Ion mobility spectrometry – Mass spectrometry coupling for synthetic polymers

This review covers applications of ion mobility spectrometry (IMS) hyphenated to mass spectrometry (MS) in the field of synthetic polymers. MS has become an essential technique in polymer science, but increasingly complex samples produced to provide desirable macroscopic properties of high‐performance materials often require separation of species prior to their mass analysis. Similar to liquid chromatography, the IMS dimension introduces shape selectivity but enables separation at a much faster rate (milliseconds vs minutes). As a post‐ionization technique, IMS can be hyphenated to MS to perform a double separation dimension of gas‐phase ions, first as a function on their mobility (determined by their charge state and collision cross section, CCS), then as a function of their m/z ratio. Implemented with a variety of ionization techniques, such coupling permits the spectral complexity to be reduced, to enhance the dynamic range of detection, or to achieve separation of isobaric ions prior to their activation in MS/MS experiments. Coupling IMS to MS also provides valuable information regarding the 3D structure of polymer ions in the gas phase and regarding how to address the question of how charges are distributed within the structure. Moreover, the ability of IMS to separate multiply charged species generated by electrospray ionization yields typical IMS‐MS 2D maps that permit the conformational dynamics of synthetic polymer chains to be described as a function of their length.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous detection of phosphatidylcholines and glycerolipids using matrix‐enhanced surface‐assisted laser desorption/ionization‐mass spectrometry with sputter‐deposited platinum film

Matrix‐assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) allows for the simultaneous detection and imaging of several molecules in brain tissue. However, the detection of glycerolipids such as diacylglycerol (DAG) and triacylglycerol (TAG) in brain tissues is hindered in MALDI‐IMS because of the ion suppression effect from excessive ion yields of phosphatidylcholine (PC). In this study, we describe an approach that employs a homogeneously deposited metal nanoparticle layer (or film) for the detection of glycerolipids in rat brain tissue sections using IMS. Surface‐assisted laser desorption/ionisation IMS with sputter‐deposited Pt film (Pt‐SALDI‐IMS) for lipid analysis was performed as a solvent‐free and organic matrix‐free method. Pt‐SALDI produced a homogenous layer of nanoparticles over the surface of the rat brain tissue section. Highly selective detection of lipids was possible by MALDI‐IMS and Pt‐SALDI‐IMS; MALDI‐IMS detected the dominant ion peak of PC in the tissue section, and there were no ion peaks representing glycerolipids such as DAG and TAG. In contrast, Pt‐SALDI‐IMS allowed the detection of these glycerolipids, but not PC. Therefore, using a hybrid method combining MALDI and Pt‐SALDI (i.e., matrix‐enhanced [ME]‐Pt‐SALDI‐IMS), we achieved the simultaneous detection of PC, PE and DAG in rat brain tissue sections, and the sensitivity for the detection of these molecules was better than that of MALDI‐IMS or Pt‐SALDI alone. The present simple ME‐Pt‐SALDI approach for the simultaneous detection of PC and DAG using two matrices (sputter‐deposited Pt film and DHB matrix) would be useful in imaging analyses of biological tissue sections. Copyright © 2015 John Wiley & Sons, Ltd.     

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time‐of‐flight mass spectrometry

The use of hybrid quadrupole ion mobility spectrometry time‐of‐flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T_d) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent‐excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI‐X, MI‐Y and MI‐Z), inverse mobility and collision cross‐section (CCS). The correlation of T_d with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS² and MS³) were successfully performed on the N‐acetyl‐p‐benzoquinoneimine glutathione (NAPQI‐GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time‐of‐flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave‐enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

An On‐Tissue Paternò–Büchi Reaction for Localization of Carbon–Carbon Double Bonds in Phospholipids and Glycolipids by Matrix‐Assisted Laser‐Desorption–Ionization Mass‐Spectrometry Imaging

Matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI) visualizes the distribution of phospho‐ and glycolipids in tissue sections. However, C=C double‐bond (db) positional isomers generally cannot be distinguished. Now an on‐tissue Paternò–Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI‐MSI‐compatible reagent is introduced. Laser‐induced postionization (MALDI‐2) was used to boost the yields of protonated PB products. Collision‐induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db‐positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB‐MALDI‐MS/MS procedure could help to better understand the physiological role of these db‐positional isomers.     

