Developing liquid chromatography ion mobility mass spectometry techniques

When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will separate according to differences in their mobilities through the gas. This separation forms the basis of the analytical method known as ion mobility spectroscopy and is highly efficient, in that it can be carried out in a very short time frame (micro- to milliseconds). Recently, efforts have been made to couple the approach with liquid-phase separations and mass spectrometry in order to create a high-throughput and high-coverage approach for analyzing complex mixtures. This article reviews recent work to develop this approach for proteomics analyses. The instrumentation is described briefly. Several multidimensional data sets obtained upon analyzing complex mixtures are shown in order to illustrate the approach as well as provide a view of the limitations and required future work.     

Development of high throughput dispersive LC-ion mobility-TOFMS techniques for analysing the human plasma proteome.

A technique that combines ion mobility spectrometry (IMS) with reversed-phase liquid chromatography (LC), collision-induced dissociation (CID) and mass spectrometry (MS) has been developed. The approach is described as a high throughput means of analysing complex mixtures of peptides that arise from enzymatic digestion of protein mixtures. In this approach, peptides are separated by LC and, as they elute from the column, they are introduced into the gas phase and ionised by electrospray ionisation. The beam of ions is accumulated in an ion trap and then the concentrated ion packet is injected into a drift tube where the ions are separated again in the gas phase by IMS, a technique that differentiates ions based on their mobilities through a buffer gas. As ions exit the drift tube, they can be subjected to collisional activation to produce fragments prior to being introduced into a mass spectrometer for detection. The IMS separation can be carried out in only a few milliseconds and offers a number of advantages compared with LC-MS alone. An example of a single 21-minute LC-IMS-(CID)-MS analysis of the human plasma proteome reveals approximately 20,000 parent ions and approximately 600,000 fragment ions and evidence for 227 unique protein assignments.     

Application and evaluation of limited mass monitoring with a gas chromatograph mass spectrometer computer system

The technique of scanning a preselected set of ions employing a combined gas chromatography mass spectrometer computer system has been investigated to ascertain the advantages and disadvantages of such a procedure. This technique allows one to determine gas chromatographic retention data with with a high degree of precision and accuracy, in rapid temperature programming operation, due to shortening of the mas spectral scanning interval. Signal-to-noise ratio in ion abundance recordings can be enhanced by increasing the dwell time for as many as 100 ions without lenghtening the scanning interval. The utility of such an approach was demonstrated by analysis of complex mixtures isolated form human urine and cerebrospinal fluid.     

Development of field modulation in a split-field drift tube for high-throughput multidimensional separations

A field modulation approach for high-throughput ion mobility/time-of-flight analyses of complex mixtures has been developed using a split-field drift tube. In this approach, complex mixtures of peptides, such as those that arise from tryptic digestion of protein mixtures, are separated by nanocolumn liquid chromatography, ionized by electrospray ionization, and analyzed by ion mobility/time-of-flight techniques. The split-field drift tube allows parent ions to be separated based on differences in their low-field mobilities through the first-field region before entering the second region. For increased throughput, the magnitude of the field in the second region can be modulated throughout an LC separation in order to favor transmission of different types of ions: parent ions at low fields; fragments from primarily [M+3H]3+ peptides at moderate fields; or, fragmentation of [M+3H]3+ and [M+2H]2+ species at higher fields. We demonstrate the approach with two examples: a mixture of tryptic peptides from digestion of hemoglobin; and a complex mixture of tryptic peptides from digestion of human plasma.     

Development of high-throughput liquid chromatography injected ion mobility quadrupole time-of-flight techniques for analysis of complex peptide mixtures

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.     

Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantitation of tBDMS derivatives using ammonia positive chemical ionization

The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.     

Toward plasma proteome profiling with ion mobility-mass spectrometry

Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMS-MS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.     

Ion/ion chemistry as a top-down approach for protein analysis

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins.     

Generating consensus sequences from partial order multiple sequence alignment graphs

MOTIVATION: Consensus sequence generation is important in many kinds of sequence analysis ranging from sequence assembly to profile-based iterative search methods. However, how can a consensus be constructed when its inherent assumption-that the aligned sequences form a single linear consensus-is not true? RESULTS: Partial Order Alignment (POA) enables construction and analysis of multiple sequence alignments as directed acyclic graphs containing complex branching structure. Here we present a dynamic programming algorithm (heaviest_bundle) for generating multiple consensus sequences from such complex alignments. The number and relationships of these consensus sequences reveals the degree of structural complexity of the source alignment. This is a powerful and general approach for analyzing and visualizing complex alignment structures, and can be applied to any alignment. We illustrate its value for analyzing expressed sequence alignments to detect alternative splicing, reconstruct full length mRNA isoform sequences from EST fragments, and separate paralog mixtures that can cause incorrect SNP predictions. AVAILABILITY: The heaviest_bundle source code is available at http://www.bioinformatics.ucla.edu/poa     

Design and analysis of mixture systems: Applications in hydroponic,plant nutrition research

This study demonstrates that nutrient solutions can be defined as mixture systems. A general methodology for design and analysis of mixture optimization experiments is developed. The emphasis is centered on multivariate investigation of the zone of optimal solution properties as a function of the ion composition and the total ionic strength of the solution. The study of the effects of ion interaction on well-defined solution properties is also possible by this multivariate approach. This work is a valuable tool in mineral nutritional research, because for the first time the chemical feasibility conditions of such solution, combined with additional chemical, physiological or economical constraints, form the foundation of the statistical experimental design theory, which makes the optimization of complex mixtures of ions in relation to well-defined response variables possible.     

