Charged Peptide Ions Research Articles

Application of de novo sequencing tools to study abiogenic peptide formations by tandem mass spectrometry. The case of homo‐peptides from glutamic acid complicated by substitutions of hydrogen by sodium or potassium atoms

Peptides and proteins are among the most important components of living systems. Different attempts have been made to experimentally model the formation of peptides from amino acid monomers in investigation of the origin of life. Detailed characterization of peptides formed under various conditions in such reactions is very important for understanding processes of abiogenic peptide formation. We used liquid chromatography coupled with tandem mass spectrometry (MS/MS) for an accurate study of homo-peptides formed in a model reaction: glutamic acid oligomerization catalyzed by 1,1'-carbonyldiimidazole in aqueous solution with 1 M of sodium or potassium chloride and without any salts. We used de novo sequencing software for peptide identification. In addition we propose an approach that uses more spectral information for de novo sequencing then standard methods. Peptides up to 9 amino acids long were found in the experiments with KCl, while in experiments with NaCl and without salts only peptides of up to 7 amino acids were detected. Due to high salt concentrations in samples a high number of singly charged peptide ions with up to 4 substitutions of hydrogen atoms by sodium or potassium atoms were observed. De novo sequencing software provided correct identifications even for peptide ions with substitutions. Multiple substitutions of hydrogen by alkali metal atoms in peptide ions strongly change their fragmentation patterns. Proposed approach for de novo sequencing was found very effective, even for ions with substitutions. So, it may be useful in more complicated cases like sequencing abiogenic peptides consisting of different amino acids.

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio‐frequency quadrupole ion trap

Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups-aspartic and glutamic acid side-chains as well as C-termini-were derivatized with an average reaction efficiency of 99%. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40% for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70% for modified versus only 43% for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50% increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Liquid AP‐UV‐MALDI Enables Stable Ion Yields of Multiply Charged Peptide and Protein Ions for Sensitive Analysis by Mass Spectrometry

All charged up: High and prolonged yields of multiply charged peptide and protein ions can be formed in MALDI mass spectrometry using liquid UV-MALDI matrices and a heated ion-transfer tube. The key features are low laser energies of 1–10 μJ, resulting in fluences of less than 200 to 2000 J m−2 and low sample ablation, high sensitivity, and continuous ion generation over tens of thousands of laser shots.

Renaissance of cation-radicals in mass spectrometry.

This brief overview addresses the topic that was presented in the Thomson Medal Award session at the 19th International Mass Spectrometry Conference in Kyoto, Japan. Mass spectrometry of cation-radicals has enjoyed a remarkable renaissance thanks to the development of new methods for electron attachment to multiply charged peptide ions. The charge-reduced ions that are odd-electron species exhibit interesting reactivity that is useful for peptide and protein sequencing. The paper briefly reviews the fundamental aspects of the formation, energetics, and backbone dissociations of peptide cation-radicals.

Laserspray ionization imaging of multiply charged ions using a commercial vacuum MALDI ion source.

This is the first report of imaging mass spectrometry (MS) from multiply charged ions at vacuum. Laserspray ionization (LSI) was recently extended to applications at vacuum producing electrospray ionization-like multiply charged ions directly from surfaces using a commercial intermediate pressure matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) MS instrument. Here, we developed a strategy to image multiply charged peptide ions. This is achieved by the use of 2-nitrophloroglucinol as matrix for spray deposition onto the tissue section and implementation of "soft" acquisition conditions including lower laser power and ion accelerating voltages similar to electrospray ionization-like conditions. Sufficient ion abundance is generated by the vacuum LSI method to employ IMS separation in imaging multiply charged ions obtained on a commercial mass spectrometer ion source without physical instrument modifications using the laser in the commercially available reflection geometry alignment. IMS gas-phase separation reduces the complexity of the ion signal from the tissue, especially for multiply charged relative to abundant singly charged ions from tissue lipids. We show examples of LSI tissue imaging from charge state +2 of three endogenous peptides consisting of between 1 and 16 amino acid residues from the acetylated N-terminal end of myelin basic protein: mass-to-charge (m/z) 795.81 (+2) molecular weight (MW) 1589.6, m/z 831.35 (+2) MW 1660.7, and m/z 917.40 (+2) MW 1832.8.

