Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates

Gas-phase ion/molecule chemistry has been combined with ion mobility separation and time-of-flight mass spectrometry to enable the characterization of large poly(ethylene glycol)s (PEGs) and PEGylated molecules (>40 kDa). A facile method is presented in which gas-phase superbases are reacted in the high-pressure source region of commercial TOF mass spectrometers to manipulate the charge states of large ions generated by electrospray ionization (ESI). Charge stripping decreases the spectral congestion typically observed in ESI mass spectra of high molecular weight polydisperse PEGylated molecules. From these data, accurate average molecular weights and molecular weight distributions for synthetic polymers and PEGylated proteins are determined. The average MW measured for PEGylated Granulocyte colony-stimulating factor (rh-GCSF, 40 726.2 Da) is in good agreement with the theoretical value, and a 16 Da mass shift is easily observed in the spectrum of an oxidized form of the heterogeneous PEGylated protein. Ion mobility separations can fractionate PEGs of different chain length; when coupled with charge stripping ion/molecule reactions, ion mobility mass spectrometry (IMMS) offers several analytical advantages over mass spectrometry alone for the characterization of large PEGylated molecules including enhanced dynamic range, increased sensitivity, and specificity. Low abundance free PEG in a PEGylated peptide preparation, which is not directly detectable by mass spectrometry, can be easily observed and accurately quantified with gas-phase ion/molecule chemistry combined with ion mobility mass spectrometry.     

Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis-electrospray ionization-mass spectrometry

Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A time-of-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of "O" by "NH". An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and alpha1-acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure.     

Gradient tandem mass spectrometry interfaced with ion mobility separation for the characterization of supramolecular architectures

Traveling wave ion mobility mass spectrometry (TWIM MS) was combined with gradient tandem mass spectrometry (gMS(2)) to deconvolute and characterize superimposed ions with different charges and shapes formed by electrospray ionization (ESI) of self-assembled, hexameric metallomacrocycles composed of terpyridine-based ligands and Cd(II) ions. ESI conditions were optimized to obtain intact hexameric cation assemblies in a low charge state (2+), in order to minimize overlapping fragments of the same mass-to-charge ratio. With TWIM MS, intact hexameric ions could be separated from remaining fragments and aggregates. Collisional activation of these hexameric ions at varying collision energies (gMS(2)), followed by TWIM separation, was then performed to resolve macrocyclic from linear hexameric species. Because of the different stabilities of these architectures, gMS(2) changes their relative amounts, which can be monitored individually after subsequent ion mobility separation. On the basis of this unique strategy, hexameric cyclic and linear isomers have been successfully resolved and identified. Complementary structural information was gained by the gMS(2) fragmentation pattern of the metallosupramolecules, acquired by collisionally activated dissociation after TWIM dispersion. TWIM MS interfaced with gMS(2) should be particularly valuable for the characterization of a variety of supramolecular polymers, which often contain isomeric architectures that yield overlapping fragments and aggregates upon ESI MS analysis.     

Coupling capillary electrophoresis and high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of complex lipopolysaccharides

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technology for atmospheric pressure, room temperature separation of gas-phase ions. The FAIMS system acts as an ion filter that can continuously transmit one type of ion, independent of mass-to-charge ratio (m/z). Capillary electrophoresis-electrospray mass spectrometry (CE-MS) has been extensively used for the analysis of complex bacterial lipopolysaccharides (LPS). The coupling of FAIMS to CE-MS provides a sensitive technique for the characterization of these complex glycolipids, permitting the separation of trace-level LPS oligosaccharide glycoforms for subsequent structural characterization using tandem mass spectrometry. This was demonstrated for LPS from nontypeable Haemophilus influenzae strain 375 following O-deacylation with anhydrous hydrazine. This strain of H. influenzae can express a triheptosyl-containing glycoform to which four hexose residues are linked forming the outer-core region of the molecule. This has been referred to as the Hex4 glycoform. Glycoforms have been identified which differ in the number of phosphoethanolamine substituents in the inner-core. With the use of CE-FAIMS, isomeric Hex4 glycoforms containing two PEtn groups were separated and characterized by MS/MS. FAIMS provided a significant reduction in mass spectral noise, leading to improved detection limits ( approximately 70 amol of the major glycoform). The extracted mass spectrum showed that the apparent noise was virtually eliminated. In addition to the reduction of chemical background, the ion current was increased by as much as 7.5 times as a result of the atmospheric pressure ion-focusing effect provided by the FAIMS system. The linearity of response of the CE-FAIMS-MS system was also studied. The calibration curve is linear for approximately 3 orders of magnitude, over a range of 40 pg/microL to 10 ng/microL.     

