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Diketopiperazine (DKP) formation is an important degradation pathway for peptides and proteins. It can occur during synthesis and storage in either solution or the solid state. The kinetics of peptide cleavage through DKP formation have been analyzed for the model peptides Xaa

We present simple considerations of how differences in time scales of motions of protons, the lightest and fastest chemical moiety, and the much longer time scales associated with the dynamics of proteins, among the heaviest and slowest analytes, may allow many protein conformations from solution to be kinetically trapped during the process of electrospraying protein solutions into the gas phase. In solution, the quantum nature of protons leads them to change locations by tunneling, an instantaneous process; moreover, the Grotthuss mechanism suggests that these small particles can respond nearly instantaneously to the dynamic motions of proteins that occur on much longer time scales. A conformational change is accompanied by favorable or unfavorable variations in the free energy of the system, providing the impetus for solvent ↔ protein proton exchange. Thus, as thermal distributions of protein conformations interconvert, protonation states rapidly respond, as specific acidic and basic sites are exposed or protected. In the vacuum of the mass spectrometer, protons become immobilized in locations that are specific to the protein conformations from which they were incorporated. In this way, conformational states from solution are preserved upon electrospraying them into the gas phase. These ideas are consistent with the exquisite sensitivity of electrospray mass spectra to small changes of the local environment that alter protein structure in solution. We might remember this approximation for the protonation of proteins in solution with the colloquial expression—protons are fast and smart; proteins are slow and dumb.

Glioblastoma multiforme (GBM), a malignant, highly aggressive, grade IV brain tumor, which rapidly infiltrates into the nearby tissue, has drawn a significant amount of attention because of its poor prognosis and the limited treatment options available. In GBM, nearly all tumor cells exhibit aberrant cell-surface glycosylation patterns due to the alteration of their biosynthesis or postsynthesis modification process. Since gangliosides (GGs) are acknowledged as tumor- associated antigens, we have carried out here a comprehensive profiling of native ganglioside mixtures extracted and purified from GBM specimens. For this purpose, high performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of GBM-specific structures and the assessment of their roles as tumor markers or possible associated antigens. GG separation by IMS according to the charge state, carbohydrate chain length, degree of sialylation, and ceramide composition led to the identification of no less than 160 distinct components, which represents 3- fold the number of structures identified before. The detected GGs and asialo-GGs were found characterized by a high heterogeneity in their ceramide and glycan compositions, encompassing up five Neu5Ac residues. The tumor was found dominated in equal and high proportions by GD3 and GT1 forms, with a particular incidence of C24:1 fatty acids in the ceramide. By the occurrence of only one mobility feature and the diagnostic fragment ions, the IMS tandem MS conducted using collision-induced dissociation (CID) disclosed for the first time the presence of GT1c(d18:1/24:1) newly proposed here as a potential GBM marker.

Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.0) was monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the stability and investigate structural changes involved in unfolding. We utilize two experimental approaches to induce thermal denaturation: a variable-temperature electrospray ionization (vT-ESI) source that heats the bulk solution in the ESI emitter, and a variable-power 10.6 μm CO(2) laser that rapidly heats nanodroplets produced by ESI. These two approaches sample different time scales of the denaturation process; long time scales (seconds to minutes) where the system is at equilibrium using the vT-ESI approach and shorter time scales (μs) by rapid droplet heating in which the system is in a pre-equilibrium state. Increasing the solution temperature (from 28 to 95 °C in the vT-ESI experiments) shifts the charge state distribution from low charge states ([M + 7H](7+) to [M + 9H](9+)) to more highly charged species. This is accompanied by loss of the heme group to yield the apomyoglobin (aMb) species, indicating that the protein has unfolded. Monitoring the formation of aMb and the shift in average charge states of aMb and hMb with solution temperature allows for relative quantitation of their individual stabilities, highlighting the stabilizing effects of heme binding. We compare the degree of unfolding induced by heating the bulk solution (using vT-ESI) to the laser droplet heating approach and find that the rapid nature of the laser heating approach allows for transient pre-equilibrium states to be sampled.

Variable-temperature nano-electrospray ionization coupled with ion mobility spectrometry-mass spectrometry is used to investigate the thermal denaturation of the tetrameric protein concanavalin A. As the solution temperature is increased, changes in mass spectra and collision cross section distributions provide evidence for discrete structural changes that occur at temperatures that are ~40 to 50 degrees below the temperature required for tetramer dissociation. The subtle structural changes are associated with four distinct tetramer conformations with unique melting temperatures. Gibbs-Helmholtz analysis of the free energies determined with respect to the most abundant “native” state yields heat capacities of ΔC(p) = 1.6 ± 0.3, −2.2 ± 0.4, and −2.9 ± 1.6 kJ·K(−1)·mol(−1), and temperature dependent enthalpies and entropies for the three non-native conformations. Analysis of the thermochemistry indicates that the high-temperature products are entropically stable until the threshold for tetramer dissociation, and changes in heat capacity are consistent with increases in solvation of polar residues. Our findings suggest these high-temperature non-native states result from an increase in disorder at surface exposed regions. Such studies provide valuable insight towards the structural details of non-native states.

