Simulating, Predicting, and Minimizing False Peaks for Hadamard Transform Ion Mobility Spectrometry.

Multiplexing techniques, including the Hadamard transform, are widely used in the recovery of weak signals from high-level noise. Hadamard transform ion mobility spectrometry (HT-IMS), however, can suffer serious drawbacks due to false peaks. False peaks in HT-IMS are generally attributed to nonperfect gating behavior. This paper confirmed that the origin of false peaks in HT-IMS is not generally due to ion gating but rather to peak shifts by Coulombic repulsion of the ion packets inside the drift tube. The amplitudes of these false peaks are determined by the number of ions inside the ion packets. This phenomenon is simulated and confirmed by the convolution of the spectrum with a shifted s-sequence to reproduce the artifact peaks with the exact position, amplitude, and profile. Two approaches, including preoffset sequence modulation and post-data processing, were evaluated to mitigate the false peaks in HT-IMS, and both methods can work effectively.

High Accuracy Ion Mobility Spectrometry for Instrument Calibration.

Ion mobility spectrometry (IMS) is widely used to characterize compounds of interest (COIs) based on their reduced mobility ( K0) values. In an attempt to increase the accuracy and agreement of studies, the most recommended method has been to use a reference compound with a known K0 value to calibrate the instrument and calculate COI K0 values from normalized spectra. Researchers are limited by the accuracy of previous K0 value reference measurements on which to base their calibrations. Any inaccuracy in these reference K0 values, typically ±2%, will propagate through to the calculated K0 value of the COI. For this reason, there is a need to standardize reference K0 values with improved accuracy. Through improvement of the accuracy of reference measurements, a lower degree of error will propagate through new K0 value calculations. The K0 values of the ammonium reactant ion, the potential reference standard dimethyl methylphosphonate (DMMP), and three explosive COIs were characterized at multiple drift gas temperatures, drift gas water contents, and electric field strengths on an accurate ion mobility spectrometry instrument. K0 values reported here are known to ±0.1% as a result of reducing the error of all instrumental parameters.

Determination of E/N Influence on K0 Values within the Low Field Region of Ion Mobility Spectrometry.

The established theory of ion motion within weak electric fields predicts that reduced ion mobility (K0) remains constant as a function of the ratio of electric field strength to drift gas number density (E/N). However, upon increasing the accuracy and precision of K0 value measurements during a previous study, a new relationship was seen in which the K0 values of ions decreased as a function of increasing E/N at field strengths below 4 Td. Here the effect of E/N on the K0 value of an ion has been investigated in order to validate the reality of the phenomenon and determine its cause. The pertinent measurements of voltage and drift time were verified in order to ensure the authenticity of the trend and that it was not a result of a systematic error in parametric measurements. The trend was also replicated on a separate ion mobility spectrometer drift tube in order to further validate its authenticity. As a result, the theory of ion motion within weak electric fields should be revised to reflect the behavior seen here.

Ambient Pressure Inverse Ion Mobility Spectrometry Coupled to Mass Spectrometry.

Although higher resolving powers are often achieved using ambient pressure drift tube ion mobility mass spectrometry (DT-IMMS) systems, lower duty cycles are often required which directly impacts sensitivity. Moreover, the mechanism of ion gating using Bradbury-Nielsen or Tyndall-Gate configurations routinely results in ion gate depletion effects which discriminate against low mobility ions. This paper reports a new method of ambient pressure ion mobility operation in which inverse ion mobility spectrometry is coupled to a time-of-flight mass spectrometer to improve sensitivity and minimize the effects of ion gate depletion. In this mode of operation, the duty cycle is improved to approximate 99% from a typical value of less than 1%, improving the signal intensity by over 2 orders of magnitude. Another advantage of inverse ion mobility mass spectrometry is a reduction of the impact of ion gate depletion on low mobility molecules that translates into higher sensitivity for this class of analytes. To demonstrate these benefits afforded by this instrumental mode of operation differences in sensitivity, resolving power, and ion discrimination are compared between the inverse and normal modes of operation using tetraalkylammonium standards. These results show that the ion throughput is significantly increased for analytes with a broad range of mobilities with little impact on resolving power. While the mobility-based discrimination is minimized using the inverse mode of operation, the noise level in the inverse mode is highly dependent upon the stability of ionization source.

