Quantification of enantiomers and blind identification of erythro-sphingosine non-racemates by cold ion spectroscopy.

Enantiomers of a lipid erythro-sphingosine have been quantified with ≈4% accuracy by UV cold ion spectroscopy of their non-covalent complexes with a chiral aromatic molecule. The diastereomeric configuration of such complexes enables the quantification using just a single enantiomeric lipid standard and the identification of non-racemic solutions with no standards at all.

Ion Spectroscopy Reveals Structural Difference for Proteins Microhydrated by Retention and Condensation of Water.

Protein ubiquitin in its +7 charge state microhydrated by 5 and 10 water molecules has been interrogated in the gas phase by cold ion UV/IR spectroscopy. The complexes were formed either by condensing water onto the unfolded bare proteins in a temperature-controlled ion trap or by incomplete dehydration of the folded proteins. In the case of cryogenic condensation, the UV spectra of the complexes exhibit a resolved vibrational structure, which looks similar to the spectrum of bare unfolded ubiquitin. The spectra become, however, broad-band with no structure when complexes of the same size are produced by incomplete dehydration under soft conditions of electrospray ionization. We attribute this spectroscopic dissimilarity to the structural difference of the protein: condensing a few water molecules cannot refold the gas-phase structure of the bare ubiquitin, while the retained water preserves its solution-like folded motif through evaporative cooling. This assessment is firmly confirmed by IR spectroscopy, which reveals the presence of free NH and carboxylic OH stretching vibrations only in the complexes with condensed water.

Revealing the Structure of Tryptophan in Microhydrated Complexes by Cold Ion Spectroscopy.

The geometry of biomolecules isolated in the gas phase usually differs substantially from their native structures in aqueous solution, which are the only ones truly relevant to life science. To connect the high resolution of cold ion spectroscopy that can be achieved in the gas phase and the key role of intermolecular hydrogen bonds that shape biomolecules in water, we study protonated tryptophan microhydrated by 1-6 water molecules. IR/UV spectra measured with the same instrument under similar conditions appear to be identical for the complexes of the same size produced by soft dehydration and cryogenic condensation methods. This observation points to the lack of kinetic trapping in the dehydration/rehydration processes. Quantum chemistry computations allow for the unambiguous assignment of the measured IR spectra to the most stable conformers of the complexes. The calculations reveal that retaining as few as four water molecules still conserves most of the TrpH+ native structural features.

Salt Bridge Structure of Microhydrated Arginine Kinetically Trapped in the Gas Phase by Evaporative Cooling.

Amino acids and peptides generally exhibit zwitterionic forms with salt bridge (SB) structures in solution but charge-solvated (CS) motifs in the gas phase. Here, we report a study of non-covalent complexes of the protonated amino acid arginine, ArgH+(H2O)n (n = 1-5), produced in the gas phase from an aqueous solution with a controlled number of retained water molecules. These complexes were probed by cold ion spectroscopy and treated by quantum chemistry. The spectroscopic changes induced upon gradual dehydration of arginine were assigned by structural calculations to the transition from SB to CS geometries. SB conformers appear to be present for the complexes with as few as 3 retained water molecules, although energetically CS structures should become prevailing already for ArgH+ with 7-8 water molecules. We attribute the revealed kinetic trapping of arginine in native-like zwitterionic forms to evaporative cooling of the hydrated complexes to as low as below 200 K.

Gentle nano-electrospray ion source for reliable and efficient generation of microsolvated ions.

We present herein the design of a nano-electrospray ion source capable of reliable generation of large quantities of microsolvated ions. The source is based on a triple molecular skimmer scheme and can be quickly tuned to generate bare ions or their ionic complexes with up to more than 100 solvent molecules retained from solution. The performance of this source is illustrated by recording the mass spectra of distributions of ionic complexes of protonated water, amino acids, and a small protein ubiquitin. Protonated water complexes with more than 110 molecules and amino acids with more than 45 water molecules could be generated. Although the commercial ion source based on the double ion funnel design with orthogonal injection, which we used in our laboratory, is more efficient in generating ions than our triple skimmer ion source, they both exhibit comparable short-term stability in generating bare ions. In return, only the new source is capable of generating microsolvated ions.

Identification of Isomeric Biomolecules by Infrared Spectroscopy of Solvent-Tagged Ions.

The difference in functionality of many isomeric biomolecules requires their analytical identification for life science studies. We present a universal approach for quantitative identification of different small- to medium-sized isomeric biomolecules that can be brought to the gas phase from solution by electrospray ionization (ESI). The method involves infrared (IR) fragment cold ion spectroscopy of analyte molecules that are incompletely desolvated by soft ESI. The use of solvent molecules as natural tags removes a need for adding to solutions any special compounds, which may interfere with liquid chromatography or mass spectrometric measurements. The tested peptides and especially monosaccharides and lipids exhibit highly isomer-specific IR fragment spectra of such noncovalent complexes, which were produced from water, methanol, acetonitrile, and 2-butanol solutions. The relative concentrations in solution mixtures of, for instance, two isomeric dipeptides can be quantified with the accuracy of 1.6% and 2.9% for the acquisition time of 25 min and, potentially, 5 s, respectively; for three isomeric phospho-octapeptides, the accuracy becomes 4.1% and 11% for 17 min and, potentially, 10 s measurements, respectively.

Tracking local and global structural changes in a protein by cold ion spectroscopy.

Characterization of native structures of proteins in the gas phase remains challenging due to the unpredictable conformational changes the molecules undergo during desolvation and ionization. We spectroscopically studied cryogenically cooled protonated protein ubiquitin and its microhydrated complexes prepared in the gas phase in a range of charge states under different ionization conditions. The UV spectra appear vibrationally resolved for the unfolded protein, but become redshifted and smooth for the native-like structures of ubiquitin. This spectroscopic change results from the H-bonding of the hydroxyl of Tyr to the amide group of Glu-51 in the compact structures; the minimum length of this bond was estimated to be ∼1.7 Å. IR spectroscopy reflects the global structural change by observing redshifts of free NH/OH-stretch vibrational transitions. Evaporative cooling of microhydrated complexes of ubiquitin keeps the protein chilly during ionization, enabling native-like conformers with up to eight protons to survive in the gas phase.

Identification of Isomeric Lipids by UV Spectroscopy of Noncovalent Complexes with Aromatic Molecules.

The tremendous structural and isomeric diversity of lipids enables a wide range of their functions in nature but makes the identification of these biomolecules challenging. We distinguish and quantify isomeric lipids using cold ion UV fragmentation spectroscopy of their noncovalent complexes with aromatic amino acids and dipeptides. On the basis of structural simulations, specific isomer-sensitive aromatic "sensors" have been preselected for lipids of each studied class. Tyrosine appeared to be a good "sensor" to distinguish steroids and prostaglandins, which are rich in functional groups, while diphenylalanine is a better choice for sensing largely hydrophobic phospholipids. With this sensor, the relative concentrations of two isomeric glycerophospholipids mixed in solution have been determined with 3.3% accuracy, which should degrade only to 3.7% for a 14 s express measurement.

Accelerating photofragmentation UV Spectroscopy-Mass spectrometry fingerprinting for quantification of isomeric peptides.

Identification of isomeric biomolecules remains a challenging analytical problem. A recently developed spectroscopic method that combines UV photofragmentation and mass spectrometry for fingerprinting of cold ions (2D UV-MS), has already demonstrated its high performance in the library-based identification and quantification of different types of biomolecular isomers. The practical use of the method has been hindered by a slow rate of data acquisition, which makes the fingerprinting incompatible with high-throughput analysis and online liquid chromatography (LC) separation. Herein we demonstrate how the use of a few pre-selected wavelengths can accelerate the method by two orders of magnitude without a significant loss of accuracy. As a proof of principle, 2D UV-MS fingerprinting was coupled to online LC separation and tested for quantification of isomeric peptides containing either Asp or isoAsp residues. The relative concentrations of the peptides mixed in solution have been determined, on average, with better than 4% and 6% accuracy for resolving and non-resolving gradients of LC separation, respectively.