Variable temperature electrospray ionization (ESI) is coupled with mass spectrometry techniques in order to investigate structural transitions of monoclonal antibody immunoglobulin G (IgG) in a 100 mM ammonium acetate (pH 7.0) solution from 26 to 70 °C. At 26 °C, the mass spectrum for intact IgG shows six charge states +22 to +26. Upon increasing the solution temperature the fraction of low-charge states decreases and new, higher-charge state ions are observed. Upon analysis it appears that heating the solution aids in desolvation of the intact IgG precursor. Above, ~50 °C a cleavage event between the light and heavy chains is observed. An analysis of the kinetics for this processes at different temperatures yields transition state thermochemistry of ΔH(‡) = 95 ± 10 kJ·mol(−1), ΔS(‡) = 8 ± 1 J·mol(−1)·K(−1), and ΔG‡ = 92 ± 11 kJ·mol(−1). The mechanism for light chain dissociation appears to involve disulfide bond scrambling that ultimately results in a non-native Cys(199)-Cys(217) disulfide bond in the light chain product. Above ~70 °C, we are unable to produce a stable ESI signal. The loss of signal is ascribed to aggregation that is primarily associated with the remaining portion of the antibody after having lost the light chain.

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H](3+) ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential-energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H](3+) in the absence of solvent are on the order of ~40 kJ∙mol(−1), substantially lower than the ~90 kJ∙mol(−1) required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas-phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited.

Rationale Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial-resolution measurements similar to matrix-assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher-mass protein applications directly from surfaces on high-performance mass spectrometers. Studying a well-interrogated protein by ion mobility spectrometry-mass spectrometry (IMS-MS) to access effects on structures using a solid vs. solvent matrix may provide insights. Methods Ubiquitin was studied by IMS-MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift-tube instrument; MS™ sources). Mass-to-charge and drift-time (td )-measurements are compared for ubiquitin ions obtained by inlet and vacuum ionization using laserspray ionization (LSI), matrix- (MAI) and solvent-assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable. Results Using the same solution conditions with SYNAPT G2(S) instruments, td -distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift-tube instrument, within the elongated distribution of structures, both similar and different td -distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI-generated ions are frequently narrower in their td -distributions. Conclusions Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI- or MAI-generated ions and not with the solution structures.

Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures,...

Ion mobility spectrometry and circular dichroism spectroscopy are used to examine the populations of the small model peptide, polyproline-13 in water, methanol, ethanol, and 1-propanol over a range of solution temperatures (from 288 to 318 K). At low temperatures, the less-polar solvents (1-propanol and ethanol) favor the all-cis polyproline I helix (PPI); as the temperature is increased, the trans-configured polyproline II helix (PPII) is formed. In polar solvents (methanol and water), PPII is favored at all temperatures. From the experimental data, we determine the relative stabilities of the eight structures in methanol, ethanol, and 1-propanol, as well as four in water, all with respect to PPII. Although these conformers show relatively small differences in free energies, substantial variability is observed in the enthalpies and entropies across the structures and solvents. This requires that enthalpies and entropies be highly correlated: in 1-propanol, cis-configured PPI conformations are energetically favorable but entropically disfavored. In more polar solvents, PPI is enthalpically less favorable and entropy favors trans-configured forms. While either ΔH(0) or ΔS(0) can favor different structures, no conformation in any solvent is simultaneously energetically and entropically stabilized. These data present a rare opportunity to examine the origin of conformational stability.

Profiling of complex biological samples (e.g., serum) using mass spectrometry continues to be an active area of research with a large and growing literature. Pattern recognition techniques can be effective methods for the analysis of complex data sets generated in these types of studies. Currently, we are investigating the discrimination of disease phenotypes associated with esophageal adenocarcinoma by analysis of single N-linked glycans using matrix assisted laser desorption ionization-ion mobility spectrometry-mass spectrometry (MALDI-IMS-MS). The glycans were extracted from sera of healthy (normal) controls (NC) and patients diagnosed with Barrett's Esophagus (BE), high grade dysplasia (HGD), and esophageal adenocarcinoma (EAC). MALDI-IMS-MS spectral images were collected in duplicate for these 58 serum samples: BE (14 individuals), HGD (7 individuals), EAC (20 individuals) and NC (17 individuals). Ion mobility distributions of N-linked glycans that possessed sufficient signal to noise in all 116 spectra were extracted from the images by box selection across a specific drift bin and m/z range corresponding to a single linked N-glycan ion. A composite ion mobility distribution profile was obtained for each image by sequentially splicing together the mobility distributions of each N-linked glycan across an arbitrary drift bin axis. Wavelet preprocessing of the composite ion mobility distribution profiles was performed using the discrete wavelet transform, which was coupled to a genetic algorithm for variable selection to identify a subset of wavelet coefficients within the data set that optimized the separation of the four classes (BE, HGD, EAC, and NC) in a plot of the two largest principal components of the wavelet transformed data. A discriminant developed from the wavelet coefficients identified by the pattern recognition GA correctly classified all ion mobility distribution profiles in the training set (45 individuals and 87 distribution profiles) and 23 of 26 blinds (13 individuals and 26 distribution profiles) in the prediction set. The proposed MALDI-IMS-MS and pattern recognition methodology has the potential to exploit molecules in serum samples that can serve as the basis of a potential method for cancer prescreening.

