A well-characterised peak identification list of MALDI MS profile peaks for human blood serum

MALDI MS profiling, using easily available body fluids such as blood serum, has attracted considerable interest for its potential in clinical applications. Despite the numerous reports on MALDI MS profiling of human serum, there is only scarce information on the identity of the species making up these profiles, particularly in the mass range of larger peptides. Here, we provide a list of more than 90 entries of MALDI MS profile peak identities up to 10 kDa obtained from human blood serum. Various modifications such as phosphorylation were detected among the peptide identifications. The overlap with the few other MALDI MS peak lists published so far was found to be limited and hence our list significantly extends the number of identified peaks commonly found in MALDI MS profiling of human blood serum

Development of a MALDI MS-based platform for early detection of acute kidney injury

Purpose: Septic acute kidney injury (AKI) is associated with poor outcome. This can partly be attributed to delayed diagnosis and incomplete understanding of the underlying pathophysiology. Our aim was to develop an early predictive test for AKI based on the analysis of urinary peptide biomarkers by MALDI-MS. Experimental design: Urine samples from 95 patients with sepsis were analyzed by MALDI-MS. Marker search and multimarker model establishment were performed using the peptide profiles from 17 patients with existing or within the next 5 days developing AKI and 17 with no change in renal function. Replicates of urine sample pools from the AKI and non-AKI patient groups and normal controls were also included to select the analytically most robust AKI markers. Results: Thirty-nine urinary peptides were selected by cross-validated variable selection to generate a support vector machine multidimensional AKI classifier. Prognostic performance of the AKI classifier on an independent validation set including the remaining 61 patients of the study population (17 controls and 44 cases) was good with an area under the receiver operating characteristics curve of 0.82 and a sensitivity and specificity of 86% and 76%, respectively. Conclusion and clinical relevance: A urinary peptide marker model detects onset of AKI with acceptable accuracy in septic patients. Such a platform can eventually be transferred to the clinic as fast MALDI-MS test format

Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

Advancing liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry toward ultra-high-throughput analysis

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labelling, which in principle can also be used in combination with liquid AP-MALDI MS

Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase

We thank EPSRC and the Scottish Imaging Network (SINAPSE) for grants. DO’H thanks the Royal Society for a Wolfson Research Merit Award and ST is grateful to the John and Kathleen Watson Scholarship for financial support. We are grateful to Dr Catherine Botting and Dr Sally Shirran of the St Andrews Mass Spectrometry Service for MALDI-MS acquisitions. We also thank Dr Sally Pimlott of the University of Glasgow for the use of radiochemistry facilities. Open access via RSC Gold for Gold.Peer reviewedPublisher PD

Using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling in order to predict clinical outcomes of patients with heart failure

Background Current risk prediction models in heart failure (HF) including clinical characteristics and biomarkers only have moderate predictive value. The aim of this study was to use matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling to determine if a combination of peptides identified with MALDI-MS will better predict clinical outcomes of patients with HF. Methods A cohort of 100 patients with HF were recruited in the biomarker discovery phase (50 patients who died or had a HF hospital admission vs. 50 patients who did not have an event). The peptide extraction from plasma samples was performed using reversed phase C18. Then samples were analysed using MALDI-MS. A multiple peptide biomarker model was discovered that was able to predict clinical outcomes for patients with HF. Finally, this model was validated in an independent cohort with 100 patients with HF. Results After normalisation and alignment of all the processed spectra, a total of 11,389 peptides (m/z) were detected using MALDI-MS. A multiple biomarker model was developed from 14 plasma peptides that was able to predict clinical outcomes in HF patients with an area under the receiver operating characteristic curve (AUC) of 1.000 (p = 0.0005). This model was validated in an independent cohort with 100 HF patients that yielded an AUC of 0.817 (p = 0.0005) in the biomarker validation phase. Addition of this model to the BIOSTAT risk prediction model increased the predictive probability for clinical outcomes of HF from an AUC value of 0.643 to an AUC of 0.823 (p = 0.0021). Moreover, using the prediction model of fourteen peptides and the composite model of the multiple biomarker of fourteen peptides with the BIOSTAT risk prediction model achieved a better predictive probability of time-to-event in prediction of clinical events in patients with HF (p = 0.0005). Conclusions The results obtained in this study suggest that a cluster of plasma peptides using MALDI-MS can reliably predict clinical outcomes in HF that may help enable precision medicine in HF

DNA methylation profiling of the human major histocompatibility complex: A pilot study for the Human Epigenome Project

The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine-guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data

Apteniols A-F, oxyneolignans from the leaves of Aptenia cordifolia.

Abstract—Investigation of the organic extract of Aptenia cordifolia leaves revealed six new oxyneolignans named apteniols A–F. The structures were determined by means of spectroscopic methods. The C6C3 units are linked by an oxygen atom at C4–C40 or C4–C20 and they are dihydrophenylpropanoid acid units. Their effects on germination and growth of Lactuca sativa L. have been studied in the range concentration 10K4–10K7 M

Introduction of 4-chloro-alpha-cyanocinnamic acid liquid matrices for high sensitivity UV-MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) is a key ionization technique in mass spectrometry (MS) for the analysis of labile macromolecules. An important area of study and improvements in relation to MALDI and its application in high-sensitivity MS is that of matrix design and sample preparation. Recently, 4-chloro-alpha-cyanocinnamic acid (ClCCA) has been introduced as a new rationally designed matrix and reported to provide an improved analytical performance as demonstrated by an increase in sequence coverage of protein digests obtained by peptide mass mapping (PMM) (Jaskolla, T. W.; et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12200-12205). This new matrix shows the potential to be a superior alternative to the commonly used and highly successful alpha-cyano-4-hydroxycinnamic acid (CHCA). We have taken this design one step further by developing and optimizing an ionic liquid matrix (ILM) and liquid support matrix (LSM) using ClCCA as the principle chromophore and MALDI matrix compound. These new liquid matrices possess greater sample homogeneity and a simpler morphology. The data obtained from our studies show improved sequence coverage for BSA digests compared to the traditional CHCA crystalline matrix and for the ClCCA-containing ILM a similar performance to the ClCCA crystalline matrix down to 1 fmol of BSA digest prepared in a single MALDI sample droplet with current sensitivity levels in the attomole range. The LSMs show a high tolerance to contamination such as ammonium bicarbonate, a commonly used buffering agent

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities