Cerium ion-chelated magnetic silica microspheres for enrichment and direct determination of phosphopeptides by matrix-assisted laser desorption ionization mass spectrometry

In this study, we employed, for the first time, the Ce4+-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before. After enrichment, the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet. With the optimized enrichment conditions, the performance of the Ce4+-chelated magnetic microspheres was compared with the Fe3+-chelated microspheres using tryptic digested peptides originating from ovalbumin, a five protein mixture containing phosphoproteins and nonphosphoproteins, as well as a mixture of beta-casein and BSA with a molar ratio of 1:50. Compared to Fe3+, Ce4+-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures. Even when the amount of the tryptic digest product of BSA is 50 times higher than that of beta-casein in the sample solution, the trace phosphopeptides derived from beta-casein can still be concentrated effectively by the Ce4+-chelated magnetic microspheres in only 30 s. Furthermore, we initially utilized the Ce4+-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment, which opens up a possibility for their further application in phosphoproteomics.     

Novel Fe3O4@TiO2 core-shell microspheres for selective enrichment of phosphopeptides in phosphoproteome analysis

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe 3O 4@TiO 2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of beta-casein, and the high specificity of the Fe 3O 4@TiO 2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of alpha-casein and beta-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and beta-casein). The Fe 3O 4@TiO 2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe 3O 4@TiO 2 core-shell microspheres.     

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.     

High-throughput proteomics using matrix-assisted laser desorption/ ionization mass spectrometry

It has become evident that the mystery of life will not be deciphered just by decoding its blueprint, the genetic code. In the life and biomedical sciences, research efforts are now shifting from pure gene analysis to the analysis of all biomolecules involved in the machinery of life. One area of these postgenomic research fields is proteomics. Although proteomics, which basically encompasses the analysis of proteins, is not a new concept, it is far from being a research field that can rely on routine and large-scale analyses. At the time the term proteomics was coined, a gold-rush mentality was created, promising vast and quick riches (i.e., solutions to the immensely complex questions of life and disease). Predictably, the reality has been quite different. The complexity of proteomes and the wide variations in the abundances and chemical properties of their constituents has rendered the use of systematic analytical approaches only partially successful, and biologically meaningful results have been slow to arrive. However, to learn more about how cells and, hence, life works, it is essential to understand the proteins and their complex interactions in their native environment. This is why proteomics will be an important part of the biomedical sciences for the foreseeable future. Therefore, any advances in providing the tools that make protein analysis a more routine and large-scale business, ideally using automated and rapid analytical procedures, are highly sought after. This review will provide some basics, thoughts and ideas on the exploitation of matrix-assisted laser desorption/ ionization in biological mass spectrometry – one of the most commonly used analytical tools in proteomics – for high-throughput analyses.     

Tracking and quantification of P-labeled phosphopeptides in liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC–MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.     

Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.     

Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities

Quantitative matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) was applied for the determination of concentrations of low-molecular-weight (< 400Da) substrates and products of enzyme-catalyzed reactions. Isotope-labeled and fluorinated internal standards were used for the quantification. Automated quantitative MALDI-ToF MS analysis of quenched samples allowed the direct and simultaneous observation of time-dependent decrease of substrate concentration and increase of product concentration without any need for prepurification or desalting steps. The results showed good agreement with established but more elaborate analytical methods. MALDI-ToF MS thus is an interesting alternative tool for the determination of enzyme activities. Due to automated and miniaturized measurement it is especially suitable for the screening of biocatalysts.     

Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry

Protein phosphorylation is a ubiquitous post-translational modification that affects a significant subset of the proteome and plays an especially important role in signal transduction and cell cycle control in eukaryotic organisms. Recently developed methods that couple multidimensional liquid chromatography to electrospray mass spectrometers can be used to analyze entire phosphoproteomes. However, they require considerable investments and technical skills that are only available in a few highly specialized laboratories. These methods also appear to be biased. Statistical analyses show that peptides from abundant proteins and multiply phosphorylated peptides are disproportionately identified. We describe an economic alternative that utilizes a phospho-affinity step to isolate the intact phosphoproteins. These are subsequently characterized by electrophoresis and identified by direct de novo sequencing using tandem mass spectrometry. We applied this technique to probe signal-induced changes in the phosphoproteome of human U937 cells, and found that the pools of two cancer-related phosphoproteins implicated in intracellular hormones signaling are dramatically altered in the course of monocyte to macrophage differentiation.     