The solution-favored conformations of the 13-residue disordered peptide, indolicidin (Ile(1)-Leu(2)-Pro(3)-Trp(4)-Lys(5)-Trp(6)-Pro(7)-Trp(8)-Trp(9)-Pro(10)-Trp(11)-Arg(12)-Arg(13)), are evaluated using electrospray ionization (ESI) coupled to ion mobility spectrometry-mass spectrometry (IMS-MS). The ESI-IMS-MS distributions for the dominant [M+4H](4+) ions indicate that three populations of structures coexist in a range of aqueous to non-aqueous solutions (water:dioxane, water:trifluoroethanol, and water:hexafluoroisopropanol). Conformer types and their relative abundances change in response to different solution environments suggesting that the gas phase conformers reflect on the solution populations present in different solvent environments. Collisional activation of isolated gas phase conformations with IMS-IMS-MS experiments provides additional insight about the relative stabilities of different structural types in the absence of solvent. Simulated annealing studies suggest that proline configuration may be important for the presence of multiple conformations.

A combination of liquid chromatography, ion mobility spectrometry, mass spectrometry, and database searching techniques were used to characterize the proteomes of four biological replicates of adult Drosophila melanogaster heads at seven time points across their lifespans. Based on the detection of tryptic peptides, the identities of 1281 proteins were determined. An estimate of the abundance of each protein, based on the three most intense peptide ions, shows that the quantified species vary in concentration over a factor of ~103. Compared to initial studies in the field of Drosophila proteomics, our current results show an eight-fold higher temporal protein coverage with increased quantitative accuracy. Across the lifespan, we observe a range of trends in the abundance of different proteins, including: an increase in abundance of proteins involved in oxidative phosphorylation, and the tricarboxylic acid cycle; a decrease in proteasomal proteins, as well as ribosomal proteins; and, many types of proteins, which remain relatively unchanged. For younger flies, proteomes are relatively similar within their age group. For older flies, proteome similarity decreases within their age group. These combined results illustrate a correlation between increasing age and decreasing proteostasis.

Ion mobility mass spectrometry (IMS-MS) techniques were used to generate a database of 2288 collision cross sections of transition-metal-coordinated tryptic peptide ions. This database consists of cross sections for 1253 [Pep + X]2+ and 1035 [Pep + X + H]3+, where X2+ corresponds to Mn2+, Co2+, Ni2+, Cu2+, or Zn2+. This number of measurements enables the extraction of structural trends for transition-metal-coordinated peptide ions. The range of structures and changes in collision cross sections for X2+-coordinated species (compared with protonated species of the same charge state) is similar to Mg2+-coordinated species. This suggests that the structures are largely determined by similarities in cation size with differences among the cross section distributions presumably caused by X2+ interactions with specific functional groups offered by the residue R-groups or the peptide backbone. Cross section contributions for individual residues upon X2+ solvation are assessed with the derivation of intrinsic size parameters (ISPs). The comparison of the [Pep + X]2+ ISPs with those previously reported for [Pep + Mg]2+ ions displays a lower contribution to the cross section for His, carboxyamidomethylated Cys, and Met, and is consistent with specific metal-residue interactions identified within protein X-ray crystallography databases.

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa–Pro–Gly–Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa–Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa–Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

N-Linked glycans, extracted from patient sera and healthy control individuals, are analyzed by Matrix-assisted laser desorption ionization (MALDI) in combination with ion mobility spectrometry (IMS), mass spectrometry (MS) and pattern recognition methods. MALDI-IMS-MS data were collected in duplicate for 58 serum samples obtained from individuals diagnosed with Barrett's esophagus (BE, 14 patients), high-grade dysplasia (HGD, 7 patients), esophageal adenocarcinoma (EAC, 20 patients) and disease-free control (NC, 17 individuals). A combined mobility distribution of 9 N-linked glycans is established for 90 MALDI-IMS-MS spectra (training set) and analyzed using a genetic algorithm for feature selection and classification. Two models for phenotype delineation are subsequently developed and as a result, the four phenotypes (BE, HGD, EAC and NC) are unequivocally differentiated. Next, the two models are tested against 26 blind measurements. Interestingly, these models allowed for the correct phenotype prediction of as many as 20 blinds. Although applied to a limited number of blind samples, this methodology appears promising as a means of discovering molecules from serum that may have capabilities as markers of disease.

Determining the affinity between a lectin and its target glycans is an important goal, both for understanding the biological functions of a given lectin as well as enabling the use of that lectin for targeted enrichment of glycosylated species from complex samples. While the overall selectivities of many lectins have been characterized, such studies generally require individually purified lectins and glycans. From these analyses, it is clear that a given lectin does not bind all of its target glycans with the same affinity. Rather, lectins display a continuum of affinities for the range of glycan structures they may encounter. Because of this continuum, it is not straightforward in practice to determine which set of structures will be enriched using a lectin as an affinity reagent. Here we describe the development of glycan affinity chromatography coupled directly to electrospray mass spectrometry, which enables direct analysis of interactions of lectins with both glycans and glycoconjugates from complex mixtures. By observing the elution behavior of individual species, we are able to determine exactly which set of glycoconjugates would be enriched for a given lectin. Furthermore, this approach allows for the direct assessment of affinity constants between an individual lectin and a large number of glycans in a single experiment, which can be conducted using a complex mixture of unpurified glycans of varying concentrations.

