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). 相似文献
Collision induced dissociation (CID) combined with matrix assisted laser desorption ionization-ion mobility-mass spectrometry
(MALDI-IM-MS) is described. In this approach, peptide ions are separated on the basis of mobility in a 15 cm drift cell. Following
mobility separation, the ions exit the drift cell and enter a 5 cm vacuum interface with a high field region (up to 1000 V/cm)
to undergo collisional activation. Ion transmission and ion kinetic energies in the interface are theoretically evaluated
accounting for the pressure gradient, interface dimensions, and electric fields. Using this CID technique, we have successfully
fragmented and sequenced a number of model peptide ions as well as peptide ions obtained by a tryptic digest. This instrument
configuration allows for the simultaneous determination of peptide mass, peptide-ion sequence, and collision-cross section
of MALDI-generated ions, providing information critical to the identification of unknown components in complex proteomic samples. 相似文献
A new, two-dimensional overtone mobility spectrometry (OMS-OMS) instrument is described for the analysis of complex peptide
mixtures. OMS separations are based on the differences in mobilities of ions in the gas phase. The method utilizes multiple
drift regions with modulated drift fields such that only ions with appropriate mobilities are transmitted to the detector.
Here we describe a hybrid OMS-OMS combination that utilizes two independently operated OMS regions that are separated by an
ion activation region. Mobility-selected ions from the first OMS region are exposed to energizing collisions and may undergo
structural transitions before entering the second OMS region. This method generates additional peak capacity and allows for
higher selectivity compared with the one-dimensional OMS method. We demonstrate the approach using a three-protein tryptic
digest spiked with the peptide Substance P. The [M + 3H]3+ ion from Substance P can be completely isolated from other components in this complex mixture prior to introduction into
the mass spectrometer. 相似文献
Two polystyrene-based capillary monolithic columns of different length (50 and 250 mm) were used to evaluate the effects of
column length on gradient separation of protein digests. A tryptic digest of a 9-protein mixture was used as a test sample.
Peak capacities were determined from selected extracted ion chromatograms, and tandem mass spectrometry data were used for
database matching using the MASCOT search engine. Peak capacities and protein identification scores were higher for the long
column with all gradients. Peak capacities appear to approach a plateau for longer gradient times; maximum peak capacity was
estimated to be 294 for the short column and 370 for the long column. Analyses with similar gradient slope produced a ratio
of the peak capacities of 3.36 for the long and the short column, which is slightly higher than the expected value of the
square root of the column length ratio. The use of a longer monolith improves peptide separation, as reflected by higher peak
capacity, and also increases protein identification, as observed from higher identification scores and a larger number of
identified peptides. Attention has also been paid to the peak production rate (PPR, peak capacity per unit time). For short
analysis times, the short column produces a higher PPR, while for analysis times longer than 40 min, the PPR of the 250-mm
column is higher. 相似文献
Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using
either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively
low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum
albumin digest with an LC–UV–mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak
capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar
gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square
root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively,
and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also
higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15-
and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and
increased reliability of protein identification. 相似文献
A hybrid ion mobility-mass spectrometer (IM-MS) incorporating a variable-temperature (80–400 K) drift tube is presented. The
instrument utilizes an electron ionization (EI) source for fundamental small molecule studies. Ions are transferred to the
IM-MS analyzer stages through a quadrupole, which can operate in either broad transmission or mass-selective mode. Ion beam
modulation for the ion mobility experiment is accomplished by an electronic shutter gate. The variable-temperature ion mobility
spectrometer consists of a 30.2 cm uniform field drift tube enclosed within a thermal envelope. Subambient temperatures down
to 80 K are achievable through cryogenic cooling with liquid nitrogen, while elevated temperatures can be accessed through
resistive heating of the envelope. Mobility separated ions are mass analyzed by an orthogonal time-of-flight (TOF) mass spectrometer.
This report describes the technological considerations for operating the instrument at variable temperature, and preliminary
results are presented for IM-MS analysis of several small mass ions. Specifically, mobility separations of benzene fragment
ions generated by EI are used to illustrate significantly improved (greater than 50%) ion mobility resolution at low temperatures
resulting from decreased diffusional broadening. Preliminary results on the separation of long-lived electronic states of
Ti+ formed by EI of TiCl4 and hydration reactions of Ti+ with residual water are presented. 相似文献
Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics.
