Challenges and opportunities in mass spectrometric analysis of proteins from dried blood spots

Dried blood spots (DBS) have been used as a clinical sample format for over 50 years, and have been analyzed for small molecules and metabolites by mass spectrometry (MS) since the early 1990s. In the meantime, MS has become the tool of choice in proteomics. Despite this obvious avenue of scientific investigation, the marriage of MS and DBS protein analysis has been comparatively recent. DBS are a potentially rich source of protein biomarkers that remain to be exploited. This article focuses on the progress made in the mass spectrometric analysis of proteins from DBS and discusses the benefits and challenges facing this emerging field.     

Metabolomics – an overview. From basic principles to potential biomarkers (part 2)

The metabolome, which represents the complete set of molecules (metabolites) of a biological sample (cell, tissue, organ, organism), is the final downstream product of the metabolic cell process that involves the genome and exogenous sources. The metabolome is characterized by a large number of small molecules with a huge diversity of chemical structures and abundances. Exploring the metabolome requires complementary analytical platforms to reach its extensive coverage. The metabolome is continually evolving, reflecting the continuous flux of metabolic and signaling pathways. Metabolomic research aims to study the biochemical processes by detecting and quantifying metabolites to obtain a metabolic picture able to give a functional readout of the physiological state. Recent advances in mass spectrometry (one of the mostly used technologies for metabolomics studies) have given the opportunity to determine the spatial distribution of metabolites in tissues. In a two-part article, we describe the usual metabolomics technologies, workflows and strategies leading to the implementation of new clinical biomarkers. In this second part, we first develop the steps of a metabolomic analysis from sample collection to biomarker validation. Then with two examples, autism spectrum disorders and Alzheimer's disease, we illustrate the contributions of metabolomics to clinical practice. Finally, we discuss the complementarity of in vivo (positron emission tomography) and in vitro (metabolomics) molecular explorations for biomarker research.     

Analysis of Polymerase Chain Reaction-Amplified DNA Products by Mass Spectrometry Using Matrix-Assisted Laser Desorption and Electrospray: Current Status

Recent advances in molecular biology are making it possible to diagnose genetic diseases and identify pathogens through the analysis of DNA. As clinical applications for molecular diagnosis increase, rapid, reliable methods for determination of DNA size will be needed. Mass spectrometry offers the potential of analyzing amplified DNA quickly and reliably, without the need for gel-based separation and sample labeling steps that are conventionally employed. Both electrospray ionization and matrix-assisted laser desorption/ionization have been evaluated for the size analysis of DNA using both synthetic oligonucleotides and PCR-amplified samples corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. Both technologies have been demonstrated to have mass range and sensitivity required for the analysis of PCR-amplified DNA in this size range using minimal sample preparation. Steps required to incorporate either ionization technique into a reliable analytical scheme for the rapid, routine analysis of DNA are outlined.     

Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.     

Methods for determining the absolute configurations of marine ladder-shaped polyethers

Marine dinoflagellates produce a unique family of bioactive substances featuring multiple ether rings aligned in a ladder shape. These are large, complex molecules with potent bioactivity. Targeted chiral centers sit on either the skeletal ladders or on the side chains of these compounds. However, the laborious steps of isolation and purification severely diminish the amount of sample available for assigning these chiral centers via structural investigations. Three important methods were used to assign the stereochemistry of the molecules, (a) circular dichroism (CD) spectroscopy, (b) labeling with fluorescent chiral reagents for high-performance liquid chromatography (HPLC) analysis, and (c) derivatization with anisotropic reagents for nuclear magnetic resonance (NMR) analysis. The addition of fluorescent chiral reagents allowed for the use of much less material than typically required. In this review, we present examples of the determination of absolute configurations in ladder-shaped polyethers. The targeted compounds include ciguatoxins (CTXs), gymnocin-B, gambieric acids, prymnesin-2, maitotoxin, yessotoxins, gambierol, brevisamide, and brevisin.     