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn(2+) to the nine-residue peptide hormone oxytocin (OT, Cys(1)-Tyr(2)-Ile(3)-Gln(4)-Asn(5)-Cys(6)-Pro(7)-Leu(8)-Gly(9)-NH2, having a disulfide bond between Cys(1) and Cys(6) residues). Zn(2+) binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn(2+), we find evidence for two primary OT conformations, which arise because the Cys(6)-Pro(7) peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn(2+), we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 μM(-1), for the trans- and cis-configured populations, respectively. The Zn(2+) bound form of OT, having a cross section of Ω = 235 Å(2), has Pro(7) in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn(2+) binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro(7)-Leu(8)-Gly(9)-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn(2+). In this way, the proline residue regulates Zn(2+) binding to OT and, hence, is important in receptor binding. Graphical Abstract ᅟ.

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an eleven-residue neuropeptide, with the sequence Arg(1)-Pro(2)-Lys(3)-Pro(4)-Gln(5)-Gln(6)-Phe(7)-Phe(8)-Gly(9)-Leu(10)-Met(11)-NH(2), in ethanol. At elevated solution temperatures (55 to 75 °C) several reactions are observed, including: a protonation event, i.e., [subP+2H](2+) + H(+) → [subP+3H](3+), that appears to be regulated by a configurational change; and, two sequential bond cleavages (the Pro(2)-Lys(3) peptide bond is cleaved to form the smaller nonapeptide Lys(3)-Met(11)-NH(2) [subP((3–11))], and subsequently, subP((3–11)) is cleaved at the Pro(4)-Gln(5) peptide bond to yield the heptapeptide Gln(5)-Met(11)-NH(2) [subP((5–11))]). Each of the product peptides [subP((3–11)) and subP((5–11))] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg(1)-Pro(2) (cRP) for the first cleavage, and cyclo-Lys(3)-Pro(4) (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg(1)-Pro(2) peptide bond. The subP((3–11)) product has a long lifetime (t(½) ~ 30 hrs at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP((3–11)) is initially formed with a Lys(3)-trans-Pro(4) peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH(‡) = 41 to 85 kJ·mol(−1) and ΔS(‡) = −43 to −157 J·mol(−1)·K(−1) for each step in the reaction.

The strong synergy arising from coupling two orthogonal analytical techniques such as ion mobility and mass spectrometry can be used to separate complex mixtures and determine structural information of analytes in the gas phase. A tandem study is performed using two systems with different gases and pressures to ascertain gas-phase conformations of homopolymer ions. Aside from spherical and stretched configurations, intermediate configurations formed by a multiply charged globule and a "bead-on-a-string" appendix are confirmed for polyethylene-glycol (PEG), polycaprolactone (PCL), and polydimethylsiloxane (PDMS). These intermediate configurations are shown to be ubiquitous for all charge states and masses present. For each charge state, configurations evolve in two distinctive patterns: an inverse evolution which occurs as an elementary charge attached to the polymer leaves the larger globule and incorporates itself into the appendage, and a forward evolution which reduces the globule without relinquishing a charge while leaving the appendix relatively constant. Forward evolutions are confirmed to form self-similar family shapes that transcend charge states for all polymers. Identical structural changes occur at the same mass over charge regardless of the system, gas or pressure strongly suggesting that conformations are only contingent on number of charges and chain length, and start arranging once the ion is at least partially ejected from the droplet, supporting a charge extrusion mechanism. Configurational changes are smoother for PDMS which is attributed to the larger steric hindrance caused by protruding pendant groups. This study has implications in the study of the configurational space of more complex homopolymers and heteropolymers. Graphical Abstract.

Clustered into the so-called "glycosynaptic" microdomains in the central nervous system (CNS), gangliosides (GGs) are involved in the formation of functional synapses and neural circuits. Therefore, GGs are important biomarkers in the early diagnosis of CNS pathologies, which are the focus of our research as potential therapeutic targets. A series of neuropsychiatric disorders, including Alzheimer's disease and schizophrenia, are characterized by amnesia and disorientation caused by hippocampal atrophy and diminished cholinergic activity. Based on ion mobility mass spectrometry (IM-MS) capability for the reliable determination of glycopatterns, the changes in the diversity and number of GGs with age and the occurrence of neurological disorders, we report here the development of a high performance IM-MS strategy for assessing the GG profile in a complex mixture extracted from a 20 year old hippocampus. IM separation of GGs based on the charge state, carbohydrate chain length and degree of sialylation led to the detection and identification of 140 species, the largest number of GGs ever reported in an adult hippocampus. Moreover, the obtained data support the concept of GG cholinergic activity. IM tandem MS experiments using collision induced dissociation (CID) confirmed the incidence of GD1b(d18:1/24:1) in the investigated hippocampus specimen.

Rationale Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. Methods IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. Results IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. Conclusions The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates. Copyright © 2015 John Wiley & Sons, Ltd.