Correcting the fundamental ion mobility equation for field effects.

The fundamental ion mobility equation computes the energy-averaged collision cross section as a function of measured drift velocity, electric field strength, ion and neutral masses, and drift gas state parameters. As field strength approaches zero, in particular when the drift velocity drops below about 4% of the average ion-neutral thermal speed, the fundamental equation takes on an especially simple form because the collision frequency and average momentum transfer become indistinguishable from their thermal values. However, in modern high-performance IMS-MS instruments, ion drift velocities may be 10-50% or more of thermal speed, and analysis using the zero-field equation gives rise to erroneously large cross sections. We address this problem by developing correction factors for the zero-field equation from an improved momentum-transfer (MT) theory for ion mobility, corrected and completed herein, and from the well-known two-temperature (2T) theory. The corrected and uncorrected equations are compared by their ability to recover known hard-sphere cross sections from accurately-computed mobility data. Both MT and 2T expressions adjust for the field-driven increase in collision frequency and are noticeably superior to zero-field expression whenever the ion drift velocity is greater than ∼4% of thermal speed. The MT expression also adjusts for the mass and field dependent change in average momentum transfer, and is more accurate than the 2T first approximation whenever the mass of the ionic species is greater than about four times that of drift gas molecules, as is the case in most analytical applications of IMS coupled to MS.

Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

E/N effects on K0 values revealed by high precision measurements under low field conditions.

Ion mobility spectrometry (IMS) is used to detect chemical warfare agents, explosives, and narcotics. While IMS has a low rate of false positives, their occurrence causes the loss of time and money as the alarm is verified. Because numerous variables affect the reduced mobility (K0) of an ion, wide detection windows are required in order to ensure a low false negative response rate. Wide detection windows, however, reduce response selectivity, and interferents with similar K0 values may be mistaken for targeted compounds and trigger a false positive alarm. Detection windows could be narrowed if reference K0 values were accurately known for specific instrumental conditions. Unfortunately, there is a lack of confidence in the literature values due to discrepancies in the reported K0 values and their lack of reported error. This creates the need for the accurate control and measurement of each variable affecting ion mobility, as well as for a central accurate IMS database for reference and calibration. A new ion mobility spectrometer has been built that reduces the error of measurements affecting K0 by an order of magnitude less than ±0.2%. Precise measurements of ±0.002 cm(2) V(-1) s(-1) or better have been produced and, as a result, an unexpected relationship between K0 and the electric field to number density ratio (E/N) has been discovered in which the K0 values of ions decreased as a function of E/N along a second degree polynomial trend line towards an apparent asymptote at approximately 4 Td.

Neuronal metabolomics by ion mobility mass spectrometry in cocaine self-administering rats after early and late withdrawal.

The neuronal metabolomes in rat striatum (STR), prefrontal cortex (PFC), and nucleus accumbens (NAC) were analyzed by Hadamard transform ion mobility mass spectrometry (HT-IMMS) in order to reveal global and specific metabolic changes induced by cocaine self-administration after 1-day or 3-week withdrawal. Metabolite features were comprehensively separated and detected using HPLC-IMMS within minutes. Global metabolic differences were observed by PCA for comparisons between cocaine and saline treatments at 1-day withdrawal time. Metabolite features that were significantly changed were selected using PCA loadings' plot and unpaired LLL test and then tentatively identified by accurate m/z, yielding a complete profile of metabolic changes induced by cocaine self-administration. The majority of these changes were found at the 1-day withdrawal time, but several of them endured even after 3-week withdrawal from cocaine, and these changes were generally brain region specific. Putatively identified metabolites associated with oxidative stress and energy metabolism were also specifically investigated. We discovered that the dysregulation of creatine/creatinine was different between the STR and NAC, demonstrating that metabolic alterations are brain region specific. Glutathione and adenosine were also changed in their abundance, and the results agreed with previous studies. In general, this study provided a high-throughput analytical platform to perform metabolomics analyses with putative identifications for altered metabolite features induced by cocaine treatment, therefore revealing additional metabolic targets of cocaine-induced changes after early and extended withdrawal times.

Multidimensional Separation of Natural Products Using Liquid Chromatography Coupled to Hadamard Transform Ion Mobility Mass Spectrometry.

A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC. Graphical Abstract ᅟ.

Characterizing metabolic changes in human colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer death worldwide, despite the fact that it is a curable disease when diagnosed early. The development of new screening methods to aid in early diagnosis or identify precursor lesions at risk for progressing to CRC will be vital to improving the survival rate of individuals predisposed to CRC. Metabolomics is an advancing area that has recently seen numerous applications to the field of cancer research. Altered metabolism has been studied for many years as a means to understand and characterize cancer. However, further work is required to establish standard procedures and improve our ability to identify distinct metabolomic profiles that can be used to diagnose CRC or predict disease progression. The present study demonstrates the use of direct infusion traveling wave ion mobility mass spectrometry to distinguish metabolic profiles from CRC samples and matched non-neoplastic epithelium as well as metastatic and primary tumors at different stages of disease (T1-T4). By directly infusing our samples, the analysis time was reduced significantly, thus increasing the speed and efficiency of this method compared to traditional metabolomics platforms. Partial least squares discriminant analysis was used to visualize differences between the metabolic profiles of sample types and to identify the specific m/z features that led to this differentiation. Identification of the distinct m/z features was made using the human metabolome database. We discovered alterations in fatty acid biosynthesis and oxidative, glycolytic, and polyamine pathways that distinguish tumors from non-malignant colonic epithelium as well as various stages of CRC. Although further studies are needed, our results indicate that colonic epithelial cells undergo metabolic reprogramming during their evolution to CRC, and the distinct metabolites could serve as diagnostic tools or potential targets in therapy or primary prevention. Graphical Abstract Colon tissue biopsy samples were collected from patients after which metabolites were extracted via sonication. Two-dimensional data were collected via IMS in tandem with MS (IMMS). Data were then interpreted statistically via PLS-DA. Scores plots provided a visualization of statistical separation and groupings of sample types. Loading plots allowed identification of influential ion features. Lists of these features were exported and analyzed for specific differences. Direct comparisons of the ion features led to the identification and comparative analyses of candidate biomarkers. These differences were then expressed visually in charts and tables.

HMGA1 drives metabolic reprogramming of intestinal epithelium during hyperproliferation, polyposis, and colorectal carcinogenesis.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

Rapid profiling and identification of anthocyanins in fruits with Hadamard transform ion mobility mass spectrometry.

The use of Hadamard transform ion mobility mass spectrometry (HT-IMMS) in the profiling of anthocyanins from different fruits is presented. Samples extracted with acidic methanol and purified with solid phase extraction were analyzed with direct IMMS infusion. The separation of various anthocyanins was achieved within 30s with resolving powers up to 110. The ion mobility drift times correlated with their mass-to-charge ratios with a correlation coefficient of 0.979 to produce a trend line that was characteristic for anthocyanins. Isomers with the same anthocyanidin but different hexoses were differentiated by ion mobility spectrometry. Furthermore, mobility separated ions underwent collision induced dissociation at the IMMS interface to provide MS/MS spectra. These fragmentation spectra aided in the identification of anthocyanidins via the loss of the saccharide groups. IMMS appears to be a rapid and efficient approach for profiling and identifying anthocyanins.

Determining the isomeric heterogeneity of neutral oligosaccharide-alditols of bovine submaxillary mucin using negative ion traveling wave ion mobility mass spectrometry.

Negative ions produced by electrospray ionization were used to evaluate the isomeric heterogeneity of neutral oligosaccharide-alditols isolated from bovine submaxillary mucin (BSM). The oligosaccharide-alditol mixture was preseparated on an off-line high-performance liquid chromatography (HPLC) column, and the structural homogeneity of individual LC fractions was investigated using a Synapt G2 traveling wave ion mobility spectrometer coupled between quadupole and time-of-flight mass spectrometers. Mixtures of isomers separated by both chromatography and ion mobility spectrometry were studied. Tandem mass spectrometry (MS/MS) of multiple mobility peaks having the same mass-to-charge ratio (m/z) demonstrated the presence of different structural isomers and not differences in ion conformations due to charge site location. Although the oligosaccharide-alditol mixture was originally separated by HPLC, multiple ion mobility peaks due to structural isomers were observed for a number of oligosaccharide-alditols from single LC fractions. The collision-induced dissociation cells located in front of and after the ion mobility separation device enabled oligosaccharide precursor or product ions to be separated by ion mobility and independent fragmentation spectra to be acquired for isomeric carbohydrate precursor or product ions. MS/MS spectra so obtained for independent mobility peaks at a single m/z demonstrated the presence of structural variants or stereochemical isomers having the same molecular formula. This was observed both for oligosaccharide precursor and product ions. In addition, mobilities of both [M - H](-) and [M + Cl](-) ions, formed by adding NH4OH or NH4Cl to the electrospray solvent, were examined and compared for selected oligosaccharide-alditols. Better separation among structural isomers appeared to be achieved for some [M + Cl](-) anions.

Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.

Desorption electrospray ionization (DESI) was coupled to an ambient pressure drift tube ion mobility time-of-flight mass spectrometer (IM-TOFMS) for the direct analysis of active ingredients in pharmaceutical samples. The DESI source was also coupled with a standalone IMS demonstrating potential of portable and inexpensive drug-quality testing platforms. The DESI-IMS required no sample pretreatment as ions were generated directly from tablets and cream formulations. The analysis of a range of over-the-counter and prescription tablet formations was demonstrated for amphetamine (methylphenidate), antidepressant (venlafaxine), barbiturate (Barbituric acid), depressant (alprazolam), narcotic (3-methylmorphine) and sympatholytic (propranolol) drugs. Active ingredients from soft and liquid formulations, such as Icy Hot cream (methyl salicylate) and Nyquil cold medicine (acetaminophen, dextromethorphan, doxylamine) were also detected. Increased sensitivity for selective drug responses was demonstrated through the formation of sodiated adduct ions by introducing small quantities of NaCl into the DESI solvent. Of the drugs and pharmaceuticals tested in this study, 68% (22 total samples) provided a clear ion mobility response at characteristic mobilities either as (M + H)(+), (M - H)(-), or (M + Na)(+) ions.

Metabolic analysis of striatal tissues from Parkinson's disease-like rats by electrospray ionization ion mobility mass spectrometry.

Electrospray ionization ion mobility mass spectrometry (ESI-IMMS) was used to study the striatal metabolomes in a Parkinson's like disease (PD-like) rat model. Striatal tissue samples from Berlin Druckrey IV (BD-IV) with PD-like disease 20 dpn-affected and 15 dpn-affected rats (dpn: days postnatal) were investigated and compared with age-matched controls. An ion mobility mass spectrometer (IMMS) produced multidimensional spectra with mass to charge ratio (m/z), ion mobility drift time, and intensity information for each individual metabolite. Principle component analysis (PCA) was applied in this study for pattern recognition and significant metabolites selection (68% data was modeled in PCA). Both IMMS spectra and PCA results showed that there were clear global metabolic differences between PD-like samples and healthy controls. Nine metabolites were selected by PCA and identified as potential biomarkers using the Human Metabolome Database (HMDB). One targeted metabolite in this study was dopamine. Selected-mass mobility analysis indicated the absence of dopamine in PD-like striatal metabolomes. A major discovery of this work, however, was the existence of an isomer of dopamine. By using ion mobility spectrometry, the dopamine isomer, which has not previously been reported, was separated from dopamine.

Evaluation of Hadamard transform atmospheric pressure ion mobility time-of-flight mass spectrometry for complex mixture analysis.

