MALDI mass spectrometry in prostate cancer biomarker discovery

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

Analysis of derivatised steroids by matrix-assisted laser desorption/ionisation and post-source decay mass spectrometry

Neutral steroids are difficult to analyse using desorption ionisation methods coupled with mass spectrometry (MS). However, steroids with an unhindered ketone group can readily be derivatised with the Girard P (GP) reagent to give GP hydrazones. Steroid GP hydrazones contain a quaternary nitrogen atom and are readily desorbed in the matrix-assisted laser desorption/ionisation (MALDI) process, giving an improvement in sensitivity of two orders of magnitude. Steroids without a ketone group, but with a 3beta-hydroxy-Delta5 function, can be readily converted to 3-oxo-Delta4 steroids and subsequently derivatised to GP hydrazones for MALDI analysis. In addition to giving strong [M]+ ions upon MALDI, steroid GP hydrazones give informative post-source decay (PSD) spectra. By using the accurate mass of the precursor-ion measured by MALDI-MS, in combination with the structural information encoded in its PSD spectrum, steroid structures can readily be determined.     

Advances in ionisation techniques for mass spectrometry-based omics research

Omics analysis by mass spectrometry (MS) is a vast field, with proteomics, metabolomics and lipidomics dominating recent research by exploiting biological MS ionisation techniques. Traditional MS ionisation techniques such as electrospray ionisation have limitations in analyte-specific sensitivity, modes of sampling and throughput, leading to many researchers investigating new ionisation methods for omics research. In this review, we examine the current landscape of these new ionisation techniques, divided into the three groups of (electro)spray-based, laser-based and other miscellaneous ionisation techniques. Due to the wide range of new developments, this review can only provide a starting point for further reading on each ionisation technique, as each have unique benefits, often for specialised applications, which promise beneficial results for different areas in the omics world.     

FAB, ESI and MALDI Mass Spectrometric methods in the study of metallo-drugs and their biomolecular interactions

This review surveys the soft ionisation mass spectrometric methods that are most commonly used for the investigation of the mechanism of interaction between metallo-drugs and biomolecules. In the first part of the review, an overview of the applications of transition metal complexes in the therapy of various diseases (arthritis, cancer, diabetes) is given, whereas the second part focuses on the obtained results dealing with various aspects of the interaction between metal complexes and different types of biomolecules. Possibilities and limitations of each mass spectrometric method-namely, fast atom bombardment (FAB), electrospray ionisation (ESI) and matrix-assisted laser desorption and ionisation (MALDI)--are discussed in the third part of this review along with the examples of their application for the analysis of metal complexes, as well as of the products of their interaction with biomolecules.     

Characterisation of castor oil by on-line and off-line non-aqueous reverse-phase high-performance liquid chromatography-mass spectrometry (APCI and UV/MALDI)

A non-aqueous reverse-phase HPLC method, based on two columns in series, has been used to separate the major triacylglycerols (TAGs) from commercial castor oil and to perform either on-line negative ion atmospheric pressure chemical ionisation (APCI), or off-line positive ion matrix-assisted laser desorption ionisation (MALDI)/MS. The resulting Mass Spectra showed chloride-attached TAG molecules [M + CI]- in the case of negative-ion APCI, and sodium-attached TAG molecules [M + Na]+ in the case of positive-ion MALDI. For MALDI time-of-flight (TOF)/MS, a liquid binary matrix system consisting of sodium ferrocyanide and glycerol was applied, resulting in excellent TAG sensitivity, which was necessary for the determination of trace amounts of TAGs in castor oil. Both techniques allowed unambiguous molecular mass determination of the intact TAG molecules with no thermal degradation. Furthermore, seamless post source decay (PSD) fragment ion analysis by means of a curved field reflector TOF mass spectrometer allowed the determination of the fatty acid composition of each individual TAG. Castor oil contained eight different TAGs which were successfully determined by both APCI and MALDI techniques. In each TAG, at least two units of 12-hydroxy-9-octadecenoic acid (ricinoleic acid) were present. The following fatty acids were determined by seamless PSD fragment ion analysis and APCI/MALDI molecular mass determination as TAG substructures: ricinoleic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, dihydroxy stearic acid and eicosenoic acid. Triricinolein was the dominating TAG.     

Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis

Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.     

Glycoproteomics based on tandem mass spectrometry of glycopeptides

Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.     

Mass spectrometric techniques for characterisation of platinum–humic substance complexes in soil and street dust samples

Abstract

Increased platinum concentrations have been detected in environmental samples since the introduction of catalytic converters in Europe. It was previously believed that PGE are relatively inert and that the soluble fraction of automobile catalyst emitted PGE is only a few percent. However, new studies suggested that metallic Pt is oxidised in the soil and that the majority of the Pt species could be humic substance complexes.

The aim of the present paper is the characterisation of platinum-humic acid complexes in spiked soil and street dust samples. A number of different soft ionisation mass spectrometric techniques, such as ESI-Q-MS, ESI-ION TRAP-MS and MALDI-TOF-MS have been tested. The ESI mass spectra of the soil humic acids were characterised by high complexity and irreproducibility.

MALDI-TOF-MS was used for humic acid complexes characterisation in positive and negative modes. Positive ionisation was less effective and produced more simple and less informative spectra than negative ionisation. Two different MALDI matrices (CHCA, DHBA) were tested for use with humic substances. DHBA yielded better results, exhibiting superior ionisation efficiency, low spectral background and the matrix peaks were almost completely suppressed causing no interferences with identified analyte peaks. The application of MALDI-TOF-MS shows its suitability for humic substance characterisation. The comparison of the MALDI-spectra of humic acids, extracted from spiked and native soil proved that Pt undergoes transformation in the environment to humic acid complexes.     

Oxidative changes of lipids monitored by MALDI MS

Oxidation processes of lipids are of paramount interest from many viewpoints. For instance, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by oxidation of lipids or free fatty acids. Additionally, many inflammatory diseases are accompanied by lipid oxidation and, therefore, oxidation products are also useful disease (bio)markers. Thus, there is also considerable interest in methods of (oxidized) lipid analysis.Nowadays, soft ionization mass spectrometric (MS) methods are regularly used to study oxidative lipid modifications due to their high sensitivities and the extreme mass resolution. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are increasing. This review aims to summarize the so far available data on MALDI analyses of oxidized lipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids under in vivo conditions, particularly the oxidation of (human) lipoproteins. It is not the aim of this review to praise MALDI as the “best” method but to provide a critical survey of the advantages and drawbacks of this method.     

Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry

Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein-RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide-RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species. We demonstrate the feasibility of this strategy by MS analysis of enriched peptide-RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Sample preparation issues for tissue imaging by imaging MS

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.     

Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.     

Selective analysis of phenolic compounds in propolis by HPLC-MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy. Copyright (c) 2007 John Wiley & Sons, Ltd.