Sample preparation for mass spectrometry imaging: small mistakes can lead to big consequences

Mass spectrometry imaging (MSI) enables the direct analysis of molecules from the surface of a wide variety of samples, allowing the multiplex measurement of both abundance and distribution of small molecules, lipids, peptides and proteins. As the technology has been refined an increasing number of ionization methods and mass analyzers has been used that enable increased spatial and spectral resolution measurements to be made at an increased speed. Alongside the instrumentation improvements there has been optimization of sample preparation procedures that allow the highest quality data to be obtained, reproducibly, from an ever increasing diversity of samples. This review will consider the development and standardization of sample preparation methods applicable to MSI, describing the stages and procedures undertaken from the instance of sample collection, through storage, preparation and on through final processing prior to analysis. Recent technical advancements will be highlighted and areas where further experimentation and optimization may well be required will be described. All aspects of the sample preparation pipeline will be considered in detail, with examples from the literature used to emphasize why rigorous sample preparation for MSI is vital to achieve the most accurate, reproducible and validated MSI data possible.     

Laser desorption/ionization—Mass spectrometry using mesoporous silicate as matrix for the analysis of various molecules

In this study, mesoporous silicate was applied as a matrix for the analysis of various molecules from small molecules to medium sized peptides in laser desorption/ionization mass spectrometry. In contrast with conventional matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), the proposed approach desorption/ionization on mesoporous silicate mass spectrometry (DIOM-MS), significantly reduces the problem of matrix interference in low mass region and can be applied to the analysis of versatile chemicals including amino acids, synthetic drugs, peptides and others. In addition, distinctive advantage of DIOM-MS showed higher salt tolerance and could be applied to identify the proteins from the analysis of tryptically digested peptides. DIOM-MS has several availabilities such as easy sample preparation, rapid analysis of small molecules without noise, peptide analysis without organic matrix, high salt tolerance, versatile coupling with other separation techniques, and high throughput manner.     

Capillary electrophoresis and microdialysis: current technology and applications

Microdialysis sampling has become an important method for the continuous monitoring from an in vivo environment. This technique has been used to monitor many endogenous molecules, such as neurotransmitters, as well as exogenous species such as drug substances. Microdialysis samples have traditionally been analyzed by liquid chromatographic (LC) methods to gain resolution and quantification of the molecules of interest. However, LC separations have a relatively large injection volume requirement which, as a consequence, increases microdialysis sampling times. Capillary electrophoresis (CE), with its very small sample volume requirements and high resolving power, has therefore gained popularity as an alternative to LC. Reviewed here are many of the technologies currently available for CE and examples of how this technique has been effectively applied to the analysis of microdialysis samples.     

Growth hormone determination in children using an immunofunctional assay in comparison to conventional assays

Conventional assays for determination of growth hormone (GH) in serum measure immunoreactive molecules of a blood sample. The immunofunctional assay (IFA), on the other hand, is able to determine biologically active molecules. In our study, we evaluated GH determination in children with IFA to compare these data with clinical reliable data of conventional assay systems, since there is only insufficient data concerning the clinical use of IFA in children. The comparison of GH determinations by IFA and two immunoradiometric assays showed different results for the same serum sample. Peak and trough concentration levels determined by these three different assays were always measured at the same time, but absolute GH concentration levels varied. Statistic analysis verified a linear regression of our data and allowed a conversion of data measured by IFA to predict values of the other assays used and vice versa. The traditional cut-off level for the diagnosis of GH deficiency of 10 ng/ml was based originally on results of polyclonal radioimmunoassays. This internationally applied cut-off level has been converted based on the regression analysis for prediction of this study and we found the 95% confidence interval on the mean measurements by IFA to be between 3.11 and 3.28 ng/ml.     

Miniaturized and direct spectrophotometric multi-sample analysis of trace metals in natural waters