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

Sterically hindered phenols in negative ion mobility spectrometry–mass spectrometry

Negative corona discharge atmospheric pressure chemical ionization (APCI) was used to investigate phenols with varying numbers of tert‐butyl groups using ion mobility spectrometry–mass spectrometry (IMS‐MS). The main characteristic ion observed for all the phenolic compounds was the deprotonated molecule [M–H]⁻. 2‐tert‐Butylphenol showed one main mobility peak in the mass‐selected mobility spectrum of the [M–H]⁻ ion measured under nitrogen atmosphere. When air was used as a nebulizer gas an oxygen addition ion was seen in the mass spectrum and, interestingly, this new species [M–H+O]⁻ had a shorter drift time than the lighter [M–H]⁻ ion. Other phenolic compounds primarily produced two IMS peaks in the mass‐selected mobility spectra measured using the [M–H]⁻ ion. It was also observed that two isomeric compounds, 2,4‐di‐tert‐butylphenol and 2,6‐di‐tert‐butylphenol, could be separated with IMS. In addition, mobilities of various characteristic ions of 2,4,6‐trinitrotoluene were measured, since this compound was previously used as a mobility standard. The possibility of using phenolic compounds as mobility standards is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Orthogonal organic and aqueous‐based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous‐based buffer for tissue section preparation before matrix‐assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water–acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14‐day mouse fetus whole‐body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on‐tissue MALDI analysis compared with solely conventional organic rinsing. Copyright © 2013 John Wiley & Sons, Ltd.     

Multidimensional Mass Spectrometry of Synthetic Polymers and Advanced Materials

Multidimensional mass spectrometry interfaces a suitable ionization technique and mass analysis (MS) with fragmentation by tandem mass spectrometry (MS²) and an orthogonal online separation method. Separation choices include liquid chromatography (LC) and ion‐mobility spectrometry (IMS), in which separation takes place pre‐ionization in the solution state or post‐ionization in the gas phase, respectively. The MS step provides elemental composition information, while MS² exploits differences in the bond stabilities of a polymer, yielding connectivity and sequence information. LC conditions can be tuned to separate by polarity, end‐group functionality, or hydrodynamic volume, whereas IMS adds selectivity by macromolecular shape and architecture. This Minireview discusses how selected combinations of the MS, MS², LC, and IMS dimensions can be applied, together with the appropriate ionization method, to determine the constituents, structures, end groups, sequences, and architectures of a wide variety of homo‐ and copolymeric materials, including multicomponent blends, supramolecular assemblies, novel hybrid materials, and large cross‐linked or nonionizable polymers.     

IMSPeptider: A computational peptide collision cross‐section area calculator based on a novel molecular dynamics simulation protocol

Introduction of ion mobility mass spectrometry (IMS/MS) into the proteomic workflow provides an orthogonal separation to the widely used LC‐MS platforms. IMS also provides structural information that could facilitate peptide identification. However, the lack of tools capable of predictive power in a high‐throughput fashion makes peptide global profiling quite challenging. To target this issue, a computational workflow was developed based on biophysical principles to predict the collision cross‐section area (CCS) of peptides as measured from IMS/MS experiments. Hosted on a web server, it allows the user to input a primary sequence (query) and retrieve information on peptide structure, sequence, and corresponding CCS. The current version is designed to identify peptide sequences up to 23 residues in length, in its higher charge state, based on a match of the molecule m/z and CCS. The protocol was validated against a 128‐sequences‐dataset and CCS predicted within 2.8% average error. © 2013 Wiley Periodicals, Inc.     

