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.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI–IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI–IMS. This is achieved by combining information from IMS and shotgun proteomics (LC–MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC–MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.     

A simple solid phase mass tagging approach for quantitative proteomics

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.     

Direct profiling and identification of peptide expression differences in the pancreas of control and ob/ob mice by imaging mass spectrometry

Imaging mass spectrometry (IMS) technology utilizes MALDI MS to map molecules of interest in thin tissue sections. In this study, we have evaluated the potential of MALDI IMS to study peptide expression patterns in the mouse pancreas under normal and pathological conditions, and to in situ identify peptides of interest using MS/MS. Different regions of the pancreas of both control and ob/ob mice were imaged, resulting in peptide-specific profiles. The distribution of ions of m/z 3120 and 3439 displayed a striking resemblance with Langerhans islet's histology and, following MS/MS fragmentation and database searching were identified as C-peptide of insulin and glicentin-related polypeptide, respectively. In addition, a significant increase of the 3120 peak intensity in the obese mice was observed. This study underscores the potential of MALDI IMS to study the contribution of peptides to pancreas pathology.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.     

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples² (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity². Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson''s disease (PD). PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age^3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA⁵. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)^1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA⁷. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control^7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson''s disease and L-DOPA induced dyskinesia. MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin''s opioid-receptor binding capacity⁹. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.     

Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

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.     

Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Identification of Monosialylated N-glycoforms in the CDG Urinome by Ion Mobility Tandem Mass Spectrometry: The Potential for Clinical Applications

Introduction

A novel approach of ion mobility tandem mass spectrometry (IMS-MS/MS) is applied to analysis of human glycourinome to obtain carbohydrate pattern data of congenital disorders of glycosylation patient. Overlapping of the complex carbohydrate mass range landscape has been highly reduced upon IMS-MS procedure, allowing more efficient identification by mapping and sequencing of glycan precursor ions, following their separation by mobility, according to difference in drift time through the traveling wave IMS cell. Intact and truncated N- and O-glycan structures modified by sialylation and fucosylation were identified according to their drift time separated molecular ions and submitted to fragmentation in a narrow mass window.

IMS CID MS/MS Analysis

The fragmentation spectra generated from the IMS separated precursor ions contain series of fragment ions maintaining the same mobility as their parent ions, and the assignment accuracy can be significantly enhanced.

Conclusion

According to the specific fragment ion patterns, carbohydrate epitopes described to be involved in pathological processes were assigned. A high potential of this glycomics-based strategy for clinical applications can be presented.     

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.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging

Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotope-coded glycosylation site-specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column-mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, 18O18O, specifically at the N-glycosylation site; and (4) identification of 18O-tagged peptides by liquid chromatography-coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.