We report the first results from a new instrument capable of acquiring infrared spectra of mobility-selected ions. This demonstration involves using ion mobility to first separate the protonated peptide Gly-Pro-Gly-Gly (GPGG) into two conformational families with collisional cross-sections of 93.8 and 96.8 Å2. After separation, each family is independently analyzed by acquiring the infrared predissociation spectrum of the H2-tagged molecules. The ion mobility and spectroscopic data combined with density functional theory (DFT) based molecular dynamics simulations confirm the presence of one major conformer per family, which arises from cis/trans isomerization about the proline residue. We induce isomerization between the two conformers by using collisional activation in the drift tube and monitor the evolution of the ion distribution with ion mobility and infrared spectroscopy. While the cis-proline species is the preferred gas-phase structure, its relative population is smaller than that of the trans-proline species in the initial ion mobility drift distribution. This suggests that a portion of the trans-proline ion population is kinetically trapped as a higher energy conformer and may retain structural elements from solution. Graphical Abstract ᅟ

Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

Collisional activation of selected conformations by multidimensional ion mobility spectrometry (IMS–IMS), combined with mass spectrometry (MS), is described as a method to determine semi-quantitative activation energies for interconversion of different structures of the nonapeptide bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). This analysis is based on a calibration involving collision-induced dissociation measurements of ions with known dissociation energies (i.e., “thermometer” ions) such as leucine enkephalin, BK, and amino acid–metal cation systems. The energetic barriers between six conformations of [BK + 3H]3+ range from 0.23 ± 0.01 to 0.55 ± 0.03 eV. Prior results indicate that the major peaks in the IMS distributions correspond to specific combinations of cis and trans configurations of the three proline residues in the peptide sequence. The analysis allows us to directly assess pathways for specific transitions. The combination of structural assignments, experimentally determined barrier heights, onset of the quasi-equilibrium region, and dissociation threshold are used to derive a semi-quantitative potential energy surface for main features of [BK + 3H]3+.

Ion mobility spectrometry-mass spectrometry and Fourier transform infrared spectroscopy (FTIR) techniques were combined with quantum chemical calculations to examine the origin of icosahedral clusters of the amino acid proline. When enantiopure proline solutions are electrosprayed (using nanospray) from 100 mM ammonium acetate, only three peaks are observed in the mass spectrum across a concentration range of five orders of magnitude: a monomer [Pro+H](+) species, favored from 0.001 to 0.01 mM proline concentrations; a dimer [2Pro+H](+) species, the most abundant species for proline concentrations above 0.01 mM; and, the dimer and dodecamer [12Pro+2H](2+) for 1.0 mM and more concentrated proline solutions. Electrospraying racemic D/L-proline solutions from 100 mM ammonium acetate leads to a monomer at low proline concentrations (0.001 to 0.1 mM), and a dimer at higher concentrations (>0.09 mM), as well as a very small population of 8 to 15 Pro clusters that comprise

The structural characterization of glycans by mass spectrometry is particularly challenging. This is because of the high degree of isomerism in which glycans of the same mass can differ in their stereochemistry, attachment points, and degree of branching. Here we show that the addition of cryogenic vibrational spectroscopy to mass and mobility measurements allows one to uniquely identify and characterize these complex biopolymers. We investigate six disaccharide isomers that differ in their stereochemistry, attachment point of the glycosidic bond, and monosaccharide content, and demonstrate that we can identify each one unambiguously. Even disaccharides that differ by a single stereogenic center or in the monosaccharide sequence order show distinct vibrational fingerprints that would clearly allow their identification in a mixture, which is not possible by ion mobility spectrometry/mass spectrometry alone. Moreover, this technique can be applied to larger glycans, which we demonstrate by distinguishing isomeric branched and linear pentasaccharides. The creation of a database containing mass, collision cross section, and vibrational fingerprint measurements for glycan standards should allow unambiguous identification and characterization of these biopolymers in mixtures, providing an enabling technology for all fields of glycoscience. Graphical Abstract ᅟ.

Ion mobility mass spectrometry (IMS-MS) is used to investigate the abundance pattern, n z (m) of Poly-(ethyleneglycol) (PEG) electrosprayed from water/methanol as a function of mass and charge state. We examine n z (m) patterns from a diversity of solution cations, primarily dimethylammonium and triethylammonium. The ability of PEG chains to initially attach to various cations in the spraying chamber, and to retain them (or not) on entering the MS, provide valuable clues on the ionization mechanism. Single chains form in highly charged and extended shapes in most buffers. But the high initial charge they hold under atmospheric pressure is lost on transit to the vacuum system for large cations. In contrast, aggregates of two or more chains carry in all buffers at most the Rayleigh charge of a water drop of the same volume. This shows either that they form via Dole’s charge residue mechanism, or that highly charged and extended aggregates are ripped apart by Coulombic repulsion. IMS-IMS experiments in He confirm these findings, and provide new mechanistic insights on the stability of aggregates. When collisionally activated, initially globular dimers are stable. However, slightly nonglobular dimers projecting out a linear appendix are segregated into two monomeric chains. The breakup of a charged dimer is therefore a multi-step process, similar to the Fenn-Consta polymer extrusion mechanism. The highest activation barrier is associated to the first step, where a short chain segment carrying a single charge escapes (ion-evaporates) from a charged drop, leading then to gradual field extrusion of the whole chain out of the drop.

