Genotools SNP manager: a new software for automated high-throughput MALDI-TOF mass spectrometry SNP genotyping

Analysis of single nucleotide polymorphisms (SNPs) is a rapidly growing field of research that provides insights into the most common type of differences between individual genomes. The resulting information has a strong impact in the fields of pharmacogenomics, drug development, forensic medicine, and diagnostics of specific disease markers. The technique of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a highly suitable tool for the analysis of DNA. It supplies a very versatile method for addressing a high-throughput SNP genotyping approach. Here, we present the Bruker genotools SNP MANAGER, a new software tool suitable for highly automated MALDI-TOF MS SNP genotyping. The genotools SNP MANAGER administers the sample preparation data, calculates masses of allele-specific primer extension products, performs genotyping analysis, and displays the results. In the current study, we have used the genotools SNP MANAGER to perform an automated duplex SNP analysis of two biallelic markers from the promoter of the gene encoding the inflammatory mediator interleukin-6.     

Microbead device for isolating biotinylated oligonucleotides for use in mass spectrometric analysis

We describe a prototypical device for isolating biotinylated oligonucleotides for use in mass spectrometric analysis. It consists of monomeric avidin-coated microbeads trapped in a pipette tip and has been used for genotyping single nucleotide polymorphisms (SNPs) with the previously developed solid phase capture-single base extension (SPC-SBE) method. The device reduces processing time for genotyping by SPC-SBE and allows direct spotting of sample for rapid analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, it allows simultaneous processing of multiple samples and can be reused after regeneration of beads with no carryover effects. These results indicate that the microbead device is a low-cost tool that enhances sample cleanup prior to MS for SNP genotyping.     

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.     

Estimation of allele frequency in pooled DNA by using PCR–RFLP combined with microchip electrophoresis

Biallelic marker, most commonly single nucleotide polymorphism (SNP), is widely utilized in genetic association analysis, which can be speeded up by estimating allele frequency in pooled DNA instead of individual genotyping. Several methods have shown high accuracy and precision for allele frequency estimation in pools. Here, we explored PCR restriction fragment length polymorphism (PCR–RFLP) combined with microchip electrophoresis as a possible strategy for allele frequency estimation in DNA pools. We have used the commercial available Agilent 2100 microchip electrophoresis analysis system for quantifying the enzymatically digested DNA fragments and the fluorescence intensities to estimate the allele frequencies in the DNA pools. In this study, we have estimated the allele frequencies of five SNPs in a DNA pool composed of 141 previously genotyped health controls and a DNA pool composed of 96 previously genotyped gastric cancer patients with a frequency representation of 10–90% for the variant allele. Our studies show that accurate, quantitative data on allele frequencies, suitable for investigating the association of SNPs with complex disorders, can be estimated from pooled DNA samples by using this assay. This approach, being independent of the number of samples, promises to drastically reduce the labor and cost of genotyping in the initial association analysis.     

Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.     

Bacterial Sub-Species Typing Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry: What Is Promising?

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification. It would be highly beneficial to also be able to use the technology as a fast way to detect clinically relevant clones of bacterial species. However, studies to this aim have often had limited success. The methods used for data acquisition, processing and data interpretation are highly diverse amongst studies on MALDI-TOF MS sub-species typing. In addition to this, feasibility may depend on the bacterial species and strains investigated, making it difficult to determine what methods may or may not work. In our paper, we have reviewed recent research on MALDI-TOF MS typing of bacterial strains. Although we found a lot of variation amongst the methods used, there were approaches shared by multiple research groups. Multiple spectra of the same isolate were often combined before further analysis for strain distinction. Many groups used a protein extraction step to increase resolution in their MALDI-TOF MS results. Peaks at a high mass range were often excluded for data interpretation. Three groups have found ways to determine feasibility of MALDI-TOF MS typing for their set of strains at an early stage of their project.     

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

MALDI-TOF MS analysis of the extracellular polysaccharides released by the diatom Thalassiosira pseudonana

The extracellular polysaccharides (ECPS) released by diatoms have significant roles in marine ecosystems and have potential applications including drug-discovery and biopharmaceutical precursors. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology was used in the structural analysis of the ECPS released by Thalassiosira pseudonana (Bacillariophyta). Three different deproteinization methods, the Sevag method, the trichloroacetic acid (TCA) method, and the enzymolysis method, were compared in the purification of ECPS. Our results suggested that TCA was the best deproteinization method among the three methods for subsequent MALDI-TOF MS investigation because of its high ECPS yield, protein removal ability and reliable MALDI-TOF MS fingerprint. The degree of polymerization (d.p.) profiles, the molecular weight of the ECPS and the distribution pattern of the polymers with different molecular mass were described from the MALDI-TOF MS spectra. This work represents the whole-level composition of the ECPS released by the diatom and has improved our knowledge of the structural characterization of ECPS.     