USE OF CHEMICAL SENSOR ARRAYS FOR FOOD AND FRAGRANCE ANALYSIS

Many workers have recently developed and demonstrated the use of gas sensor arrays to detect and recognize a variety of compounds. In general, a pattern recognition approach is used in which the signature of an unknown sample is compared to a library of signatures of known substances. All of these efforts have shown varying degrees of success when applied to pure compounds. Unfortunately, flavors and fragrances are never pure compounds. A prototype gas sensor array has been constructed and tested with various mixtures of interest. The results are discussed in terms of their implication to the food and fragrance industry. Early experiments on gasoline-alcohol mixtures demonstrated that the signatures do not follow a rule of mixing, but were more strongly influenced by the more volatile and/or more reactive component. This led us to examine the behavior of alcohol-water mixtures for possible quality control in the distilling industry. We discovered that the signature of alcohol dominated even at concentrations as low as ten percent. Preliminary experiments were also performed to see if the prototype sensor array could be used to automatically detect rancidity in dairy products. Flavorings and essential oils are in many cases even more complex mixtures. The behavior of representative classes of these substances were characterized in an attempt to understand their signatures on the basis of their constituents.     

Enhancement of structural information in FAB ionized carbohydrate samples by neutral gas collision

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.     

Biocatalytic carbon capture via reversible reaction cycle catalyzed by isocitrate dehydrogenase

The practice of carbon capture and storage (CCS) requires efficient capture and separation of carbon dioxide from its gaseous mixtures such as flue gas, followed by releasing it as a pure gas which can be subsequently compressed and injected into underground storage sites. This has been mostly achieved via reversible thermochemical reactions which are generally energy-intensive. The current work examines a biocatalytic approach for carbon capture using an NADP(H)-dependent isocitrate dehydrogenase (ICDH) which catalyzes reversibly carboxylation and decarboxylation reactions. Different from chemical carbon capture processes that rely on thermal energy to realize purification of carbon dioxide, the biocatalytic strategy utilizes pH to leverage the reaction equilibrium, thereby realizing energy-efficient carbon capture under ambient conditions. Results showed that over 25 mol of carbon dioxide could be captured and purified from its gas mixture for each gram of ICDH applied for each carboxylation/decarboxylation reaction cycle by varying pH between 6 and 9. This work demonstrates the promising potentials of pH-sensitive biocatalysis as a green-chemistry route for carbon capture.     

Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.     

Hydrocracking of vacuum gas oil-vegetable oil mixtures for biofuels production

Hydrocracking of vacuum gas oil (VGO) - vegetable oil mixtures is a prominent process for the production of biofuels. In this work both pre-hydrotreated and non-hydrotreated VGO are assessed whether they are suitable fossil components in a VGO-vegetable oil mixture as feed-stocks to a hydrocracking process. This assessment indicates the necessity of a VGO pre-hydrotreated step prior to hydrocracking the VGO-vegetable oil mixture. Moreover, the comparison of two different mixing ratios suggests that higher vegetable oil content favors hydrocracking product yields and qualities. Three commercial catalysts of different activity are utilized in order to identify a range of products that can be produced via a hydrocracking route. Finally, the effect of temperature on hydrocracking VGO-vegetable oil mixtures is studied in terms of conversion and selectivity to diesel, jet/kerosene and naphtha.     

Nanospray FAIMS fractionation provides significant increases in proteome coverage of unfractionated complex protein digests

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments.     

Joint Modeling of Multiple Social Networks to Elucidate Primate Social Dynamics: I. Maximum Entropy Principle and Network-Based Interactions

In a complex behavioral system, such as an animal society, the dynamics of the system as a whole represent the synergistic interaction among multiple aspects of the society. We constructed multiple single-behavior social networks for the purpose of approximating from multiple aspects a single complex behavioral system of interest: rhesus macaque society. Instead of analyzing these networks individually, we describe a new method for jointly analyzing them in order to gain comprehensive understanding about the system dynamics as a whole. This method of jointly modeling multiple networks becomes valuable analytical tool for studying the complex nature of the interaction among multiple aspects of any system. Here we develop a bottom-up, iterative modeling approach based upon the maximum entropy principle. This principle is applied to a multi-dimensional link-based distributional framework, which is derived by jointly transforming the multiple directed behavioral social network data, for extracting patterns of synergistic inter-behavioral relationships. Using a rhesus macaque group as a model system, we jointly modeled and analyzed four different social behavioral networks at two different time points (one stable and one unstable) from a rhesus macaque group housed at the California National Primate Research Center (CNPRC). We report and discuss the inter-behavioral dynamics uncovered by our joint modeling approach with respect to social stability.