The solution dependence of gas-phase unfolding for ubiquitin [M + 7H](7+) ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed.

The interaction of a series of doubly charged metal cations (M2+ = Ca, Co, Ni, Cu, and Zn) with angiotensin I (AngI, Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10) is examined by collision-induced dissociation (CID) and ion mobility spectrometry–mass spectrometry (IMS–MS). The series of CID patterns combined with IMS–MS data for [AngI+M+H]3+ ions provides information about the metal–peptide binding sites. Overall, Ca2+ favors association with oxygen atoms spanning the peptide backbone; whereas, the transition metals favor binding at a site that involves association at the His6 and His9 sites. From these experiments, it is possible to derive insight into the populations of different metal coordination sites that are sampled in solution prior to introduction of species into the gas phase.

When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (>90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII → PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a "dry" environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions-indicating that some transitions observed in water (i.e., "wet" folding, are accessible (albeit inefficient) in vacuo. Lastly, these "dry" folding experiments show that PPI (all cis) is favored under "dry" conditions, which underscores the role of water as the major factor promoting preference for trans proline. Graphical Abstract ᅟ.

Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/trans orientation of Xaa-Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences.

Ion mobility/mass spectrometry techniques have been used to generate a cross section database containing 1772 entries (147 singly-, 1325 doubly-, and 300 triply-charged) for protonated and alkalated tryptic peptide ions. Such a large number of values make it possible to assess the influence of alkali metal cations [where the cation (M+) corresponds to Li+ ,N a + ,K + ,o r Cs +] on peptide ion conformation. Peptide ion sizes generally increase with increasing cation size relative to the respective singly- or doublyprotonated species. Intrinsic size parameters for individual amino acid residues for alkali [Pep+M+H] 2+

Recently, we reported that 157 nm vacuum ultraviolet irradiation (VUV) of proton-bound peptide dimers trapped in a vacuum, results in the elimination of water and formation of a peptide bond [J. Am. Chem. Soc. 133 (2011) 15834–15837]. Here, we further explore the ability to form a covalent bond between biomolecular ions with photoexcitation. Photoexcitation of long-lived charge-bound complexes appears to be a general phenomenon, resulting in the loss of water and the formation of covalent bonds between many types of molecules. Several examples are described, including: the linear coupling of amino acid chains that produce octapeptides from tetrapeptide complexes; inter-molecular cross-linking of amino acid side chains, and glycosidic bond formation between disaccharide complexes. Simple mechanisms for each case are proposed.

Comparative analyses utilizing collision induced dissociation (CID) and vacuum ultraviolet photodissociation (VUVPD) for seven isobaric disaccharides have been performed in order to differentiate the linkage type and anomeric configuration of the isomers. Although an individual CID spectrum of a disaccharide ion provides information related to its structure, CID does not sufficiently differentiate mixture components due to the identical mass-to-charge values of most of the intense fragments. In contrast to the ambiguity of the CID analyses for the disaccharide mixture, VUVPD (157 nm) generates unique fragments for each disaccharide ion that are useful for distinguishing individual components from the mixture. When combined with a gas-phase ion mobility separation of the ions, the identification of each component from the mixture can be obtained.

Increasingly comprehensive questions related to the biosynthesis of lipids relevant to understanding new signaling pathways have created daunting tasks for their chemical analysis. Here, ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques combined with electrospray ionization have been used to examine mixtures of closely related lipid structures. The drift time distributions of sphingomyelins show baseline separations for ethylene chain length differences (Δ ∼ 1.2 ms) and partial separations in single unsaturation differences (Δ ∼ 0.3 ms) revealing that the most compact structures are observed with shorter chains and increasing unsaturation. Drift time distributions of different ionizations frequently fall into families with the same drift times (isodrifts) indicating that the ion attached to the lipid has little structural influence. The present data show that phospholipids, especially phosphatidylinositol, aggregate to form inverted micelles. Phospholipids (phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol) are effectively separated according to their polar head groups. This method also provides information about the mixture composition of the chemically different lipids N-palmitoyl glycine, N-arachidonoyl ethanolamide, and phosphatidylcholine existing over an array of charge states and sizes (inverted micelles) depending on mixture concentration. Multidimensional IMS3-MS introduces an additional dimension to fragmentation analysis by separating the fragmented ions into groups related to size, shape and charge and allows determination of sn-1 and sn-2 substitution as is shown for phosphatidylglycerols. This contribution provides evidence for extending the targeted approach to global lipidomics analysis using the high-efficiency gas-phase separation afforded by multidimensional IMS-MS.