Microhydration of Biomolecules: Revealing the Native Structures by Cold Ion IR Spectroscopy.

The native-like structures of protonated glycine and peptide Gly3H+ were elucidated using cold ion IR spectroscopy of these biomolecules hydrated by a controlled number of water molecules. The complexes were generated directly from an aqueous solution using gentle electrospray ionization. Already with a single retained water molecule, GlyH+ exhibits the native-like structure characterized by a lack of intramolecular hydrogen bonds. We use our spectra to calibrate the available data for the same complexes, which are produced by cryogenic condensation of water onto the gas-phase glycine. In some conformers of these complexes, GlyH+ adopts the native-like structure, while in the others, it remains "kinetically" trapped in the intrinsic state. Upon condensation of 4-5 water molecules, the embedded amino acid fully adopts its native-like structure. Similarly, condensation of one water molecule onto the tripeptide is insufficient to fully eliminate its kinetically trapped intrinsic states.

Identification and Quantification of Any Isoforms of Carbohydrates by 2D UV-MS Fingerprinting of Cold Ions.

Biological functionality of isomeric carbohydrates may differ drastically, making their identifications indispensable in many applications of life science. Because of the large number of isoforms, structural assignment of saccharides is challenging and often requires a use of different orthogonal analytical techniques. We demonstrate that isomeric carbohydrates of any isoforms can be distinguished and quantified using solely the library-based method of 2D ultraviolet fragmentation spectroscopy-mass spectrometry (2D UV-MS) of cold ions. The two-dimensional "fingerprint" identities of UV transparent saccharides were revealed by photofragmentation of their noncovalent complexes with aromatic molecules. We assess the accuracy of the method by comparing the known relative concentrations of isomeric carbohydrates mixed in solution with the concentrations that were mathematically determined from the measured in the gas-phase fingerprints of the complexes. For the tested sets with up to five isomers of di- to heptasaccharides, the root-mean-square deviation of 3-5% was typically achieved. This indicates the expected level of accuracy in analysis of unknown mixtures for isomeric carbohydrates of similar complexity.

Revealing Single-Bond Anomeric Selectivity in Carbohydrate-Protein Interactions.

The noncovalent binding of proteins to glycans is amazingly selective to the isoforms of carbohydrates, including α/ß anomers that coexist in solution. We isolate in the gas phase and study at the atomic level the simplest model system: noncovalent complexes of monosaccharide α/ß-GalNAc and protonated aromatic molecule tyramine. IR/UV cold ion spectroscopy and quantum chemistry calculations jointly solve the structures of the two complexes. Although the onsets of the measured UV absorptions of the complexes differ significantly, the networks of H bonds in both complexes appear identical and do not include the anomeric hydroxyl. The detailed analysis reveals that, through inductive polarization, the α- to ß-reorientation of this group nevertheless reduces the length of one remote short intermolecular H-bond by 0.03 Å. Although small, this change substantially strengthens the bond, thus contributing to the anomeric selectivity of the binding.

Spectroscopic Evidence for Peptide-Bond-Selective Ultraviolet Photodissociation.

We study the photodissociation induced by ultraviolet excitation of amide bonds in gas-phase protonated peptides. Jointly, mass spectrometry and cold ion spectroscopy provide evidence for a selective nonstatistical dissociation of specific peptide bonds in the spectral region of the formally forbidden n → π* transition of amide groups. Structural analysis reveals that the activation of this transition, peaked at 226 nm, originates from the nonplanar geometry of the bond. In contrast, the statistical dissociation in the electronic ground state appears to be the main outcome of the π → π* excitation of the peptide bonds at 193 nm. We propose a tentative model that explains the difference in the fragmentation mechanisms by the difference in localization of the electronic transitions and the higher amount of vibrational energy released in the electronic excited state upon absorption at 193 nm.

Gas-phase structures reflect the pain-relief potency of enkephalin peptides.