Correlating ETD fragment ion intensities with peptide ion conformational and electronic structure

Amino acid residues affect the formation of backbone fragments upon electron transfer dissociation of doubly charged peptide ions of the AAXAR type. Peptide ions having non-polar residues (X=A, V, L, M, W) undergo backbone dissociations near the protonated N- and C-termini, forming mainly z1 and z4 fragment ions. Polar residues (X=H, D) prefer backbone dissociations occurring next to the polar residue, forming z2 and z3 fragment ions. Computational analysis, combining molecular dynamics, semi-empirical, density functional theory, and Møller–Plesset theory of precursor ion conformational space, identified the thermodynamically most stable tautomers and conformers for (AAVAR+2H)2+, (AAHAR+2H)2+, and (AADAR+2H)2+ precursor ions. Analysis of electronic states and potential energy surfaces for charge-reduced cation-radicals was used to interpret the ETD fragmentations. Electron attachment to (AAVAR+2H)2+ and (AAHAR+2H)2+ forms zwitterionic tautomers of charge-reduced cation-radicals as local energy minima in the ground electronic state. These are dynamically unstable with respect to exothermic ammonium proton migration forming aminoketyl intermediates. Direct NCα bond cleavage in the zwitterion requires an energy barrier which makes it noncompetitive with the proton migration. This bears on the long-standing chicken-and-egg problem of the ExD dissociation mechanisms according to the Utah–Washington model. Migration of a proton from a proximate neutral amide group was found to form a dynamically unstable enolimidate intermediate that cannot undergo NCα bond cleavage according to the Murrell–Laidler rule of reaction dynamics. According to electronic structure analysis in a conformationally defined peptide ion, the non-polar side chain in charge-reduced (AAVAR+2H)+ has only a spectator role in isomerizations and backbone dissociations. This casts doubt on the “hydrophobic” effect of amino acid residues on electron-based fragmentations proposed previously.

ECD-like peptide fragmentation at atmospheric pressure

Fragmentation of multiply charged peptide ions via interaction with products of gas discharge at atmospheric pressure conditions was studied using ion mobility separation – fragmentation cell – linear ion trap mass spectrometer. The observed fragmentation spectra mainly consisted of c-type ions that are specific to electron capture dissociation. Experiments with different gases flowing through the discharge and different discharge polarities suggested that fragmentation proceeds via capture of free electrons. Fragmentation of a model phosphorylated peptide using this technique produced c-type fragments with an intact phosphorylation group. High field asymmetric waveform ion mobility separation of a peptide mixture prior to the fragmentation cell demonstrated the feasibility of conducting MS/MS-like experiments at atmospheric pressure conditions.

Improvement of Phosphoproteome Analyses Using FAIMS and Decision Tree Fragmentation. Application to the Insulin Signaling Pathway in Drosophila melanogaster S2 Cells

This report examines the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography mass spectrometry (LC-MS) for phosphoproteomics analyses. The ability of FAIMS to separate multiply charged peptide ions from chemical interferences confers a unique advantage in phosphoproteomics by enhancing the detection of low abundance phosphopeptides. LC-FAIMS-MS experiments performed on TiO(2)-enriched tryptic digests from Drosophila melanogaster provided a 50% increase in phosphopeptide identification compared to conventional LC-MS analysis. Also, FAIMS can be used to select different population of multiply charged phosphopeptide ions prior to their activation with either collision activated dissociation (CAD) or electron transfer dissociation (ETD). Importantly, FAIMS enabled the resolution of coeluting phosphoisomers of different abundances to facilitate their unambiguous identification using conventional database search engines. The benefits of FAIMS in large-scale phosphoproteomics of D. melanogaster are further investigated using label-free quantitation to identify differentially regulated phosphoproteins in response to insulin stimulation.

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.

Charge States of y Ions in the Collision-Induced Dissociation of Doubly Charged Tryptic Peptide Ions

Bonds that break in collision-induced dissociation (CID) are often weakened by a nearby proton, which can, in principle, be carried away by either of the product fragments. Since peptide backbone dissociation is commonly charge-directed, relative intensities of charge states of product y- and b-ions depend on the final location of that proton. This study examines y-ion charge distributions for dissociation of doubly charged peptide ions, using a large reference library of peptide ion fragmentation generated from ion-trap CID of peptide ions from tryptic digests. Trends in relative intensities of y(2+) and y(1+) ions are examined as a function of bond cleavage position, peptide length (n), residues on either side of the bond and effects of residues remote from the bond. It is found that y(n-2)/b(2) dissociation is the most sensitive to adjacent amino acids, that y(2+)/y(1+) steadily increase with increasing peptide length, that the N-terminal amino acid can have a major influence in all dissociations, and in some cases other residues remote from the bond cleavage exert significant effects. Good correlation is found between the values of y(2+)/y(1+) for the peptide and the proton affinities of the amino acids present at the dissociating peptide bond. A few deviations from this correlation are rationalized by specific effects of the amino acid residues. These correlations can be used to estimate trends in y(2+)/y(1+) ratios for peptide ions from amino acid proton affinities.

