dissociation of these species (<30.5 kcal/mol); the corresponding microcanonical rate constants for isomerization obtained from RRKM calculations were all considerably lower than the dissociation rates at all internal energies. Thus, interconversion between the GGW isomers examined in this study cannot compete with their fragmentations.

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.

In this paper, we demonstrate for the first time the formation of radical anionic peptides [M − 2H]·− through a one-electron transfer mechanism upon low-energy collision-induced dissociation (CID) of gas-phase singly charged [MnIII(salen)(M − 2H)]·− complex ions [where salen is N,N′-ethylenebis(salicylideneiminato) and M is an angiotensin III derivative]. The types of fragment ions formed from [M − 2H]·− share some similarities with those from the cationic radical peptides M·+ and [M + H]·2+, but differ significantly from those of the corresponding deprotonated peptides [M − H]−. Fragmentation of [M − 2H]·− radical anionic angiotensin III derivatives leads preferentially to product ions of side-chain cleavage of amino acid residues, z-type and minor x-type fragment ions, most of which are types rarely observed in low-energy CID spectra of deprotonated analogs. The degree of competitive dissociation of the complexes is highly dependent on the nature of the substituted salen derivatives. The yields of anionic peptide radicals were enhanced to the greatest extent when electron withdrawing groups were positioned at the 5 and 5′ positions, but the effect was rather modest when such groups resided at the 3 and 3′ positions. Substituting a cyclohexyl unit of a salen with phenyl or naphthyl moieties at the 8 and 8′ positions also facilitated electron-transfer pathways.

Molecular radical cations of tripeptides of the form glycylglycyl(residue X) (GGX•+) are produced by the collision-induced, intramolecular one-electron transfer of [Cu(II)(L)GGX]•2+ complexes (L = triamine ligand). We demonstrate, for the first time, the formation of molecular radical cations of all of the aliphatic, basic, aromatic, acidic, and some heteroatom-bearing GGX tripeptides, albeit inefficiently in some cases, by altering the structure of the auxiliary polyamine ligand bound to the copper atom. The design of the ligand allows exquisite control over the nature of the dissociation pathway. Steric hindrance of bulky groups in the ligand affects the binding of the peptide to the copper ion; this interaction is an important factor in determining whether the electron transfer pathway predominates.

The first example of the formation of hydrogen-deficient radical cations of the type [M + H]·2+ is demonstrated to occur through a one-electron-transfer mechanism upon low-energy collision-induced dissociation (CID) of gas-phase triply charged [CuII(terpy)(M + H)]·3+ complex ions (where M is an angiotensin III or enkephalin derivative; terpy = 2,2′:6′,2″-terpyridine). The collision-induced dissociation of doubly charged [M + H]·2+ radical cations generates similar product ions to those prepared through hot electron capture dissociation (HECD). Isomeric isoleucine and leucine residues were distinguished by observing the mass differences between [zn + H]·+ and wn+ ions (having the same residue number, n) of the Xle residues. The product ion spectrum of [zn + H]·+ reveals that the wn+ ions are formed possibly from consecutive fragmentations of [zn + H]·+ ions. Although only the first few [M + H]·2+ species have been observed using this approach, these hydrogen-deficient radical cations produce fragment ions that have more structure-informative patterns and are very different from those formed during the low-energy tandem mass spectrometry of protonated peptides.

Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn − H]˙+ ions with [b2 − H − 17]˙+ and [c1 − 17]+ ions, the dominant products in the dissociation of [zn − H]˙+, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [Yπ˙GG]+ establishes that in the formation of [b2 − H − 17]˙+ ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 − 17]+ ion derived from [Yπ˙GG]+ shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Yπ˙GG]+ using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn − H]˙+ ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 − 17]+ ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 − H − 17]˙+ ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 − 17]+ ions.

Fragmentation of radical cationic peptides [R(G)n−2X(G)7−n]˙+ and [R(G)m−2XG]˙+ (X = Phe or Tyr; m = 2–5; n = 2–7) leads selectively to an+ product ions through in situ Cα–C peptide backbone cleavage at the aromatic amino acid residues. In contrast, substituting the arginine residue with a less-basic lysine residue, forming [K(G)n−2X(G)7−n]˙+ (X = Phe or Tyr; n = 2–7) analogs, generates abundant b–y product ions; no site-selective Cα–C peptide bond cleavage was observed. Studying the prototypical radical cationic tripeptides [RFG]˙+ and [KFG]˙+ using low-energy collision-induced dissociation and density functional theory, we have examined the influence of the basicity of the N-terminal amino acid residue on the competition between the isomerization and dissociation channels, particularly the selective Cα–C bond cleavage via β-hydrogen atom migration. The dissociation barriers for the formation of a2+ ions from [RFG]˙+ and [KFG]˙+via their β-radical isomers are comparable (33.1 and 35.0 kcal mol−1, respectively); the dissociation barrier for the charge-induced formation of the [b2 − H]˙+ radical cation from [RFG]˙+via its α-radical isomer (39.8 kcal mol−1) was considerably higher than that from [KFG]˙+ (27.2 kcal mol−1). Thus, the basic arginine residue sequesters the mobile proton to promote the charge-remote selective Cα–C bond cleavage by energetically hindering the competing charge-induced pathways.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal–ligand–peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen–nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol−1, much lower than the barriers to their dissociation (40.3 kcal mol−1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 − H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.