Direct analysis of liquid samples by desorption electrospray ionization-mass spectrometry (DESI-MS)

Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the direct analysis of liquid samples. Several interesting results were found. First, in contrast to the previous DESI analysis of dried solid samples that was limited to proteins with MW ≤25 kDa (Anal. Chem. 2007, 79, 3514), bovine serum albumin (BSA, 66 kDa) was successfully ionized from solutions by DESI with observation of corresponding multiply charged ions. Second, direct DESI analysis of protein tryptic digest solutions without chromatographic separation, sample clean-up, and the sample drying step was demonstrated, providing reasonably good sequence coverage of 52% to 97%. Third, direct analysis of biofluids such as an undiluted urine sample without sample pretreatment is possible, emphasizing the high tolerance of DESI with salt. These results suggest that a charged droplet pick-up mechanism is responsible for desorption and ionization of liquid samples by DESI. Also, unlike in electrospray ionization (ESI), inhibition of electrochemical reduction in the negative ion mode was observed for liquid sample DESI. In addition, reactive DESI can be performed with ion/ion reactions of Zn(II) complexes for the selective binding of phosphoserine in the presence of serine. DESI experiment can also be carried out directly to liquid samples flowing out of a pumped syringe needle tip, allowing rapid analysis. Furthermore, on-line coupling of electrochemical cell with DESI-MS was demonstrated, in which perylene radical cations generated in the cell were successfully transferred to the gas-phase for MS detection by DESI. This study extended the scope of DESI-MS applications, which could have potentials in bioanalytical and forensic analysis.     

Ion mobility augments the utility of mass spectrometry in the identification of human hemoglobin variants

The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Use of imaging multivariate analysis to improve biochemical and anatomical discrimination in desorption electrospray ionisation mass spectrometry imaging

Desorption electrospray ionisation (DESI) mass spectrometry images usually contain a large amount of information that can be difficult to interpret in an objective manner. We explore the use of imaging multivariate analysis (MVA) on DESI images of protein spots and rat brain sections to automatically assign peaks and improve discrimination of spatially important features. DESI parameters were optimised on an ion trap mass spectrometer for (a) consistent imaging of dried single and mixture spots of insulin, myoglobin and BSA from a Permanox slide, and (b) to produce a MS image of rat brain coronal section at 100 μm resolution. Multivariate curve resolution (MCR), an imaging MVA technique was applied to these images after appropriate data binning. MCR analysis on DESI images of protein mixture spots allowed the multiply charged peaks of a number of proteins to be distinctly separated. Application of MCR to a DESI image of a rat brain coronal section deconvoluted the image into components that showed biologically important features. Further application of MCR to a subsection of the image produced a component that clearly separated out the substantia nigra region, which allowed us to produce a biochemical anatomy for this area of the brain. We have demonstrated the ability of imaging MVA to automatically and objectively analyse DESI images of standardised and complex biological samples, and have shown its capacity for detailed spatial profiling of biomolecules in specific morphological regions. We propose the routine use of this technique for future DESI imaging experiments.     

Rapid accurate mass desorption electrospray ionisation tandem mass spectrometry of pharmaceutical samples

Desorption electrospray ionisation (DESI) has been successfully combined with a hybrid quadrupole time-of-flight mass spectrometer to provide mass spectra and product ion mass spectra of active ingredients formulated in pharmaceutical tablets, gels and ointments. Accurate mass data has been obtained from the DESI mass spectra and of the product ion fragments of selected ions, greatly enhancing the selectivity and information content of the experiment. This accurate mass information only takes seconds to acquire since the DESI technique does not require any sample preparation or extraction prior to mass analysis.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.     

Direct analysis of pharmaceutical formulations from non‐bonded reversed‐phase thin‐layer chromatography plates by desorption electrospray ionisation ion mobility mass spectrometry

The direct analysis of pharmaceutical formulations and active ingredients from non‐bonded reversed‐phase thin layer chromatography (RP‐TLC) plates by desorption electrospray ionisation (DESI) combined with ion mobility mass spectrometry (IM‐MS) is reported. The analysis of formulations containing analgesic (paracetamol), decongestant (ephedrine), opiate (codeine) and stimulant (caffeine) active pharmaceutical ingredients is described, with and without chromatographic development to separate the active ingredients from the excipient formulation. Selectivity was enhanced by combining ion mobility and mass spectrometry to characterise the desorbed gas‐phase analyte ions on the basis of mass‐to‐charge ratio (m/z) and gas‐phase ion mobility (drift time). The solvent composition of the DESI spray using a step gradient was varied to optimise the desorption of active pharmaceutical ingredients from the RP‐TLC plates. The combined RP‐TLC/DESI‐IM‐MS approach has potential as a rapid and selective technique for pharmaceutical analysis by orthogonal gas‐phase electrophoretic and mass‐to‐charge separation. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry

Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. Proteins are converted to gas-phase, multiply-charged, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. M/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.     