Trends in the analysis of trace metals in natural waters are mainly based on the development of sample treatment methods to isolate and pre-concentrate the metal from the matrix in a simpler extract for further instrumental analysis. However, direct analysis is often possible using more accessible techniques such as spectrophotometry. In this case a proper ligand is required to form a complex that absorbs radiation in the ultraviolet–visible (UV–Vis) spectrum. In this sense, the hydrazone derivative, di-2-pyridylketone benzoylhydrazone (dPKBH), forms complexes with copper (Cu) and vanadium (V) that absorb light at 370 and 395 nm, respectively. Although spectrophotometric methods are considered as time- and reagent-consuming, this work focused on its miniaturization by reducing the volume of sample as well as time and cost of analysis. In both methods, a micro-amount of sample is placed into a microplate reader with a capacity for 96 samples, which can be analyzed in times ranging from 5 to 10 min. The proposed methods have been optimized using a Box–Behnken design of experiments. For Cu determination, concentration of phosphate buffer solution at pH 8.33, masking agents (ammonium fluoride and sodium citrate), and dPKBH were optimized. For V analysis, sample (pH 4.5) was obtained using acetic acid/sodium acetate buffer, and masking agents were ammonium fluoride and 1,2-cyclohexanediaminetetraacetic acid. Under optimal conditions, both methods were applied to the analysis of certified reference materials TMDA-62 (lake water), LGC-6016 (estuarine water), and LGC-6019 (river water). In all cases, results proved the accuracy of the method.     

Bias, precision and heritability of self-reported and clinically measured height in Australian twins

Many studies of quantitative and disease traits in human genetics rely upon self-reported measures. Such measures are based on questionnaires or interviews and are often cheaper and more readily available than alternatives. However, the precision and potential bias cannot usually be assessed. Here we report a detailed quantitative genetic analysis of stature. We characterise the degree of measurement error by utilising a large sample of Australian twin pairs (857 MZ, 815 DZ) with both clinical and self-reported measures of height. Self-report height measurements are shown to be more variable than clinical measures. This has led to lowered estimates of heritability in many previous studies of stature. In our twin sample the heritability estimate for clinical height exceeded 90%. Repeated measures analysis shows that 2–3 times as many self-report measures are required to recover heritability estimates similar to those obtained from clinical measures. Bivariate genetic repeated measures analysis of self-report and clinical height measures showed an additive genetic correlation >0.98. We show that the accuracy of self-report height is upwardly biased in older individuals and in individuals of short stature. By comparing clinical and self-report measures we also showed that there was a genetic component to females systematically reporting their height incorrectly; this phenomenon appeared to not be present in males. The results from the measurement error analysis were subsequently used to assess the effects of error on the power to detect linkage in a genome scan. Moderate reduction in error (through the use of accurate clinical or multiple self-report measures) increased the effective sample size by 22%; elimination of measurement error led to increases in effective sample size of 41%.     

Synthesis and cellular effects of cycloterpenals: cyclohexadienal-based activators of neurite outgrowth

An unusual class of diterpenoid natural products, 'cycloterpenals' (with a central cyclohexadienal core), that arise in nature by condensation of retinoids and other isoprenes, have been isolated from a variety of organisms including marine sponges as well as from the human eye. A milk whey protein has also demonstrated the formation of a cycloterpenal derived from beta-ionylidineacetaldehyde. Here, we generate a synthetic library of these molecules where we detail reaction conditions required to effect cross condensation of alpha,beta-unsaturated aldehydes as opposed to homodimerization. The ability of this class of molecules to activate neurite outgrowth activity is reported.     

Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma.

The advantage of using proteins and peptides as biomarkers is that they can be found readily in blood, urine, and other biological fluids. Such sample types are easily obtained and represent a potentially rich palette of biologically informative molecules. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a key tool for rapidly interrogating such sample types. The goal of clinical proteomics is to harness the power of this tool for identifying novel, condition-specific protein fingerprints that may, in turn, lead to the elucidation and use of diseasespecific biomarkers that may be used to diagnose disease as well as to evaluate disease severity, disease progression, and intervention efficacy. Here we have evaluated a simple, affordable bench-top MALDI-TOF mass spectrometer to generate protein profiles from human plasma samples of asthma patients and healthy individuals. We achieve this profiling by using C8-functionalized magnetic beads that enrich a specific subset of plasma proteins based on their absorption by this resin. This step is followed by elution, transfer onto a prestructured sample support (AnchorChip technology), and analysis in a bench-top MALDI-TOF mass spectrometer (OmniFLEX) with AutoXecute acquisition control which enables automated operation with reproducible results. Resulting spectra are compiled and analyzed through the pattern recognition component of ClinProTools software. This approach in combination with ClinProTools software permits the investigator to rapidly scan for potential biomarker peptides/proteins in human plasma. The reproducibility of plasma profiles within and between days has been evaluated. The results show that the novel and facile approach with manual magnetic-bead sample preparation and a low-cost bench-top MALDI-TOF mass spectrometer is suitable for preliminary biomarker discovery studies.     