Practical considerations in BIA/MS: optimizing the biosensor-mass spectrometry interface

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multiplexed analytical technique that utilizes a unique combination of surface plasmon resonance (SPR) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of small amounts of proteins residing in complex biological systems. In order to achieve high sensitivity during BIA/MS, certain experimental parameters and sequences of events need to be optimized and maintained. Immobilized ligand density, flow rate and biosensor control (in SPR-BIA) and matrix choice and application (in MALDI-TOF MS) have significant influence on the final outcome of the BIA/MS analysis and, consequently, need to be optimized and carefully controlled. In addition, chip washing and cutting are essential in converting the SPR-active sensor chips into target surfaces amenable to MALDI-TOF MS. Reviewed here are the prerequisites for successfully interfacing SPR-BIA with MALDI-TOF MS.     

Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments

The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.     

Towards global analysis of mosquito chorion proteins through sequential extraction, two-dimensional electrophoresis and mass spectrometry

This study describes the separation and identification of chorion proteins through two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) techniques. Due to their high hydrophobicity, chorion proteins are difficult to be solubilized and absorbed into the immobilized pH gradient strip for isoelectric focusing. By optimizing the applied conditions for chorion protein extraction and sample application, we were able to solubilize the majority of the chorion proteins and resolve them by 2-DE. Under optimized conditions, there are more than 700 protein spots resolved by 2-D analysis. Trypsin digestions of individual protein spots, MALDI-TOF MS analysis of their digested peptides, and subsequent BLAST search of peptide masses resulted in the tentative identification of 38 protein spots. Our data show that sequential extraction of the isolated chorion, 2-DE of the solubilized chorion proteins, in-gel digestion of the resolved protein and MALDI-TOF MS analysis of the protein digests is an effective overall strategy towards determination of chorion proteins in mosquitoes. The merits of the method described for the determination of mosquito chorion proteins and its feasibility for the separation and identification of membrane proteins and chorion or eggshell proteins from other insect species are discussed.     

A metal-coded affinity tag approach to quantitative proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.     

MALDI-TOF MS of phosphatidylethanolamines: Different adducts cause different post source decay (PSD) fragment ion spectra

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described.In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens.

Methodology/Principal Findings

We compared the identification efficiency of MALDI-TOF MS with that of Phoenix®, API® and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7% of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63% of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn''s kappa values: 0.9 to 0.93).

Conclusions/Significance

MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria.     

Quantitative multiplexed C-reactive protein mass spectrometric immunoassay

Reported in this work is the development and application of a high sensitivity mass spectrometric immunoassay for the quantitative analysis of C-reactive protein from human plasma. Multiplexed affinity retrieval devices and methodology were developed to simultaneously target retinol binding protein, C-reactive protein, serum amyloid P component, as well as an added exogenous internal reference standard (staphylococcal enterotoxin B) for subsequent MALDI-TOF MS analysis. This approach allows for semiquantitative analysis of both retinol binding protein and serum amyloid P component while performing absolute quantitative measurements of C-reactive protein. The ability to qualitatively differentiate between all three human proteins and their associated variants is also maintained. Standard curve, QC, and human plasma samples were analyzed in a high throughput manner, which performed with a CV < 15%. The resultant human plasma sample C-reactive protein quantitative measurements were then compared to those achieved with a high sensitivity latex immunoturbidimetric assay.     

Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult.In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method.By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.     

Multiplex SNP genotyping by MALDI-TOF mass spectrometry: Frequencies of 56 immune response gene SNPs in human populations

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic polymorphisms. Despite the progress in sequencing and postgenomic technologies, targeted SNP genotyping continues to be in highest demand in the approach to human and medical genetics. In this work, we describe the application of multiple SNP genotyping by MALDI-TOF mass spectrometry for analysis of genetic diversity of immune response genes in human populations. It was shown that MALDI-TOF mass spectrometry is a rapid, accurate, and efficient method of medium-scale SNP genotyping. Allele frequencies of 56 SNPs in 41 genes implicated in the regulation of immune response were similar in four populations studied (Russians, Komi, Khanty, and Buryats). These populations had similar levels of genetic diversity and were clustered according to their geographic location. The cost efficiency of MALDI-TOF mass spectrometry was evaluated compared to real-time PCR technology.