A drift tube mass spectrometer that utilizes back-to-back ion mobility regions separated by a collisional activation zone and new autosampling [R.T. Kurulugama, S.J. Valentine, R.A. Sowell, D.E. Clemmer, Development of a high-throughput IMS–IMS–MS approach for analyzing mixtures of biomolecules, J. Proteomics 71 (3) (2008) 318–331] and data acquisition techniques [S. Trimpin, D.E. Clemmer, Ion mobility spectrometry/mass spectrometry snapshots for assessing the molecular compositions of complex polymeric systems, Anal. Chem. 80 (23) (2008) 9073–9083; C. Becker, K.N. Qian, D.H. Russell, Molecular weight distributions of asphaltenes and deasphaltened oils studied by laser desorption ionization and ion mobility mass spectrometry, Anal. Chem. 80 (22) (2008) 8592–8597; S.I. Merenbloom, S.L. Koeniger, B.C. Bohrer, S.J. Valentine, D.E. Clemmer, Improving the efficiency of IMS–IMS by a combing technique, Anal. Chem. 80 (6) (2008) 1918–1927; M.E. Belov, B.H. Clowers, D.C. Prior, W.F. Danielson III, A.V. Liyu, B.O. Petritis, R.D. Smith, Dynamically multiplexed ion mobility time-of-flight mass spectrometry, Anal. Chem. 80 (15) (2008) 5873–5883] is used to analyze tryptic peptides from proteins obtained from 70 individual human plasma samples. The combination of methods allows ions to be efficiently separated in an initial drift region (based on differences in mobilities that arise from initial conformations), activated (in order to alter conformations), and then separated again in a second drift region, prior to detection in a mass spectrometer. The two-dimensional ion mobility separation makes it possible to create a large analytical peak capacity for the complex mixture. Additionally, the mobility distributions of the ions in different states (precursor and post-activation) provide a signature that is valuable in verifying that the same ions are being examined across the distribution of individuals. Finally, because this characterization is carried out in the gas phase it is possible to obtain information in a high-throughput fashion. Here, we develop and assess an analysis that requires ∼4 min per sample. While this prototype approach is at a very early stage several thousand peaks can be resolved and detected for each sample. From a comparison of 15 plasma samples analyzed with IMS–IMS–MS and LC–MS/MS techniques we find that an average of 262 ± 54 (1� ) unique peptides assigned in the latter analyses are also observed in the former. On average, these assigned peptides represent 63 ± 9 proteins. The utility of the approach as a means of characterizing physiologies in a state-to-state fashion is discussed.

The transport of ions through multiple drift regions is modeled in order to develop an equation that is useful for an understanding of the resolving power of an overtone mobility spectrometry (OMS) technique. It is found that resolving power is influenced by a number of experimental variables, including those that define ion mobility spectrometry (IMS) resolving power: drift field (E), drift region length (L), and buffer gas temperature (T). However, unlike IMS, the resolving power of OMS is also influenced by the number of drift regions (n), harmonic frequency value (m), and the phase number (ϕ) of the applied drift field. The OMS resolving power dependence upon the new OMS variables (n, m, and ϕ) scales differently than the square root dependence of the E, L, and T variables in IMS. The results provide insight about optimal instrumental design and operation.

Electrospray ionization, combined with two-dimensional ion mobility spectrometry and mass spectrometry, is used to produce, select, and activate distributions of elongated ions, [M + 11H]11+ to [M + 13H]13+, of ubiquitin. The analysis makes it possible to examine state-to-state transitions for structural types, and transition diagrams associated with the efficiencies of structural changes are presented. The +11 and +12 charge states can form four resolvable states while only one state is formed for [M + 13H]13+. Some conformations, which appear to belong to the same family based on mobility analysis of different charge states, undergo similar transitions, others do not. Activation of ions that exist in low-abundance conformations, having mobilities that fall in between sharp peaks associated with higher abundances species, shows that the low-abundance forms undergo efficient (approximately 90 to 100%) conversion into states associated with well-defined peaks. This efficiency is significantly higher than the approximately 10 to 60% efficiency of transitions of structures associated with well-defined peaks. The formation of sharp features from a range of low-intensity species with different cross sections indicates that large regions of conformation space must be unfavorable or inaccessible in the gas phase. These results are compared with several previous IMS measurements of this system as well as information about gas-phase structure provided by other techniques.

Ion mobility spectrometry techniques (IMS and IMS-IMS) combined with collision-induced dissociation (CID) and mass spectrometry (MS) are used to investigate the structures of singly-lithiated carbohydrate isomers. With the exception of some favorable cases, IMS-MS analyses of underivatized carbohydrates reveal that most isobaric precursor ions have similar collision cross sections (ccs). In contrast, ccs values for isomeric fragment ions obtained by IMS-CID-IMS-MS analysis are often different, and thus appear to be useful as a means of distinguishing the isomeric precursors. We report values of ccs (in He) for precursor- and associated-fragment ions for three monosaccharide isomers (glucose, galactose and fructose), ten disaccharide isomers (sucrose, leucrose, palatinose, trehalose, cellobiose, β-gentiobiose, isomaltose, maltose, lactose and melibiose), and three trisaccharide isomers (raffinose, melezitose and maltotriose). These values are discussed as a means of differentiating precursor carbohydrates.

Cross sections for 61 palmitoylated peptides and 73 cysteine-unmodified peptides are determined and used together with a previously obtained tryptic peptide library to derive a set of intrinsic size parameters (ISPs) for the palmitoyl (Pal) group (1.26 ± 0.04), carboxyamidomethyl (Am) group (0.92 ± 0.04), and the 20 amino acid residues to assess the influence of Pal- and Am-modification on cysteine and other amino acid residues. These values highlight the influence of the intrinsic hydrophobic and hydrophilic nature of these modifications on the overall cross sections. As a part of this analysis, we find that ISPs derived from a database of a modifier on one amino acid residue (Cys Pal ) can be applied on the same modification group on different amino acid residues (Ser Pal and Tyr Pal ). Using these ISP values, we are able to calculate peptide cross sections to within ±2% of experimental values for 83% of Pal-modified peptide ions and 63% of Am-modified peptide ions. We propose that modification groups should be treated as individual contribution factors, instead of treating the combination of the particular group and the amino acid residue they are on as a whole when considering their effects on the peptide ion mobility features.

