Quantitative approach to single-nucleotide polymorphism analysis using MALDI-TOF mass spectrometry

Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.     

Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry

Mass spectrometry-based amino acid sequencing is currently based almost entirely on collision-induced peptide fragmentation and analyses. Here, we describe a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, heat-loving Bacillus subtilis-derived aminopeptidase (BsuAP), which acts optimally at 70 °C and allows ‘single-shot’ sequencing to be carried out through simultaneous accumulation, and detection of sub-populations of peptides of progressively reducing length.     

Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated proteins detected by immunostaining

We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.     

Detection of peptides covalently modified with multiple fatty acids by MALDI-TOF mass spectrometry.

Analysis and characterization of membrane proteins and hydrophobic peptides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a considerable challenge because of their lower ionization efficiency. Detergents are used to solubilize hydrophobic peptides and proteins. However, in MALDI-MS, the presence of detergents can cause considerable loss of signal intensity. The extent of interference depends on the matrix/sample preparation method and experimental conditions. In the present study, we have analyzed the MALDI response of multiple fatty acylated peptides in the presence of the matrices alpha-cyano-4-hydroxy cinnamic acid (HCCA) and 2,5-dihydroxy benzoic acid (DHB). The effect of adding the nonionic detergent n-octylglucoside (OG) was also examined. The presence of OG facilitated detection of tetrapalmitoylated peptide, particularly when HCCA was used as the matrix. When DHB was used as the matrix, good signal intensity was observed in the absence of OG. Lower laser pulse rate in the linear mode of analysis resulted in good signal intensity for the tetrapalmitoylated peptide. Conditions for obtaining good signal intensities for dipalmitoylated and N-myristoyl peptides with both HCCA and DHB as matrices were also investigated.     

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.     

A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry

This protocol allows the accurate quantification of cell-penetrating peptide (CPP) cellular uptake by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Quantification is based on the use of an internal standard with same chemical structure as the analyte but labeled with a stable isotope. The analyte and the standard can both be obtained by standard solid-phase peptide synthesis using commercially available amino acids. They are functionalized by biotin to allow their easy purification before MALDI-TOF MS analysis. The method allows determination of the amount of intact internalized peptide and the identification of potential intracellular digests. It can be used to simultaneously compare the uptake of several peptides, and can also be applied to the quantification of peptidic cargoes and the study of their intracellular stability. It is therefore a potent tool to study the mechanisms of CPPs internalization and to select new carriers for drug delivery. This protocol will take approximately 5 hours for the analysis of 12 samples (not including the time for cell incubation with peptides).     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species.     

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.     

SNP and mutation discovery using base-specific cleavage and MALDI-TOF mass spectrometry

MOTIVATION: Single Nucleotide Polymorphisms (SNPs) are believed to contribute strongly to the genetic variability in living beings, in particular their disease or drug side effect predispositions. Mutation-induced sequence variations are playing an important role in the development of cancer, among others. From this, it is clear that SNP and mutation discovery is of great interest in today's Life Sciences. Currently, such discovery is often performed utilizing electrophoresis-based Sanger Sequencing. Discovery of SNPs can also be performed by multiple sequence alignment of publicly available sequence data, but recent studies indicate that only a small percentage of SNPs can be discovered using this approach and, in particular, that SNPs with low frequency are often missed. Other SNP discovery methods only indicate the presence of a SNP in a sample region, but fail to resolve its characterization and localization. RESULTS: We present a method to discover mutations and SNPs using base-specific cleavage and mass spectrometry. An amplicon of known reference sequence with length usually between 100 and 1000 nt is amplified, transcribed, and cleaved using base-specific endonucleases such as RNAse A or T1. The resulting cleavage products (or fragments) are analyzed by MALDI-TOF mass spectrometry and, comparing the measured spectra with those predicted in-silico, the goal is to discover and pinpoint sequence variations of the sample sequence compared to the reference sequence. A time-efficient algorithm for discovering sequence variations is presented that enables fast analysis of such variations even if the sample sequence differs significantly from the reference sequence.     

Direct analysis of neuropeptides by in situ MALDI-TOF mass spectrometry in the rat brain

The measure of neuropeptides is an important tool in biology to better define endocrine and neuroendocrine function. Traditionally most methods have relied on the development of specific antibodies. Newer molecular methodologies have used measures of gene expression of neuropeptide precursors, such as Northern blot, PCR or in situ hybridization analysis. Matrix-assisted laser desorption/ionization (MALDI) mass analysis is a novel powerful technique for investigation of neuropeptides. Multiple peptides and peptide forms can be detected simultaneously and with great sensitivity in tissue extracts or partially purified samples. We have now adapted a MALDI methodology for the direct measurement of neuropeptides on fresh tissue sections of rat brains. We have validated the method by examining peptidergic mass profiles of the supraoptic nucleus (SON) and caudate putamen hypothalamic regions. Interestingly, mass profiles showed that vasopressin, which is specifically present in the SON, is modulated when animals are treated with lipopolysaccharides. MALDI-MS on brain slides is a novel complementary technique for neurobiologists and endocrinologists in order to investigate the dynamic and regional distribution of neuropeptides during physiological events.     