Characterisation of the intact rainbow trout vitellogenin protein and analysis of its derived tryptic and cyanogen bromide peptides by matrix-assisted laser desorption/ionisation time-of-flight-mass spectrometry and electrospray ionisation quadrupole/time-of-flight mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals. It is being used as a biomarker for exposure to endocrine disruptors in many species. Rainbow trout Vtg has recently been sequenced by the conventional cDNA nucleotide approach. We focused on protein characterization of the intact protein and its derived tryptic and cyanogen bromide peptides by matrix-assisted laser desorption/ionisation and electrospray ionisation mass spectrometry. The molecular mass of the intact protein was found to be 183127 Da. A large number of unidentified peptide ions encourage further structural analysis to propose possible sequence variants and post-translational modifications.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Tandem mass spectrometry of half-generation PAMAM dendrimer anions

Ions derived from negative electrospray ionization of polyamidoamine (PAMAM) dendrimer generation 0.5 were subjected to ion trap tandem mass spectrometry. Ion/ion proton transfer reactions were used to manipulate the charge states of PAMAM precursor ions to form lower charge states from those initially formed by electrospray, as well as to facilitate the interpretation of the product ion mass spectra. Most of the products derived from dendrimer precursor ions could be rationalized by retro-Michael decomposition reactions. The dominant fragmentation channels are highly dependent on the composition of the counter-ions, which in this case are restricted to different numbers of sodium ions and protons, and whether the precursor ion is multiply charged or singly charged. An interpretation is given that is consistent with all of the observations made with the various anions associated with this study. The nature of the structural information that can be obtained via ion trap tandem mass spectrometry of the dendrimers is dependent on the types of precursor ions subjected to study. The tandem mass spectrometry data also provided information about the structure of faulty synthesis products present in the PAMAM dendrimer sample.     

Separation of peptides from detergents using ion mobility spectrometry

Mass spectrometry (MS) has dramatically evolved in the last two decades and has been the driving force of the spectacular expansion of proteomics during this period. However, the very poor compatibility of MS with detergents is still a technical obstacle in some studies, in particular on membrane proteins. Indeed, the high hydrophobicity of membrane proteins necessitates the use of detergents for their extraction and solubilization. Here, we address the analytical potential of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for separating peptides from detergents. The study was focused on peptides from the human integral membrane protein CD9. A tryptic peptide was mixed with the non-ionic detergents Triton X-100 or beta-D-dodecyl maltoside (DDM) as well as with the ionic detergents sodium dodecyl sulfate (SDS) or sodium deoxycholate (SDC). Although electrospray ionization (ESI) alone led to a total suppression of the peptide ion signal on mass spectra with only detection of the detergents, use of FAIMS allowed separation and clear identification of the peptide with any of the detergents studied. The detection and identification of the target compound in the presence of an excess of detergents are then feasible. FAIMS should prove especially useful in the structural and proteomic analysis of membrane proteins.     

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.     

Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm(3). Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins

Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.     

Top-down protein sequencing by CID and ECD using desorption electrospray ionisation (DESI) and high-field FTICR mass spectrometry

We report high resolution spectra for the medium molecular weight proteins myoglobin and cytochrome-c obtained using a custom desorption electrospray ionisation (DESI) source coupled to a Bruker Daltonics 12 T Apex Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The DESI source was designed for accurate alignment and reproduction of critical geometric variables. A two axis motorised stage was included to enable automated rastering of the sample under the DESI plume. Spectra for the intact proteins have been obtained under single-acquisition conditions and a top-down analysis of cytochrome-c was performed using both collision induced dissociation (CID) and electron capture dissociation (ECD) of the isolated [M+15H]¹⁵⁺ charge state. The sequence coverage is comparable to that obtained using electrospray ionisation, demonstrating the utility of top-down protein analysis by DESI FTICR-MS.     

The influence and utility of varying field strength for the separation of tryptic peptides by ion mobility-mass spectrometry

The influence of field strength on the separation of tryptic peptides by drift tube-based ion mobility-mass spectrometry is reported. Operating the ion mobility drift tube at elevated field strengths (expressed in V cm(-1) torr(-1)) reduces separation times and increases ion transmission efficiencies. Several accounts in the literature suggest that performing ion mobility separation at elevated field strength can change the selectivity of ion separation. To evaluate the field strength dependant selectivity of ion mobility separation, we examined a data set of 65 singly charged tryptic peptide ion signals (mass range 500-2500 m/z) at six different field strengths and four different drift gas compositions (He, N2, Ar, and CH4). Our results clearly illustrate that changing the field strength from low field (15 V cm(-1) torr(-1)) to high field (66 V cm(-1) torr(-1)) does not significantly alter the selectivity or peak capacity of IM-MS. The implications of these results are discussed in the context of separation methodologies that rely on the field strength dependence of ion mobility for separation selectivity, e.g., high-field asymmetric ion mobility spectrometry (FAIMS).