Ion mobility mass spectrometry (IMMS) has gained popularity in the analysis of complex mixtures such as those encountered in metabolomics and proteomics. However, the challenge that exists in conventional pulsed IMMS is its inherent low duty cycle. The first application of Hadamard transform (HT)-type signal coupled with atmospheric pressure IMMS to complex mixtures is presented. Performance of the prototype was assessed by the analysis of metabolite standard mixture. With 200 times increased IMS duty cycle in HT mode compared with conventional pulsed mode, the limit of detection (LOD) was decreased by ∼10 times. Evaluation for application to complex mixtures was achieved using the NIST Standard Reference Material 1950 Metabolites in Human Plasma. Approximately 180 metabolite ions were detected within 1 min with an IMS resolving power (Rp) of ∼100. Rapid chromatographic separation prior to IMMS analysis was also demonstrated for improving the response of metabolite ions in rat brain tissue extract.

Ion Mobility-Mass Correlation Trend Line Separation of Glycoprotein Digests without Deglycosylation.

A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electrospray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides. Peptides and glycosylated peptides with different charge states (up to 4 charges) were observed and fell on distinguishable trend lines in 2-D IMMS spectra in both positive and negative modes. The trend line separation patterns matched between both modes. Peptide sequence was identified based on the corresponding extracted mass spectra and collision induced dissociated (CID) experiments were performed for selected compounds to prove class identification. The signal-to-noise ratio of the glycopeptides was increased dramatically with ion mobility trend line separation compared to non-trend line separation, primarily due to selection of precursor ion subsets within specific mobility windows. In addition, isomeric mobility peaks were detected for specific glycopeptides. IMMS demonstrated unique capabilities and advantages for investigating and separating glycoprotein digests in this study and suggests a novel strategy for rapid glycoproteomics studies in the future.

Analysis of psychoactive cathinones and tryptamines by electrospray ionization atmospheric pressure ion mobility time-of-flight mass spectrometry.

The ability to use positive ion monitoring mode with an atmospheric pressure ion mobility time-of-flight mass spectrometer (APIM(tof)MS) to detect psychoactive cathinones and tryptamines from aqueous phase samples was evaluated. The study used a traditional electrospray ionization (ESI) source for sample introduction and ionization. A total of four cathinones (mephedrone, butylone, 4-Me-PPP, and 4-MEC) and five tryptamines (5-EtO-DPT, 5-EtO-DALT, 5-EtO-MIPT, 5-EtO-ALCHT, and 5-EtO-2MALET) were investigated, and we report on parent ions, collision induced dissociation (CID) fragment ions, reduced mobility (Ko), mass flight times, and detection limits obtained from a single instrument run for the psychoactive substances. Detection limits reported ranged from 3 to 11 µM concentration for the compounds studied. This detection limit range corresponded to 1-5 ng of material needed for improved detection on the instrument. This article demonstrates that it was possible to use a single instrument platform for the separation, detection, and identification of cathinones and tryptamines in less than 1 min. The application holds great promise for detecting and identifying a new class of drugs often referred to as "bath salts" or "legal highs" distributed over the Internet.

Metabolomics of colorectal cancer: past and current analytical platforms.

Metabolomics is coming of age as an important area of investigation which may help reveal answers to questions left unanswered or only partially understood from proteomic or genomic approaches. Increased knowledge of the relationship of genes and proteins to smaller biomolecules (metabolites) will advance our ability to diagnose, treat, and perhaps prevent cancer and other diseases that have eluded scientists for generations. Colorectal tumors are the second leading cause of cancer mortality in the USA, and the incidence is rising. Many patients present late, after the onset of symptoms, when the tumor has spread from the primary site. Once metastases have occurred, the prognosis is significantly worse. Understanding alterations in metabolic profiles that occur with tumor onset and progression could lead to better diagnostic tests as well as uncover new approaches to treat or even prevent colorectal cancer (CRC). In this review, we explore the various analytical technologies that have been applied in CRC metabolomics research and summarize all metabolites measured in CRC and integrate them into metabolic pathways. Early studies with nuclear magnetic resonance and gas-chromatographic mass spectrometry suggest that tumor cells are characterized by aerobic glycolysis, increased purine metabolism for DNA synthesis, and protein synthesis. Liquid chromatography, capillary electrophoresis, and ion mobility, each coupled with mass spectrometry, promise to advance the field and provide new insight into metabolic pathways used by cancer cells. Studies with improved technology are needed to identify better biomarkers and targets for treatment or prevention of CRC.