Thermoanalytical techniques are currently used for the analysis of additives contained in polymers that cannot be easily dissolved, extracted, or hydrolyzed. With these techniques, the polymers are heated to liberate the additives trapped in the polymer matrix. If the polymer is heated slowly, up to its thermal degradation, the technique is called temperature-programmed pyrolysis (TPPy). For TPPy experiments, mass spectrometry is generally used as the detection method. The ionization sources commonly used in mass spectrometry, such as CI and EI, can cause fragmentation during the ionization process. Fragmentation decreases the sensitivity of the molecular ions and increases the risks of interferences with the compounds coming from the matrix. An energy-tunable ionization technique, called metastable atom bombardment (MAB), is proposed for TPPy/MS experiments. With this ionization source, the energy of ionization depends on the metastable gas used. With low-energy metastable gases such as Xe or N(2), fragmentation is reduced compared to CI, whereas with medium-energy metastable gases such as Ar or Kr, the fragmentation is similar to that observed with CI. TPPy/MAB-MS was performed on an unknown polyurethane-based car paint. The detection of molecular ions and characteristic fragments with MAB(N(2)) led to the identification of two light stabilizers: Bis(1,2,2,6,6-pentamethyl-4-piperidinyl)sebacate (BPPS) and 2-(2H-benzotriazol-2-yl)-4,6- di-tert-pentylphenol (PTPP). Using MAB(Ar) to simulate CI, the molecular ion and one of the two characteristic fragments of BPPS were not detected, thus confirming the advantage of using MAB(N(2)) ionization for TPPy/MS experiments. 相似文献
A comprehensive two-dimensional system coupling ultra-performance liquid chromatography (UPLC) and ion mobility-mass spectrometry
(IM-MS) has been applied for the separation and analysis of hydroxylated polybrominated diphenyl ethers (OH-PBDEs). A complex
mixture containing 23 OH-PBDE congeners ranging from hydroxylated monobromodiphenyl ether (OH-monoBDE) to hydroxylated octabromodiphenyl
ether (OH-octaBDE) was satisfactorily separated within 16 min of analysis time. The first-dimensional reversed-phase UPLC
was performed on a sub-2 μm BEH C18 chromatographic column using acetonitrile-water gradient elution program with a flow rate ramp. It enabled excellent chromatographic
separation for both between-class and within-class OH-PBDEs based on their differences in hydrophobicity. Following the pre-ionization
resolution in the first dimension, the second-dimensional IM-MS employed a hybrid electrospray quadrupole ion mobility time-of-flight
mass spectrometer and added an extra post-ionization separation for between-class OH-PBDE congeners on account of their relative
mobility disparity during a very short period of 8.80 ms. The orthogonality of the developed two-dimensional system was evaluated
with the correlation coefficient of 0.9665 and peak spreading angle of 14.87°. The peak capacity of the system was calculated
to be approximately 2 and 15 times higher than that of the two dimensions used alone, respectively. The two-dimensional separation
plane also contributed to the removal of background interference ions and the enhanced confidence in the characterization
of OH-PBDEs of interest. 相似文献
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. 相似文献
Mass spectrometry (MS)–based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house–developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses.
A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.
In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins. 相似文献
A new experimental technique for measuring the mobilities of positive ions in their parent gases is presented. The technique was applied to the rare gases, Ar, Kr, and Xe, and, for pressures typically below 10 Torr, two different types of positive ions were observed. The reduced mobilities of these ions in their parent gases were measured as a function of E/N, the ratio of the electric field strength to the gas number density, at a temperature of 300±1 K. The results were compared with others available in the literature and the two ions were identified as being the atomic and the dimer rare gas ions. The results are in good agreement with those from other authors. Space charge and impurities effects are discussed. 相似文献
This work demonstrated the potential of using a secondary drift gas of differing polarizability from the primary drift gas for confirmation of a positive response for drugs or explosives by ion mobility spectrometry (IMS). The gas phase mobilities of response ions for selected drugs and explosives were measured in four drift gases. The drift gases chosen for this study were air, nitrogen, carbon dioxide and nitrous oxide providing a range of polarizability and molecular weights. Four other drift gases (helium, neon, argon and sulfur hexafluoride) were also investigated but design limitations of the commercial instrument prevented their use for this application. When ion mobility was plotted against drift gas polarizability, the resulting slopes were often unique for individual ions, indicating that selectivity factors between any two analytes varied with the choice of drift gas. In some cases, drugs like THC and heroin, which are unresolved in air or nitrogen, were well resolved in carbon dioxide or nitrous oxide. 相似文献
A new two-dimensional ion mobility spectrometry approach combined with mass spectrometry has been used to examine ubiquitin ions in the gas phase. In this approach ions are separated in an initial drift tube into conformation types (defined by their collision cross sections) and then a gate is used to introduce a narrow distribution of mobility-separated ions into a second drift tube for subsequent separation. The results show that upon selection a narrow peak shape is retained through the second drift tube. This requires that at 300 K the selected distribution does not interconvert substantially within the broader range of structures associated with the conformation type within the approximately 10-20 ms time scale of these experiments. For the [M + 7H]7+ ion, it appears that many ( approximately 5-10) narrow selections can be made across each of the compact, partially-folded, and elongated conformer types, defined previously (Int. J. Mass Spectrom. 1999, 187, 37-47). 相似文献