Characterization of naphthenic acids by electrospray ionization high-field asymmetric waveform ion mobility spectrometry mass spectrometry

Electrospray ionization (ESI) high-field asymmetric waveform ion mobility spectrometry (FAIMS) was combined with quadrupole, time-of-flight, and tandem mass spectrometry to characterize commercial and naturally occurring naphthenic acids (NA) mixtures. This new method provides quantitatively reliable mass and isomer distributions of NA components in approximately 3 min without extensive sample preparation. ESI-FAIMS-MS seems to be especially useful for characterization of fragile ions that cannot be detected by other methods. A unique part of this technique is separation of structural isomers that proved to be critical in determination of elemental composition and in structure elucidation. Tandem mass spectrometry of NA ions separated by FAIMS provides more information about the structure of NA than other methods in the field of NA analysis.     

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.     

Microfluidic electrocapture for separation of peptides

A separation method based on electroimmobilization and sequential release of captured molecules is reported. A microfluidic electrocapture device is utilized to immobilize peptides in a microflow stream. After capture, the electric field is decreased in a stepwise manner, causing sequential release of the captured peptides according to their electrophoretic mobility. Tryptic peptides were separated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The separation power was high enough to increase the ionization yield of several peptides not seen in the unprocessed sample. In addition to separation, simultaneous sample cleanup was demonstrated for peptides obtained by shotgun tryptic digestion of membrane protein extracts.     

Characterization of gas-phase molecular interactions on differential mobility ion behavior utilizing an electrospray ionization-differential mobility-mass spectrometer system

Differential mobility spectrometry (DMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise ratios, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and potential for rapid analyte quantitation. The introduction of a new ESI-DMS-MS system and its utilization to aid in the understanding of DMS separation theory is described. A current contribution to DMS separation theory is one of an association/dissociation process between ions/molecules in the gas phase during the differential mobility separation. A model study was designed to investigate the molecular dynamics and chemical factors influencing the theorized association/dissociation process, and the mechanisms by which these gas-phase interactions affect an ion's DM behavior. Five piperidine analogues were selected as model analytes, and three alcohol drift gas dopants/modifiers were used to interrogate the analyte ions in the gas phase. Two proposed DMS separation mechanisms, introduced as Core and Fa?ade, corresponding to strong and weak attractions between ions/molecules in the gas phase, are detailed. The proposed mechanisms provide explanation for the observed changes in analyte separation by the various drift gas modifiers. Molecular modeling of the proposed mechanisms provides supportive data and demonstrates the potential for predictive optimization of analyte separation based on drift gas modifier effects.     

Mass spectrometric characterization of functional poly(methyl methacrylate) in combination with critical liquid chromatography

State-of-the-art techniques for the mass spectrometric characterization of synthetic polymers have been applied to functional poly(methyl methacrylate), synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The polymers were first separated effectively according to functionality by liquid chromatography (LC) at the critical conditions (i.e., almost no influence of molecular weight on retention). The separated polymers were characterized off-line by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and both off-line and on-line by LC-electrospray-ionization-quadrupole-TOF-MS (LC-ESI-QTOF- MS). The on-line ESI experiments confirmed a clear baseline separation of the hydroxyl-functional prepolymers according to the number of hydroxyl groups. Labile end groups of PMMA, such as the dithioester group, were lost in the MALDI-TOF-MS experiments, while they were observed intact in the ESI-QTOF-MS spectra. This indicates that in the present case ESI is a much softer ionization technique than is MALDI. The ESI-MS experiments provided direct evidence that the RAFT polymers still exhibited living characteristics in the form of the dithio moiety.     

