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.     

BLI-MS: Combining biolayer interferometry and mass spectrometry

Biolayer interferometry (BLI) is a technology which allows to study the affinity between two interacting macro-molecules and to visualize their kinetic of interaction in real time. In this work, we combine BLI interaction measurement with mass spectrometry in order to identify the proteins interacting with the bait. We provide for the first time the proof of concept of the feasibility of BLI-MS in complex biological mixtures.     

Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410)

A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.     

Direct Analysis of Protein Mixtures by Tandem Mass Spectrometry

Methods to identify proteins contained in mixtures are described. The approach uses microcolumn liquid chromatography and automated tandem mass spectrometry in conjunction with protein and nucleotide database searching algorithms. This approach is applied to the identification of proteins obtained by immunoprecipitation reactions, interaction with a GST protein fusion products and interaction with a macromolecular complex.     

The use of a hybrid linear trap/FT-ICR mass spectrometer for on-line high resolution/high mass accuracy bottom-up sequencing.

In this work we present a hybrid linear trap/Fourier transform ion cyclotron resonance (ICR) mass spectrometer to perform protein sequencing using the bottom-up approach. We demonstrate that incorporation of the linear trap greatly enhances the overall performance of the hybrid system for the study of complex peptide mixtures separated by fast high-performance liquid chromatography gradients. The ability to detect in the linear trap enables employment of automatic gain control to greatly reduce space charging in the ICR cell irregardless of ion flux. Resulting accurate mass measurements of 2 ppm or better using external calibration are achieved for the base peak as well as ions at 2% relative abundance. The linear trap is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. The increased duty cycle allows for data-dependent mass analysis of coeluting peptides to be acquired increasing protein sequence coverage without increasing the gradient length. In addition, the linear trap could be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests. Comparisons of protein sequence coverage are presented for product ion detection in the linear trap and ICR cell.     

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.     

Comparative measurements of multicomponent phospholipid mixtures by electrospray mass spectroscopy: relating ion intensity to concentration

Electrospray mass spectrometry allows direct identification and sensitive detection of multiple phospholipids in non-derivatized cell extracts. However, quantitative analyses are not straightforward, and are confounded by analyte and mass discrimination effects, and non-linear dependence of the ion intensity on concentration. This non-linearity is particularly severe in the negative mode and precludes even comparative measurements of anion concentrations. Herein, we report a general method for relating negative electrospray ion intensity to concentration when analyzing multicomponent phospholipid samples. In this method, the intensity of individual ions is measured at several different concentrations of the total mixture and the slope (n(E)) of the double log plot of sample concentration vs. intensity for each analyte is determined. The n(E) is then used to map intensity data to a quantity proportional to concentration for each analyte. The method allows facile and accurate comparison of negative spectra of complex mixtures containing structurally different anions.     

Enrichment of N-terminal cysteinyl-peptides by covalent capture

The detection of low abundance proteins in complex biological samples is still a challenge in proteomics. To circumvent this obstacle a number of strategies involving the targeting of subsets of proteins or peptides were developed.The following work describes a new approach to simplify peptide mixtures by enrichment of N-terminal cysteinyl peptides (and to some extent N-terminal threonine peptides). The strategy is based on the use of an isolation method, so-called covalent capture (CC), which relies on the formation of a covalent bond between an N-terminal free cysteine or N-terminal free threonine and an aldehyde fixed on a solid support. The CC is highly selective. It permits extensive washes of the resin for the elimination of non-specific moieties before the release of the captured peptides. The application of the CC to proteomics was evaluated on tryptic peptides of standard proteins and test protein mixtures. The procedure demonstrated a significant reduction in sample complexity, while allowing the identification of N-terminal cysteinyl peptides hidden in the non-fractionated samples.This new strategy provides an efficient tool to existing proteomics approaches to reduce sample complexity and potentially identify less abundance proteins.     

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.     

Mass spectrometric approaches for characterizing bacterial proteomes

The emergence of advanced liquid chromatography mass spectrometry technologies for characterizing very complex mixtures of proteins has greatly propelled the field of proteomics, the goal of which is the simultaneous examination of all the proteins expressed by an organism. This research area represents a paradigm shift in molecular biology by attempting to provide a top-down qualitative and quantitative view of all the proteins (including their modifications and interactions) that are essential for an organism’s life cycle, rather than targeting a particular protein family. This level of global protein information about an organism such as a bacterium can be combined with genomic and metabolomic data to enable a systems biology approach for understanding how these organisms live and function.     

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.     

Mass spectrometry technologies for proteomics.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.