Synthesis and MS Analysis of Thiazolium and Pyridinium Derivatives of Peptide Nucleic Acids (PNAs) and Peptides

Abstract

Sensitivity of ESI-MS analysis of crude PNAs is enhanced using their pyridinium or thiazolium derivatives. Identification of the molecular ion of the product is easier when the label contains bromine, based on the isotope distribution. Study of side reactions, occurred upon the synthesis and/or cleavage, is simple with labelling. Sequencing of non-polar peptides is clear as only a_n type ions can be observed during their MS/MS analysis.     

Identification and quantification of N-linked glycoproteins using hydrazide chemistry,stable isotope labeling and mass spectrometry

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.     

Confirmatory analysis for drugs of abuse in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry with respect to criteria for compound identification

Recently, high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative confirmatory analysis of drugs of abuse and has begun to spread in the field of forensic toxicology. Guidelines for confirmatory analysis by GC/MS and LC/MS/MS have been published recently by several organizations (WADA, IOC, SOFT, GTFCh, EU). However, these guidelines have not yet been included in procedures for drug analysis with LC/MS/MS. The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to EU guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. LC/MS/MS methods for determination of several classes of drugs of abuse including some basic drugs (opiates, stimulants), cannabinoids and some of their phase-I- and phase-II-metabolites (especially glucuronides) in urine and serum of drug abusers and/or crime offenders or victims have been developed and validated following current recommendations and are presented in this paper. At least two MRM transitions for each substance were monitored to provide sufficient identification of drugs, deuterated analogues of analytes were used as internal standards for quantitation where possible and chromatographic separation has been performed on reversed-phase columns with gradient elution. Validation data obtained and the application to real samples show that the requested criteria for confirmatory analysis of drugs of abuse by EU guidelines can be fulfilled with a total number of four identification points by LC/MS/MS methods using a triple-quadrupole mass spectrometer. Furthermore, the methods are sufficiently sensitive to meet current requirements for confirmatory analysis of drugs of abuse in driving under the influence of drugs (DUID) cases established by the Society of Toxicological and Forensic Chemistry (GTFCh).     

Chi-square comparison of tryptic peptide-to-protein distributions of tandem mass spectrometry from blood with those of random expectation

Proteomics uses tandem mass spectrometers and correlation algorithms to match peptides and their fragment spectra to amino acid sequences. The replication of multiple liquid chromatography experiments with electrospray ionization of peptides and tandem mass spectrometry (LC–ESI–MS/MS) produces large sets of MS/MS spectra. There is a need to assess the quality of large sets of experimental results by statistical comparison with that of random expectation. Classical frequency-based statistics such as goodness-of-fit tests for peptide-to-protein distributions could be used to calculate the probability that an entire set of experimental results has arisen by random chance. The frequency distributions of authentic MS/MS spectra from human blood were compared with those of false positive MS/MS spectra generated by a computer, or instrument noise, using the chi-square test. Here the mechanics of the chi-square test to compare the results in toto from a set of LC–ESI–MS/MS experiments with those of random expectation is detailed. The chi-square analysis of authentic spectra demonstrates unambiguously that the analysis of blood proteins separated by partition chromatography prior to tryptic digestions has a low probability that the cumulative peptide-to-protein distribution is the same as that of random or noise false positive spectra.     

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.     

Fast atom bombardment and tandem mass spectrometry for structure determination of cysteine, N-acetylcysteine, and glutathione adducts of xenobiotics

Fast atom bombardment mass spectrometry (FAB/MS) and FAB combined with tandem mass spectrometry (MS/MS) were examined for their applicability to the structure determination of xenobiotics conjugated with the members of the glutathione family (glutathione, cysteine, and N-acetylcysteine). Comparisons between FAB/MS and thermospray MS data are made. FAB/MS is generally successful at generating molecular ion species under both positive and negative ion conditions. Upon collisional activation the adducts undergo characteristic cleavages around the sulfur providing structural information about the conjugate. The analysis of the N-acetylcysteine conjugate isolated from rat urine is presented as an example of the application of FAB/MS/MS to biological problems.     