Sample deposition device for off-line combination of supercritical fluid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new sample deposition device for off-line SFC-MALDI combination of supercritical fluid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was assembled. This device was successfully applied to the detailed characterization of synthetic silicone oils. SFC was used to separate samples of silicone oils on micropacked capillary columns and to determine their molecular mass distribution. The separated fractions for the identification studies were obtained from SFC runs at defined time intervals. Using the constructed deposition device, these fractions were sprayed directly from the restrictor on the target probe covered with a proper matrix. MALDI-TOF MS was used for the identification of individual oligomers in the separated fractions and also in the unfractionated sample. The determined molecular mass distributions based on supercritical fluid chromatography with flame ionization detector, MALDI-TOF MS, and combined SFC-MALDI measurements were compared and the results were in a good agreement. The sample deposition device is based on a common plotter unit, complemented by a microcontroller PIC16C84. The unit is connected by an RS-232 interface to a PC with the main control software running under MS Windows. The new sample deposition device made the off-line combination SFC-MALDI simpler, faster, and more sensitive.     

Screening method for isopeptides from small ubiquitin-related modifier-conjugated proteins by ion mobility mass spectrometry

Posttranslational modification by the small ubiquitin-related modifier (SUMO) is a highly regulated modification, which is often restricted to very specific cellular events. A number of analytical strategies for identification of SUMOylated proteins have been previously reported in the literature. A new screening method for SUMOylated peptides based on ion mobility mass spectrometry is presented. Using poly-SUMO2 as a model system, a two-enzyme trypsin/chymotrypsin digestion was performed to reduce the size of the isopeptide conjugated to the substrate lysine residue. Traveling wave ion mobility mass spectrometry was used to screen for peptides containing the QQQTGG isopeptide tag from SUMO, which increases the mass and size of the peptide by 618 Da. This increase in mass along with solution conditions to promote higher charge states allows the isopeptides to be separated from the typically smaller and lesser charged linear peptides. On the basis of these findings, this method can be used as a quick and easy screening method for identifying possible SUMO isopeptides.     

Nonaqueous capillary zone electrophoresis of synthetic organic polypeptides

Poly(Nepsilon-trifluoroacetyl-L-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanoVacetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of approximately 50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.     

Enhanced intestinal absorption and bioavailability of raloxifene hydrochloride via lyophilized solid lipid nanoparticles

The current oral therapy with raloxifene hydrochloride (RXH) is less effective due to its poor bioavailability (only 2%). Henceforth, an attempt was made to investigate the utility of triglyceride (trimyristin, tripalmitin and tristearin) based solid lipid nanoparticles (SLNs) for improved oral delivery of RXH. The SLN formulations prepared were evaluated for particle size, zeta potential and % entrapment and the optimized formulation was lyophilized. Solid state characterization studies unravel the transformation of RXH to amorphous or molecular state from the native crystalline form. Further the in situ perfusion studies carried out in rat intestine reveal the potential of SLN for enhanced permeation of raloxifene HCl across gastrointestinal barrier. To derive the conclusions, in vivo pharmacokinetic study was conducted in rats to assess the bioavailability of RXH from SLN formulation compared to drug suspension. Overall a twofold increase in bioavailability with SLN formulations confer their potential for improved oral delivery of RXH.     

Development of an ion mobility quadrupole time of flight mass spectrometer

We describe here a new ion mobility capable mass spectrometer which comprises a drift cell for mobility separation and a quadrapole time of flight mass spectrometer for mass analysis--the MoQTOF. A commercial QToF instrument (Micromass UK Ltd., Manchester, UK) has been modified by the inclusion of an additional chamber containing a drift cell and ancillary ion optics. The drift cell is 5.1 cm long made from a copper block and is mounted from a top hat flange in a chamber situated post source optics and prior to the quadapole analyzer. Details of this instrument are provided along with information about how it can be used to acquire mobilities of ions along with their mass to charge ratios. The MoQTOF is used to examine conformations of a series of antimicrobial peptides based on a beta-defensin template. In vivo, these cationic cystine-rich amphiphilic peptides are conformationally restrained by three or more disulfide bridges, although recent findings by several groups have cast doubt on the importance of canonical disulfide pairing to antimicrobial activities. By synthesizing a panel of variants to Defb14 (the murine orthologue of HBD3), we exploit ion mobility to distinguish conformational differences which arise due to disulfide formation and to the hydrophobicity of the peptide sequence. Our gas-phase results are interpreted in terms of the antimicrobial and chemotacic properties of beta-defensins, and this mass spectrometry based approach to discern structure may have a role in future design of novel antibiotics.     