Mass determination of low-molecular-weight proteins in phloem sap using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The low-molecular-weight (LMW), low-abundance protein composition of lupin and pea phloem exudates was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)> Phloem sap was collected from lupin inflorescence stalks and pods (using shallow incisions) or pea seedlings (by placing cut stems in an EDTA solution). Western blot analysis of phloem exudate proteins with either a polyclonal antibody raised against Ricinus communis sieve-tube exudate proteins or pea Rubisco antibody revealed that the collected exudates contained phloem sap, and that contamination with other plant fluids was negligible. Three matrix combinations were tested to assess their ability to facilitate protein ionization. Sinapinic acid in combination with trifluoroacetic acid yielded the cleanest mass spectra, and revealed an array of LMW proteins ranging from 2 to 10 kDa. For pea phloem exudate, the addition of protease inhibitors to the exudate collection solution prevented proteolysis of endogenous proteins; the inhibitors did not interfere with the detection of proteins. The sensitivity of this technique was sufficient to detect changes in LMW phloem peptides throughout plant development in lupin, or to detect differences in the phloem peptide composition of two genotypes of pea. Because only limited sample preparation is required, MALDI-TOF-MS is a useful technique for characterizing complex fluids such as phloem sap.     

Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) serves as a rapid and accurate means to determine masses of proteins independent of their shapes or interactions with other molecules. It provides one of the most fundamental characterizations of major plasma proteins. Purified proteins in saline or serum specimens were prepared for analysis by dilution, mixing with a solution of sinapinic acid, and drying on a target plate. Specimens were analyzed in a linear TOF mode with external calibration. Analyses of 24 purified plasma proteins showed predominance of singly charged ions with lesser amounts of dimer and doubly charged monomer, and provided measured masses for these proteins. A number of proteins, including albumin, transferrin, apolipoproteins A-I, A-II, C-I, C-II, and C-III, and prealbumin, could be analyzed directly in serum with appropriate dilution. Measured values for masses of major plasma proteins will assist in analysis of serum and plasma. It is possible to analyze a number of components by MALDI-TOF/MS directly in diluted serum. Extremely simple sample preparation techniques may be useful in analyzing structural variation of several major plasma proteins, particularly those with masses <30 kDa, including a number of apolipoproteins and markers of nutritional status or acute phase responses.     

Dual polarity accurate mass calibration for electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry using maltooligosaccharides

In view of the fact that memory effects associated with instrument calibration hinder the use of many mass-to-charge (m/z) ratios and tuning standards, identification of robust, comprehensive, inexpensive, and memory-free calibration standards is of particular interest to the mass spectrometry community. Glucose and its isomers are known to have a residue mass of 162.05282 Da; therefore, both linear and branched forms of polyhexose oligosaccharides possess well-defined masses, making them ideal candidates for mass calibration. Using a wide range of maltooligosaccharides (MOSs) derived from commercially available beers, ions with m/z ratios from approximately 500 to 2500 Da or more have been observed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and time-of-flight mass spectrometry (TOF-MS). The MOS mixtures were further characterized using infrared multiphoton dissociation (IRMPD) and nano-liquid chromatography/mass spectrometry (nano-LC/MS). In addition to providing well-defined series of positive and negative calibrant ions using either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), the MOSs are not encumbered by memory effects and, thus, are well-suited mass calibration and instrument tuning standards for carbohydrate analysis.     

Analysis of hemoglobin variants using nondenaturing gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry

Molecular analysis of hemoglobin variants is crucial in the diagnosis of hemoglobinopathies. Routinely used techniques for identifying variants include alkaline gel electrophoresis and automated HPLC. Sometimes comigration of variants in electrophoresis or coelution in HPLC provides ambiguous results. Due to high sequence homology between normal and variant hemoglobin, proteomic analysis using LC/ESI-MS data is also challenging. Here we describe a novel method wherein alkaline gel electrophoresis and MALDI-MS were used in combination to characterize variant samples such as Hb FSD and Hb D-Iran unambiguously. The method is rapid, efficient, and cost effective. In the future, it can be applied as a diagnostic tool.     

