Matthew S. Glover

Using Ion Mobility Data to Improve Peptide Identification: Intrinsic Amino Acid Size Parameters

A new method for enhancing peptide ion identification in proteomics analyses using ion mobility data is presented. Ideally, direct comparisons of experimental drift times (t(D)) with a standard mobility database could be used to rank candidate peptide sequence assignments. Such a database would represent only a fraction of sequences in protein databases and significant difficulties associated with the verification of data for constituent peptide ions would exist. A method that employs intrinsic amino acid size parameters to obtain ion mobility predictions that can be used to rank candidate peptide ion assignments is proposed. Intrinsic amino acid size parameters have been determined for doubly charged peptide ions from an annotated yeast proteome. Predictions of ion mobilities using the intrinsic size parameters are more accurate than those obtained from a polynomial fit to t(D) versus molecular weight data. More than a 2-fold improvement in prediction accuracy has been observed for a group of arginine-terminated peptide ions 12 residues in length. The use of this predictive enhancement as a means to aid peptide ion identification is discussed, and a simple peptide ion scoring scheme is presented.

Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG)

AbstractWe 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 Abstractᅟ

Hybrid ion mobility and mass spectrometry as a separation tool

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has seen spectacular growth over the last two decades. Increasing IMS sensitivity and capacity with improvements in MS instrumentation have driven this growth. As a result, a diverse new set of techniques for separating ions by their mobility have arisen, each with characteristics that make them favorable for some experiments and some mass spectrometers. Ion mobility techniques can be broken down into dispersive and selective techniques based upon whether they pass through all mobilities for later analysis by mass spectrometry or select ions by mobility or a related characteristic. How ion mobility techniques fit within a more complicated separation including mass spectrometry and other techniques such as liquid chromatography is of fundamental interest to separations scientists. In this review we explore the multitude of ion mobility techniques hybridized to different mass spectrometers, detailing current challenges and opportunities for each ion mobility technique and for what experiments one technique might be chosen over another. The underlying principles of ion mobility separations, including: considerations regarding separation capabilities, ion transmission, signal intensity and sensitivity, and the impact that the separation has upon the ion structure (i.e., the possibility of configurational changes due to ion heating) are discussed.

Ion Mobility-Mass Spectrometry Reveals the Energetics of Intermediates that Guide Polyproline Folding

AbstractProline favors trans-configured peptide bonds in native proteins. Although cis/trans configurations vary for non-native and unstructured states, solvent also influences these preferences. Water induces the all-cis right-handed polyproline-I (PPI) helix of polyproline to fold into the all-trans left-handed polyproline-II (PPII) helix. Our recent work has shown that this occurs via a sequential mechanism involving six resolved 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 use ion mobility-mass spectrometry to make the first detailed thermodynamic measurements of the folding intermediates, which inform us about how and why this transition occurs. It appears that early intermediates are energetically favorable because of the hydration of the peptide backbone, whereas late intermediates are enthalpically unfavorable. However, folding continues, as the entropy of the system increases upon successive formation of each new structure. When PPII is immersed in 1-propanol, the PPII→PPI transition occurs, but this reaction occurs through a very different mechanism. Early on, the PPII population splits onto multiple pathways that eventually converge through a late intermediate that continues on to the folded PPI helix. Nearly every step is endothermic. Folding results from a stepwise increase in the disorder of the system, allowing a wide-scale search for a critical late intermediate. Overall, the data presented here allow us to establish the first experimentally determined energy surface for biopolymer folding as a function of solution environment. Graphical Abstractᅟ

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

AbstractProtein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. Graphical Abstractᅟ

A Database of Alkaline-Earth-Coordinated Peptide Cross Sections: Insight into General Aspects of Structure

AbstractA database of 1470 collision cross sections (666 doubly- and 804 triply-charged) of alkaline-earth-coordinated tryptic peptide ions [where the cation (M2+) correspond to Mg2+, Ca2+, or Ba2+] is presented. The utility of such an extensive set of measurements is illustrated by extraction of general properties of M2+-coordinated peptide structures. Specifically, we derive sets of intrinsic size parameters (ISPs) for individual amino acid residues for M2+-coordinated peptides. Comparison of these parameters with existing ISPs for protonated peptides suggests that M2+ binding occurs primarily through interactions with specific polar aliphatic residues (Asp, Ser, and Thr) and the peptide backbone. A comparison of binding interactions for these alkaline-earth metals with interactions reported previously for alkali metals is provided. Finally, we describe a new analysis in which ISPs are used as probes for assessing peptide structure based on amino acid composition.

Populations of Metal-Glycan Structures Influence MS Fragmentation Patterns

AbstractThe structures and collision-induced dissociation (CID) fragmentation patterns of the permethylated glycan Man5GlcNAc2 are investigated by a combination of hybrid ion mobility spectrometry (IMS), mass spectrometry (MS), and MS/MS techniques. IMS analysis of eight metal-adducted glycans ([Man5GlcNAc2 + M]2+, where M = Mn, Fe, Co, Ni, Cu, Mg, Ca, and Ba) shows distinct conformer patterns. These conformers appear to arise from individual metals binding at different sites on the glycan. Fragmentation studies suggest that these different binding sites influence the CID fragmentation patterns. This paper describes a series of separation, activation, and fragmentation studies that assess which fragments arise from each of the different gas-phase conformer states. Comparison of the glycan distributions formed under gentle ionization conditions with those obtained after activation of the gas-phase ions suggests that these conformer binding states also appear to exist in solution. Graphical Abstractᅟ

Recent advances in ion mobility–mass spectrometry for improved structural characterization of glycans and glycoconjugates

Glycans and glycoconjugates are involved in regulating a vast array of cellular and molecular processes. Despite the importance of glycans in biology and disease, characterization of glycans remains difficult due to their structural complexity and diversity. Mass spectrometry (MS)-based techniques have emerged as the premier analytical tools for characterizing glycans. However, traditional MS-based strategies struggle to distinguish the large number of coexisting isomeric glycans that are indistinguishable by mass alone. Because of this, ion mobility spectrometry coupled to MS (IM-MS) has received considerable attention as an analytical tool for improving glycan characterization due to the capability of IM to resolve isomeric glycans before MS measurements. In this review, we present recent improvements in IM-MS instrumentation and methods for the structural characterization of isomeric glycans. In addition, we highlight recent applications of IM-MS that illustrate the enormous potential of this technology in a variety of research areas, including glycomics, glycoproteomics, and glycobiology.

Penultimate Proline in Neuropeptides

A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry

AbstractIon 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. Graphical Abstractᅟ