Characterization of khaya gum as a binder in a paracetamol tablet formulation

The influence of khaya gum, a binding agent obtained from Khaya grandifolia (Meliaceae family), on the bulk, compressional, and tabletting characteristics of a paracetamol tablet formulation was studied in comparison with the effects of two standard binders: polyvinylpyrrolidone (PVP; molecular weight 40,000) and gelatin. The relative ability of khaya gum to destroy any residual microbial contamination in the binder or in the formulation during tabletting was also studied using Bacillus subtilis spores as a model. Formulations containing khaya gum exhibited more densification than formulations containing PVP and gelatin during die filling, but less densification due to rearrangement at low pressures. The mean yield pressure of the formulation particles obtained from Heckel plots, and another pressure term, also inversely related to plasticity, obtained from Kawakita plots, showed dependence on the nature and concentration of the binder, with formulations containing khaya gum exhibiting the lowest and highest values respectively. The values of the pressure terms suggest that the yield pressure relates to the onset of plastic deformation during compression, while the Kawakita pressure relates to the total amount of plastic deformation occurring during the compression process. Tablets made from formulations containing khaya gum had the lowest tensile strength values but also the lowest tendency to laminate or cap, as indicated by their lowest brittleness. All the tablets had friability values < 1% at higher concentrations of the three binders. In addition, khaya gum demonstrated a comparable ability to destroy microorganisms in the formulation during tabletting as the two binders. The characterization of the formulations suggests that khaya gum can be developed into a commercial binding agent for particular tablets.     

Continuous Flow Deformability‐Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label‐free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label‐free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10⁴‐fold enrichment of target cells relative to leukocytes. In patients with metastatic castration‐resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.     

Nanoparticle characterization by cyclical electrical field-flow fractionation

In this work, the analytical potential of cyclical electrical field flow fractionation (CyElFFF) for nanomaterial and colloidal particle characterization has been experimentally demonstrated. Different operating parameters were investigated in order to evaluate their effect on the mechanisms of retention and fractionation power of CyElFFF. The voltage and frequency of the oscillating electrical field appeared to be the most influential parameters controlling the separation mode. Mobile phase flow rate was also found to be a key parameter controlling the fractionation efficiency. This work allowed the definition of operating conditions such that a reliable CyElFFF analysis could be performed on different nanoparticles on the basis of the direct comparison of their theoretical and experimental behavior. The results show that this technique in optimized conditions is a powerful tool for electrophoretic mobility based separation and characterization of various nanoparticles.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Characterization of a variant of the spinach PSII type I light-harvesting protein using kinetically controlled digestion and RP-HPLC-ESI-MS

A previously unknown isoform of the type I major antenna protein of photosystem II of spinach was identified, and its amino-terminal sequence was characterized by a novel kinetic digestion approach, in which sequential tryptic digestion was followed by analysis of both released peptides and truncated proteins by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Using nonpolar, monolithic, 200-microm-i.d. separation columns based on poly(styrene/divinylbenzene) copolymer and applying gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid, released peptides and truncated proteins could be separated and mass analyzed in a single chromatographic run. This enabled a straightforward identification of the fragments removed from the amino-terminal ends of the protein, which was essential for the characterization of the antenna isomers showing the most significant sequence variation in the amino-terminal region. The sequences of the amino termini were derived from the differences in molecular mass between intact and truncated proteins and were corroborated by sequencing using tandem mass spectrometry and database searching. The sequence of the 23 amino-terminal residues of the previously unknown isoform differed from that of the other two known isoforms only in one and three amino acids, respectively. Such subtle changes in amino acid sequence are supposed to play an important role in the supramolecular organization of photosynthetic antenna proteins.