Phyloproteomics: species identification of Enterobacteriaceae using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as a tool for rapid identification of common clinical bacterial isolates, we analyzed 25 carefully selected isolates of pathogenic Escherichia coli (E. coli) and additional Enterobacteriaceae members. Organisms were prepared according to clinical microbiological protocols and analyzed with minimal additional processing. Spectra were reproducible from preparation to preparation and comprised 40-100 peaks primarily representing intracellular proteins with masses up to 25 kDa. Spectra of 14 genetically diverse bacteremic isolates of E. coli were compared with isolates representing other genera within the Enterobacteriaceae family. Using a new spectrum comparison algorithm, E. coli isolates were closely related to each other and were readily distinguishable from other Enterobacteriaceae, including Salmonella and Shigella. Presently, the methodology permits the analysis of 40 unknown isolates per hour per instrument. These results suggest that MALDI-ToF MS offers a rapid and reliable approach for performing phyloproteomics i.e., identification of unknown bacterial isolates based on similarities within protein biomarker databases.     

Localization of water-soluble carbohydrates in wheat stems using imaging matrix-assisted laser desorption ionization mass spectrometry

The pool of endogenous water-soluble oligosaccharides found in the stems of wheat (Triticum aestivum) is being investigated as a potential indicator of grain yield. Techniques such as liquid chromatography with mass spectrometry (LC-MS) can profile these analytes but provide no spatial information regarding their distribution in the wheat stem. The imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique has not been utilized for the analysis of oligosaccharides in plant systems previously. Imaging MALDI mass spectrometry was used to analyse cross and longitudinal sections from the stems of Triticum aestivum. A range of oligosaccharides up to Hex(11) were observed. Water-soluble oligosaccharides were ionized as potassiated molecules, and found to be located in the stem pith that is retained predominantly around the inner stem wall. Imaging MALDI analyses provided spatial information on endogenous oligosaccharides present in wheat stems. The technique was found to offer comparable sensitivities for oligosaccharide detection to those of our established LC-MS method, and has potential for broad application in studying the in situ localization of other compound types in plant material.     

Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry

Chitin/chitosan oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D-glucopyranose (GlcN) were prepared by chemical degradation of chitin or chitosan and separated by gel permeation chromatography. Oligosaccharides obtained after enzymatic hydrolysis of chitosan [F(A) 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The composition of a hexamer D3A3 was ca. 65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X not equal Y) are mutually either D or A. This structure motif was also observed in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)(3)-D-A-A.     

Rapid discrimination of environmental Vibrio by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The aim of this study was to discriminate 30 Vibrio strains isolated from two wastewater treatment plants from Agadir, Morocco by two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Out of the 30 strains of Vibrio examined in this study, 5 isolates could not be typed by PFGE and consistently appeared as a smear on the gel. In general, high genetic biodiversity among the Vibrio strains was found regardless to the isolation source. The results of MALDI TOF analysis show a high congruence of strain grouping demonstrating the accuracy and reliability of MALDI-TOF MS.     

Characterization of propionic acid bacteria using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

It was shown that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry could be used for the diagnostic characterization of propionic acid bacteria (PABs). The spectra of proteins (whole PAB cells) with a molecular mass of 3000 to 11 000 were obtained and analyzed using three matrices: sinapinic (SA), 2,5-dihydroxibenzoic (DHB), and α-cyano-4-hydroxycinnamic acid (HCCA). The MALDI spectra of PAB revealed the protein peaks characteristic of (1) the genus Propionibacterium (3496, 5386, 5605, 10 470), (2) the groups of species sharing the common composition of their cell walls and fatty acids, and (3) a species (four species were investigated). Exemplified by the P. shermanii strains (the collection and mutant ones) producing and not producing vitamin B₁₂, the possibility of using MALDI profiles for strain differentiation was confirmed. The MALDI profiles of the propionic acid cocci of the genus Luteococcus differ substantially from the profiles of PAB strains of the genus Propionibacterium, which is an additional proof of the validity of whole-cell MALDI spectra for generic differentiation of bacteria. Our investigation shows that the bacterial groups determined using the MALDI profiles correlate with the phylogenetic 16S rRNA gene groups, thus demonstrating the high resolution of this method for the differentiation of intraspecific differences (subspecies, strains).     

Determination of photosystem II subunits by matrix-assisted laser desorption/ionization mass spectrometry

Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes.     

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.