Isomerizations of the retinal chromophore were investigated using the IMS-IMS technique. Four different structural features of the chromophore were observed, isolated, excited collisionally, and the resulting isomer and fragment distributions were measured. By establishing the threshold activation voltages for isomerization for each of the reaction pathways, and by measuring the threshold activation voltage for fragmentation, the relative energies of the isomers as well as the energy barriers for isomerization were determined. The energy barrier for a single cis–trans isomerization is (0.64±0.05) eV, which is significantly lower than that observed for the reaction within opsin proteins.

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0–90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal–ligand–monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser(1)-Pro(2)-Glu(3)-Leu(4)-Pro(5)-Ser(6)-Pro(7)-Gln(8)-Ala(9)-Glu(10)-Lys(11). We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.

High-performance liquid chromatography (HPLC) has been combined with high-resolution ion mobility separations and time-of-flight mass spectrometry (MS) for the analysis of complex biomolecular mixtures such as those that arise upon tryptic digestion of protein mixtures. In this approach, components in a mixture are separated using reversed phase HPLC. As mixtures of peptides exit the column, they are electrosprayed into an ion mobility/time-of-flight mass spectrometer. Mixtures of ions are separated based on differences in mobilities through a buffer gas, and subsequently dispersed by differences in mass-to-charge (m/z) ratios in a mass spectrometer. The multidimensional approach is feasible because of the large differences in timescales of the HPLC (minutes), ion mobility (milliseconds), and time-of-flight (microseconds) techniques. Peak capacities for the two-dimensional liquid chromatography-ion mobility separations (LC-IMS) and three-dimensional LC-IMS-MS separations are estimated to be ∼900 to 1 200 and ∼3.7 to 4.6 × 105, respectively. The experimental apparatus and data acquisition considerations are described; data for a mixture of peptides obtained upon tryptic digestion of five proteins (albumin, bovine and pig; cytochrome c, horse; hemoglobin, dog and pig) are presented to illustrate the approach.

The conformations of a series of anhydrous sodiated polyalanine ions ([Alan + 3Na]3+, where n = 18–36) have been examined in the gas phase by ion mobility measurements and molecular modeling simulations. The experimental results indicate that these ions exist as highly extended conformations. There is no strong evidence for a folded state, observed previously for a series of analogous protonated polyalanines ([Alan + 3H]3+, where n = 24–41) (A.E. Counterman, D.E. Clemmer, J. Am. Chem. Soc., submitted). Molecular dynamics simulations for the [Alan + 3Na]3+ ions also indicate that extended structures are favored. The simulations show that extensive helical regions are present; however, near the sites where Na+ ions are attached, helical regions appear to be substantially disrupted by intramolecular charge solvation of the Na+. Simulations of some [Alan + 3Na]3+ charge site assignments show evidence for structures that are similar to folded structures observed for analogous [Alan + 3H]3+ ions; however, the calculated energy gap between the folded and unfolded states for the triply sodiated system is slightly greater than the gap in analogous protonated polyalanines. We propose this as a possible explanation for the absence of experimental evidence for a folded state in the sodiated system. Keywords: Polyalanine; Ion mobility; Sodium; Stability; Electrospray

A combinatorial tripeptide library having the general form D-Glu-Xxx-Xxx-CONH2 has been synthesized using a standard mix and split synthetic protocol that is expected to produce 676 components. All components of the mixture were analyzed using a new high-resolution ion mobility/time-of-flight mass spectrometer coupled with an electrospray ionization source. In this approach ions are separated by differences in their gas-phase mobilities prior to being introduced into the mass spectrometer for mass-to-charge analysis. The peptide library includes a wide range of different sequence, structural, and stereo isomers; trends in the number of expected and resolved isomers that are observed at each m/z ratio allow specific synthetic steps that have failed to be identified, even in the presence of other isomers. Information about the relative abundances of different isomers should dramatically improve the reliability of binding affinity studies from direct analysis of mixtures.

High-resolution ion mobility/mass spectrometry methods have been used to examine the influence of solvent composition and capillary temperature on the gas-phase conformations of ubiquitin ions (+6 to +13) formed during electrospray ionization. Three general conformer types are observed: compact forms (favored for the +6 and +7 charge states); partially folded conformers (favored for the +8 and +9 ions); and, unfolded conformers (favored for the +10 to +13 charge states). The populations of different conformers are highly sensitive to solvent composition and capillary temperature used for electrospray ionization. Electrospray of “pseudonative” solutions leads to formation of some conformers that are more compact than those observed for “denatured” solutions. Studies as a function of capillary temperature show that as the capillary temperature is increased, compact and partially folded states undergo unfolding transitions. Compact states from pseudonative solutions unfold at higher temperatures than compact states from denatured solutions. The products of unfolding transitions have similar cross sections for both solution types.