MALDI-TOF mass spectrometry of oligomeric food polyphenols

The structural heterogeneity of polyphenols from cranberries, grape seed extracts, sorghum and pomegranate was characterized by MALDI-TOF MS. Polyphenolics were isolated by liquid chromatography and subjected to MALDI-TOF MS using trans-3-indoleacrylic acid as the matrix. Spectrometric analysis gave information on degree of polymerization, monomeric substitution, and the nature of intermolecular bonds. Cranberry polyflavan-3-ols had variation in interflavan bonds (A- and B-types) and contained polyflavan-3-ols linked to anthocyanins through a CH3-CH bridge. Polygalloyl-polyflavan-3-ols in grape seed extract had large variation in the degree of galloyl substitution. Sorghum polyflavans had structural heterogeneity in glycosylation and hydroxylation. Pomegranate hydrolyzable tannins that correspond to previously described structures were detected, such as punicalagin, but others that correspond to oligomeric ellgitannins in which two to five core glucose units are cross-linked by dehydrodigalloyl and or valoneoyl units were also observed. Results demonstrate that large heterogeneity occurs in degree of polymerization, intermolecular bonds, pattern of hydroxylation, and substitution with monosaccharides and gallic acid.     

Molecular characterization of human and bovine ceruloplasmin using MALDI-TOF mass spectrometry.

Using SDS-PAGE and MALDI-TOF mass spectrometry, we investigated the difference in the molecular structure between human and bovine ceruloplasmin. In both cases, we found that the protein is present in two majors forms of different molecular mass. The difference between human and bovine ceruloplasmin was more obvious when characterized by MALDI-TOF than with the SDS-PAGE analysis. Furthermore, we established that the N-glycoside content of both enzymes is dissimilar and that the N-glycosyl moieties are distributed in a distinctive fashion in two glycoproteins. Finally, it appeared that both proteins exhibited different cleavage patterns after treatment with trypsin. This study indicates that human and bovine ceruloplasmin differ not only in sugar composition but also in primary structure.     

Rapid detection of chlorinated bisbibenzyls in Bazzania trilobata using MALDI-TOF mass spectrometry

Chlorinated bisbibenzyls of the bazzanin type are detected in crude bryophyte plant extracts of Bazzania trilobata from different locations using MALDI-TOF mass spectrometry. These results show that these chlorinated compounds are not artefacts of an incidental occurrence or of the sample preparation but are genuine and produced by the liverwort or an endosymbiotic metabolism. Further experiments were performed concerning the in vitro chlorination of the halogen free basic unit isoplagiochin C.     

Identification of human liver cytochromes P450 by using MALDI-TOF mass spectrometry

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.     

In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues.     

Identification of proteins in leaf tissues of white clover using MALDI-TOF mass spectrometry

Protein extracts, made to leaves harvested from the stolons of the pasture legume white clover (Trifolium repens L.) at two developmental stages (newly initiated; onset of senescence) were purified further using reverse-phase and ionexchange chromatography. Fractions enriched with the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase were selected for each stage and the final, partially purified fraction was subjected to twodimensional gel electrophoresis (2DE). Antibodies raised against a recombinant ACC oxidase (ACO) from white clover (antiTR-ACO2) recognised a series of spots of differing pI suggesting that ACO undergoes post-translational modifications. Further, the pattern differed between the ACO proteins partially purified from newly initiated leaves with leaves at the onset of senescence suggesting that the environmental and developmental cues that operate in each tissue influences the type and/or degree of post-translational modifications of the ACO protein. MALDI-TOF mass spectrometry was used to identify protein spots associated with the ACO proteins. Protein with identities to an ACO isoform from Oryza sativa, and a phosphoribulokinase from Arabidopsis thaliana were identified in the 2DE separations from newly initiated leaves, while an isoflavone reductase from Medicago sativa was identified in the 2DE separation of the senescent leaf extract.     

Analysis of glycated insulin by MALDI-TOF mass spectrometry

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.     

Applications of MALDI-TOF mass spectrometry in clinical proteomics

Introduction: The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics.

Areas covered: This review aims to provide an updated overview of MALDI-TOF MS applications in clinical proteomics. The most relevant features of this analysis have been discussed, highlighting both pre-analytical and analytical factors that are crucial in proteomics studies. Particular emphasis is placed on biofluids proteomics for biomarkers discovery and on recent progresses in clinical microbiology, drug monitoring, and minimal residual disease (MRD).

Expert commentary: Despite some analytical limitations, the latest technological advances together with the easiness of use, the low time and low cost consuming and the high throughput are making MALDI-TOF MS instruments very attractive for the clinical practice. These features offer a significant potential for the routine of the clinical laboratory and ultimately for personalized medicine.     

Identification of fungal microorganisms by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable tool for fast identification and classification of microorganisms. In this regard, it represents a strong challenge to microscopic and molecular biology methods. Nowadays, commercial MALDI systems are accessible for biological research work as well as for diagnostic applications in clinical medicine, biotechnology and industry. They are employed namely in bacterial biotyping but numerous experimental strategies have also been developed for the analysis of fungi, which is the topic of the present review. Members of many fungal genera such as Aspergillus, Fusarium, Penicillium or Trichoderma and also various yeasts from clinical samples (e.g. Candida albicans) have been successfully identified by MALDI-TOF MS. However, there is no versatile method for fungi currently available even though the use of only a limited number of matrix compounds has been reported. Either intact cell/spore MALDI-TOF MS is chosen or an extraction of surface proteins is performed and then the resulting extract is measured. Biotrophic fungal phytopathogens can be identified via a direct acquisition of MALDI-TOF mass spectra e.g. from infected plant organs contaminated by fungal spores. Mass spectrometric peptide/protein profiles of fungi display peaks in the m/z region of 1000–20 000, where a unique set of biomarker ions may appear facilitating a differentiation of samples at the level of genus, species or strain. This is done with the help of a processing software and spectral database of reference strains, which should preferably be constructed under the same standardized experimental conditions.