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Commercial Intermediate Pressure MALDI Ion Mobility Spectrometry Mass Spectrometer Capable of Producing Highly Charged Laserspray Ionization Ions

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.

Miniaturized Ultra High Field Asymmetric Waveform Ion Mobility Spectrometry Combined with Mass Spectrometry for Peptide Analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.

Atmospheric pressure‐electron capture dissociation of peptides using a modified PhotoSpray ion source

An improved in-source atmospheric pressure-electron capture dissociation (AP-ECD) method is described. Building upon the early example of Laprévote's group, photoelectrons generated within a commercial PhotoSpray atmospheric pressure photoionization source are used to induce ECD of multiply charged peptide ions originating from an upstream heated nebulizer device. To attain high sensitivity, the method makes use of a novel electropneumatic-heated nebulizer to assist in the creation and transmission of multiply charged ions from sample solutions. Here, we demonstrate that readily interpretable AP-ECD spectra of infused peptides can be acquired from 100 fmol sample consumed, on a chromatographic time scale, using a conventional quadrupole time-of-flight (Q-ToF) mass spectrometer otherwise incapable of ECD/ETD experiments. Though much work remains to be done to develop and characterize the method, the results indicate that AP-ECD has the potential to be a practical new tool for the mass spectrometric analysis of peptides and proteins.

Electron Transfer Dissociation with Supplemental Activation to Differentiate Aspartic and Isoaspartic Residues in Doubly Charged Peptide Cations

Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z*-57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately, the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid residue difficult due to interference with side-chain loss from arginine and glutamic acid residues.

Laserspray Ionization, a New Atmospheric Pressure MALDI Method for Producing Highly Charged Gas-phase Ions of Peptides and Proteins Directly from Solid Solutions

The first example of a matrix-assisted laser desorption/ionization (MALDI) process producing multiply charged mass spectra nearly identical to those observed with electrospray ionization (ESI) is presented. MALDI is noted for its ability to produce singly charged ions, but in the experiments described here multiply charged ions are produced by laser ablation of analyte incorporated into a common MALDI matrix, 2,5-dihydroxybenzoic acid, using standard solvent-based sample preparation protocols. Laser ablation is known to produce matrix clusters in MALDI provided a threshold energy is achieved. We propose that these clusters (liquid droplets) are highly charged, and under conditions that produce sufficient matrix evaporation, ions are field-evaporated from the droplets similarly to ESI. Because of the multiple charging, advanced mass spectrometers with limited mass-to-charge range can be used for protein characterization. Thus, using an Orbitrap mass spectrometer, low femtomole quantities of proteins produce full-range mass spectra at 100,000 mass resolution with <5-ppm mass accuracy and with 1-s acquisition. Furthermore, the first example of protein fragmentation using electron transfer dissociation with MALDI is presented.

Factors that influence helical preferences for singly charged gas-phase peptide ions: the effects of multiple potential charge-carrying sites.

Ion mobility-mass spectrometry is used to investigate the structure(s) of a series of model peptide [M + H](+) ions to better understand how intrinsic properties affect structure in low dielectric environments. The influence of peptide length, amino acid sequence, and composition on gas-phase structure is examined for a series of model peptides that have been previously studied in solution. Collision cross sections for the [M + H](+) ions of Ac-(AAKAA)(n)Y-NH(2) (n = 3-6) and Ac-Y(AEAAKA)(n)F-NH(2) (n = 2-5) are reported and correlated with candidate structures generated using molecular modeling techniques. The [M + H](+) ions of the AAKAA peptide series each exhibit a single, dominant ion mobility arrival time distribution (ATD) which correlates to partial helical structures, whereas the [M + H](+) ions of the AEAAKA ion series are composed of ATDs which correlate to charge-solvated globules (i.e., the charge is coordinated or solvated by polar peptide functional groups). These data raise numerous questions concerning intrinsic properties (amino acid sequence and composition as well as charge location) that dictate gas-phase peptide ion structure, which may reflect trends for peptide ion structure in low dielectric environments, such as transmembrane segments.