Modern developments in mass spectrometry of chondroitin and dermatan sulfate glycosaminoglycans

Chondroitin sulfate (CS) and dermatan sulfate (DS) are special types of glycosaminoglycan (GAG) oligosaccharides able to regulate vital biological functions that depend on precise motifs of their constituent hexose sequences and the extent and location of their sulfation. As a result, the need for better understanding of CS/DS biological role called for the elaboration and application of straightforward strategies for their composition and structure elucidation. Due to its high sensitivity, reproducibility, and the possibility to rapidly generate data on fine CS/DS structure determinants, mass spectrometry (MS) based on either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) brought a major progress in the field. Here, modern developments in MS of CS/DS GAGs are gathered in a critical review covering the past 5 years. The first section is dedicated to protocols for CS/DS extraction from parent proteoglycan, digestion, and purification that are among critical prerequisites of a successful MS experiment. The second part highlights several MALDI MS aspects, the requirements, and applications of this ionization method to CS/DS investigation. An ample chapter is devoted to ESI MS strategies, which employ either capillary- or advanced chip-based sample infusion in combination with multistage MS (MS ⁿ ) using either collision-induced (CID) or electron detachment dissociation (EDD). At last, the potential of two versatile separation techniques, capillary electrophoresis (CE), and liquid chromatography (LC) in off- and/or on-line coupling with ESI MS and MS ⁿ , is discussed, alongside an assessment of particular buffer/solvent conditions and instrumental parameters required for CS/DS mixture separation followed by on-line mass analysis of individual components.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Image analysis tools and emerging algorithms for expression proteomics

Since their origins in academic endeavours in the 1970s, computational analysis tools have matured into a number of established commercial packages that underpin research in expression proteomics. In this paper we describe the image analysis pipeline for the established 2-DE technique of protein separation, and by first covering signal analysis for MS, we also explain the current image analysis workflow for the emerging high-throughput 'shotgun' proteomics platform of LC coupled to MS (LC/MS). The bioinformatics challenges for both methods are illustrated and compared, whereas existing commercial and academic packages and their workflows are described from both a user's and a technical perspective. Attention is given to the importance of sound statistical treatment of the resultant quantifications in the search for differential expression. Despite wide availability of proteomics software, a number of challenges have yet to be overcome regarding algorithm accuracy, objectivity and automation, generally due to deterministic spot-centric approaches that discard information early in the pipeline, propagating errors. We review recent advances in signal and image analysis algorithms in 2-DE, MS, LC/MS and Imaging MS. Particular attention is given to wavelet techniques, automated image-based alignment and differential analysis in 2-DE, Bayesian peak mixture models, and functional mixed modelling in MS, and group-wise consensus alignment methods for LC/MS.     

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.     

Identification and validation of eukaryotic aspartate and glutamate methylation in proteins

Methylation of lysine and arginine is known to be critical in cellular processes. However, methylation of other amino acidic residues has been largely overlooked. Here, we report a systematic screening for methylation of side chains of aspartate and glutamate (D/E-methylation), involving exhaustive nano-HPLC/MS/MS, a protein sequence database search, and manual verification. The putative D/E-methylated peptides were confirmed by MS/MS of synthetic peptides. Our analysis identified several D/E-methylation substrate proteins and their modification sites in human and yeast cells. To our knowledge, this is the first report conclusively identifying in vivo D/E-methylation substrates and their modification sites in eukaryotic cells, demonstrating that D/E-methylations are abundant protein modifications. The substrate proteins identified here provide a stepping stone for future biochemical characterization of protein methylation pathways.     

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Algorithms and tools for analysis and management of mass spectrometry data

Mass spectrometry (MS) is a technique that is used for biological studies. It consists in associating a spectrum to a biological sample. A spectrum consists of couples of values (intensity, m/z), where intensity measures the abundance of biomolecules (as proteins) with a mass-to-charge ratio (m/z) present in the originating sample. In proteomics experiments, MS spectra are used to identify pattern expressions in clinical samples that may be responsible of diseases. Recently, to improve the identification of peptides/proteins related to patterns, MS/MS process is used, consisting in performing cascade of mass spectrometric analysis on selected peaks. Latter technique has been demonstrated to improve the identification and quantification of proteins/peptide in samples. Nevertheless, MS analysis deals with a huge amount of data, often affected by noises, thus requiring automatic data management systems. Tools have been developed and most of the time furnished with the instruments allowing: (i) spectra analysis and visualization, (ii) pattern recognition, (iii) protein databases querying, (iv) peptides/proteins quantification and identification. Currently most of the tools supporting such phases need to be optimized to improve the protein (and their functionalities) identification processes. In this article we survey on applications supporting spectrometrists and biologists in obtaining information from biological samples, analyzing available software for different phases. We consider different mass spectrometry techniques, and thus different requirements. We focus on tools for (i) data preprocessing, allowing to prepare results obtained from spectrometers to be analyzed; (ii) spectra analysis, representation and mining, aimed to identify common and/or hidden patterns in spectra sets or in classifying data; (iii) databases querying to identify peptides; and (iv) improving and boosting the identification and quantification of selected peaks. We trace some open problems and report on requirements that represent new challenges for bioinformatics.     