Study of fragmentation pathways of lithiated α,β‐unsaturated thioesters by electrospray ionization mass spectrometry

The fragmentation pathways of lithiated α,β‐unsaturated thioesters with different substituents were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) in positive ion mode. In mass spectrometry of the α,β‐unsaturated thioesters, Ar‐CH?CH‐CO‐S‐Ph, loss of PhSLi and elimination of a thiophenol were the two major fragmentation reactions of the lithiated molecules. The elemental compositions of all the ions were confirmed by high‐resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR‐MS/MS). The thioesters studied here were para‐monosubstituted on the phenyl ring of cinnamoyl and the electron‐withdrawing groups favored loss of a thiophenol, whereas the electron‐releasing groups strongly favored the competing reaction leading to the loss of PhSLi to form a cinnamoyl cation, Ar‐CH?CHCO⁺. The intensity ratios of the two competitive product ions were well correlated with the σ substituent constants. The mechanisms of these two competing routes were further investigated by density functional theory (DFT) calculations. Copyright © 2010 John Wiley & Sons, Ltd.     

MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

Evaluation of tetracationic salts as gas‐phase ion‐pairing agents for the detection of trivalent anions in positive mode electrospray ionization mass spectrometry

In previous studies, new electrospray ionization mass spectrometry (ESI‐MS) approaches were developed for the highly sensitive detection of singly and doubly charged anions in positive mode ESI‐MS by using specially synthesized dicationic and tricationic ion‐pairing agents, respectively. By detecting the positively charged ion complex in the positive mode, limits of detection (LODs) for the anions can be lowered by several magnitudes. In this work, we used eighteen newly synthesized tetracationic ion‐pairing agents, constructed with different geometries, linkages and cation moieties, for the detection of eighteen triply charged anions of different structural motifs. The LODs for these anions were from ten to several thousand times lower in the positive selective ion monitoring (SIM) mode than in the negative mode. These tetracationic agents also were shown to be useful for the detection of ?1 and ?2 anions. In addition, the LODs for ?3 anions can be further lowered by monitoring the product fragments of the ion‐pairing complexes in the single reaction monitoring (SRM) mode. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐ tandem mass spectrometry

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.     

Evidence for electron‐based ion generation in radio‐frequency ionization

Radio‐frequency ionization (RFI) is a novel ionization method coupled to mass spectrometry (MS) for analysis of semi‐volatile and volatile organic compounds (VOCs). Despite the demonstrated capabilities of RFI MS for VOC analysis in both positive‐ and negative‐ion modes, mechanism of RFI is not completely understood. Improved understanding of the ion generation process in RFI should expand its utility in MS. Here, we studied the possibility of electron emission in RFI using both direct charged particle current measurements and indirect electron detection in a 9.4‐T Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometer. We show that RF‐generated electrons can be trapped in the ICR cell and, subsequently, reacted with neutral hexafluorobenzene (C₆F₆) molecules to generate C₆F₆^●?. Intensity of observed C₆F₆^●? species correlated with the number of trapped electrons and decreased as a function of electron quenching period. We also measured the electron attachment rate constant of hexafluorobenzene using a post‐RF electron trapping experiment. Measured electron attachment rate constant of hexafluorobenzene (1.19 (±0.53) × 10^?9 cm³ molecule^?1 s^?1) for post‐RF FT‐ICR MS agreed with the previously reported value (1.60 (±0.30) × 10^?9 cm³ molecule^?1 s^?1) from low‐pressure ICR MS measurements. Experimental results from direct and indirect electron measurements suggest that RFI process involves RF‐generated electrons under ultrahigh vacuum conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

Ion‐Mobility Spectrometry Can Assign Exact Fucosyl Positions in Glycans and Prevent Misinterpretation of Mass‐Spectrometry Data After Gas‐Phase Rearrangement

The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion‐mobility MS and well‐defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N‐acetylneuraminic acid as well as N‐acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival‐time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.