A Comparative Analysis of the Altered Levels of Human Seminal Plasma Constituents as Contributing Factors in Different Types of Male Infertility

(1) Background: The relationships between the biochemical and immunological components in seminal plasma and their physiological effects on male reproductive system have been underreported. In this study, we evaluated the potential of several seminal plasma biochemical and immunological markers in the pathophysiological developments of the infertile male patients. The study was designed to identify and assess different markers that may be associated with semen functions in different types of male infertility. (2) Methods: A total of 50 infertile male patients who underwent checkup for fertility assessment and 50 fertile controls were included in this study. The complete medical history of each recruited participant was reviewed. The infertile sub-groups (non-obstructive azoospermia (NOA), asthenozoospermia (AS), normozoospermic infertile (NI), and oligozoospermia (OZ)) were characterized based on sperm motility and concentration, while NI patients were included after a thorough check up of their female partners as well. We investigated each sample for 21 different analytes, enzymes, trace elements, and immunological markers to find crucial markers posing as contributing factors to a specific type of male infertility. (3) Results: The levels of 15 out of 21 markers, assayed from the seminal plasma of infertile males, were significantly altered in comparison to fertile controls (p < 0.05). For the first time, microprotein levels were also analyzed. The presence of monocytes, lymphocytes, and granulocytes was limited to semen from NOA patients, while a significant increase in the level of platelets was observed in AS. Hierarchical clustering and ROC-AUC analysis identified the three most significant markers (zinc, LDH, and TG) for the healthy control group and asthenozoospermic group (AUC, of 0.92 and 0.81, respectively). (4) Conclusions: The altered levels of biochemical and immunological markers in seminal plasma might be associated with the different male infertility profiles and could be required for the sperm metabolism and maintenance. However, a larger sample size and follow up analysis is required for establishing the hypothesized panel of markers as biomarkers at clinical stage.     

Single-molecule reader for proteomics and genomics

Recent developments in ultrasensitive fluorescence microscopy enabled the detection and detailed characterization of individual biomolecules in their native environment. New types of information can be obtained from studying individual molecules, which is not accessible from ensemble measurements. Moreover, this methodological advance matches the need of bioscience to downscale the sample amount required for screening devices. It is envisioned that concentrations as low as approximately 1000 molecules contained in a sample of 1 nl can be detected in a chip-based assay. In this review, we overview state-of-the-art single molecule microscopy with respect to its applicability to ultrasensitive screening. Quantitative estimations will be given, based on a novel apparatus designed for large area screening at single molecule sensitivity.     

Rates of Decline in Alzheimer Disease Decrease with Age

Age is the strongest risk factor for sporadic Alzheimer disease (AD), yet the effects of age on rates of clinical decline and brain atrophy in AD have been largely unexplored. Here, we examined longitudinal rates of change as a function of baseline age for measures of clinical decline and structural MRI-based regional brain atrophy, in cohorts of AD, mild cognitive impairment (MCI), and cognitively healthy (HC) individuals aged 65 to 90 years (total n = 723). The effect of age was modeled using mixed effects linear regression. There was pronounced reduction in rates of clinical decline and atrophy with age for AD and MCI individuals, whereas HCs showed increased rates of clinical decline and atrophy with age. This resulted in convergence in rates of change for HCs and patients with advancing age for several measures. Baseline cerebrospinal fluid densities of AD-relevant proteins, Aβ_1–42, tau, and phospho-tau_181p (ptau), showed a similar pattern of convergence with advanced age across cohorts, particularly for ptau. In contrast, baseline clinical measures did not differ by age, indicating uniformity of clinical severity at baseline. These results imply that the phenotypic expression of AD is relatively mild in individuals older than approximately 85 years, and this may affect the ability to distinguish AD from normal aging in the very old. Our findings show that inclusion of older individuals in clinical trials will substantially reduce the power to detect disease-modifying therapeutic effects, leading to dramatic increases in required clinical trial sample sizes with age of study sample.