We use cold ion spectroscopy and quantum-chemical computations to solve the structures of opioid peptides enkephalins in the gas phase. The derived structural parameters clearly correlate with the known pharmacological efficiency of the studied drugs, suggesting that gas-phase methods, perhaps, can be used for predicting the relative potency of ligand drugs that target the hydrophobic pockets of receptors.

Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions.

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Interplay of H-Bonds with Aromatics in Isolated Complexes Identifies Isomeric Carbohydrates.

The tremendous isomeric diversity of carbohydrates enables a wide range of their biological functions but makes the identification and study of these molecules difficult. We investigated the ability of intermolecular interactions to communicate structural specificity of carbohydrates to protonated aromatic molecules in non-covalent complexes, isolated and cooled in the gas phase. Our study revealed that small structural differences between carbohydrate isomers of any type, including enantiomers, are accurately communicated by these interactions to aromatic molecules as detectable changes in their electronic excitation spectra. The specific response of the aromatics to the isomers of carbohydrates is fine-tuned by the interplay of the various involved non-covalent bonds. These findings enable the gas-phase identification and relative quantification of any isomers of oligosaccharides in their solution mixtures using the 2D UV-MS fingerprinting technique.

Intrinsic structure of pentapeptide Leu-enkephalin: geometry optimization and validation by comparison of VSCF-PT2 calculations with cold ion spectroscopy.

The intrinsic structure of an opioid peptide [Ala2, Leu5]-leucine enkephalin (ALE) has been investigated using first-principles based vibrational self-consistent field (VSCF) theory and cold ion spectroscopy. IR-UV double resonance spectroscopy revealed the presence of only one highly abundant conformer of the singly protonated ALE, isolated and cryogenically cooled in the gas phase. High-level quantum mechanical calculations of electronic structures in conjunction with a systematic conformational search allowed for finding a few low-energy candidate structures. In order to identify the observed structure, we computed vibrational spectra of the candidate structures and employed the theory at the semi-empirically scaled harmonic level and at the first-principles based anharmonic VSCF levels. The best match between the calculated "anharmonic" and the measured spectra appeared, indeed, for the most stable candidate. An average of two spectra calculated with different quantum mechanical potentials is proposed for the best match with experiment. The match thus validates the calculated intrinsic structure of ALE and demonstrates the predictive power of first-principles theory for solving structures of such large molecules.

Peptide Bond Ultraviolet Absorption Enables Vibrational Cold-Ion Spectroscopy of Nonaromatic Peptides.

Peptide-bond VUV absorption is inherent to all proteins and peptides. Although widely exploited in top-down proteomics for photodissociation, this absorption has never been spectroscopically characterized in the gas phase. We have measured VUV/UV photofragmentation spectrum of a single peptide bond in a cryogenically cold protonated dipeptide. Although the spectrum appears to be very broadband and structureless, vibrational pre-excitation of this and even larger cold peptides significantly increases the UV dissociation yield for some of their photofragments. We use this effect to extend the technique of IR-UV photofragmentation vibrational spectroscopy, developed for aromatic peptides, to nonaromatic ones and demonstrate measurements of conformation-specific and nonspecific IR spectra for di- to hexa-peptides.

Identification of isoforms of aspartic acid residues in peptides by 2D UV-MS fingerprinting of cold ions.

We use 2D UV-MS cold-ion spectroscopy for the identification of l-Asp, d-Asp, l-isoAsp and d-isoAsp residues in a fragment peptide derived from the hormone protein amylin. Relative solution concentrations of all four isoforms in an equimolar quaternary mixture have been determined within 4% error. This method demonstrates that for binary mixtures of the peptides an accuracy of 2.5% can be reached in few-second measurements.

Initial Steps of Amyloidogenic Peptide Assembly Revealed by Cold-Ion Spectroscopy.

The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution. The UV spectra of the tetra- and pentamer are superimposable but differ significantly from the spectra of the monomer and trimer. Such a spectral evolution suggests that a common structural motif is formed as early as the tetramer. The presence of this stable motif is further supported by the low conformational heterogeneity of the tetra- and pentamer, revealed from their IR spectra. From comparison of the IR-spectra in the gas and condensed phases, we propose putative assignments for the dominant motif in the oligomers.