Extraction methods and chemical standardization of botanicals and herbal preparations

Botanicals and herbal preparations are medicinal preparations, containing a single or two or more medicinal plants. The focus of this review paper is on the analytical methodologies, which included the combination of sample preparation tools and chromatographic techniques for the chemical standardization of marker compounds or active ingredients in botanicals and herbal preparations. The common problems and key challenges in the chemical standardization of botanicals and herbal preparations were discussed. As sample preparation is the most important step in the development of analytical methods for the analysis of constituents present in botanicals and herbal preparations, the strength and weakness of different extraction techniques are discussed. For the analysis of compounds present in the plant extracts, the applications of common chromatographic techniques, such as HPLC, CE, HRGC/MS, HPLC/MS and HPLC/MS/MS are discussed. The strength, weakness and applicability of various separation tools are stated. Procedures for the identification of marker or active compounds in plant extracts, using HPLC/MS, were proposed. Finally, the effects of batch-to-batch variation of the medicinal plants are investigated and discussed.     

A platform for accurate mass and time analyses of mass spectrometry data

We describe an integrated suite of algorithms and software for general accurate mass and time (AMT) tagging data analysis of mass spectrometry data. The AMT approach combines identifications from liquid chromatography (LC) tandem mass spectrometry (MS/MS) data with peptide accurate mass and retention time locations from high-resolution LC-MS data. Our workflow includes the traditional AMT approach, in which MS/MS identifications are located in external databases, as well as methods based on more recent hybrid instruments such as the LTQ-FT or Orbitrap, where MS/MS identifications are embedded with the MS data. We demonstrate our AMT workflow's utility for general data synthesis by combining data from two dissimilar biospecimens. Specifically, we demonstrate its use relevant to serum biomarker discovery by identifying which peptides sequenced by MS/MS analysis of tumor tissue may also be present in the plasma of tumor-bearing and control mice. The analysis workflow, referred to as msInspect/AMT, extends and combines existing open-source platforms for LC-MS/MS (CPAS) and LC-MS (msInspect) data analysis and is available in an unrestricted open-source distribution.     

Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides

Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/M Sanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MS schemes. In the present experimental outcome, additional LC-MS/MS analysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MS based identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.     

Genetic epidemiology of cardiometabolic risk factors and their clustering patterns in Mexican American children and adolescents: the SAFARI Study

Pediatric metabolic syndrome (MS) and its cardiometabolic components (MSCs) have become increasingly prevalent, yet little is known about the genetics underlying MS risk in children. We examined the prevalence and genetics of MS-related traits among 670 non-diabetic Mexican American (MA) children and adolescents, aged 6–17 years (49 % female), who were participants in the San Antonio Family Assessment of Metabolic Risk Indicators in Youth study. These children are offspring or biological relatives of adult participants from three well-established Mexican American family studies in San Antonio, TX, at increased risk of type 2 diabetes. MS was defined as ≥3 abnormalities among 6 MSC measures: waist circumference, systolic and/or diastolic blood pressure, fasting insulin, triglycerides, HDL-cholesterol, and fasting and/or 2-h OGTT glucose. Genetic analyses of MS, number of MSCs (MSC-N), MS factors, and bivariate MS traits were performed. Overweight/obesity (53 %), pre-diabetes (13 %), acanthosis nigricans (33 %), and MS (19 %) were strikingly prevalent, as were MS components, including abdominal adiposity (32 %) and low HDL-cholesterol (32 %). Factor analysis of MS traits yielded three constructs: adipo-insulin-lipid, blood pressure, and glucose factors, and their factor scores were highly heritable. MS itself exhibited 68 % heritability. MSC-N showed strong positive genetic correlations with obesity, insulin resistance, inflammation, and acanthosis nigricans, and negative genetic correlation with physical fitness. MS trait pairs exhibited strong genetic and/or environmental correlations. These findings highlight the complex genetic architecture of MS/MSCs in MA children, and underscore the need for early screening and intervention to prevent chronic sequelae in this vulnerable pediatric population.