Identification of glycoconjugates in the urine of a patient with congenital disorder of glycosylation by high-resolution mass spectrometry

More than 150 molecular species were detected in a single glycoconjugate fraction obtained from urine of a congenital disorders of glycosylation (CDG) patient by use of high-resolution FT-ICR MS. With respect to its high-mass accuracy and resolving power, FT-ICR MS represents an ideal tool for analysis of single components in complex glycoconjugate mixtures obtained from body fluids. The presence of overlapping nearly isobaric ionic species in glycoconjugate mixtures obtained from CDG patient's urine was postulated from fragmentation data of several precursor ions obtained by nanoESI Q-TOF CID. Their existence was confirmed by high-resolution/high-mass accuracy FT-ICR MS detection. High-resolution FT-ICR mass spectra can, therefore, be generally considered for glycoscreening of complex mixture samples in a single stage. From the accurate molecular ion mass determinations the composition of glycoconjugate species can be identified. Particular enhancement of identification is offered by computer-assisted calculations in combination with monosaccharide building block analysis, which can be extended by considerations of non-carbohydrate modifications, such as amino acids, phosphates and sulfates. Taking advantage of this strategy, the number of compositions assigned to mass peaks was significantly increased in a fraction obtained from urine by size exclusion and anion exchange chromatography.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

Development and validation of a spectral library searching method for peptide identification from MS/MS

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching.     

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI‐TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.     

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

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

Characterization of the <Emphasis Type="Italic">N</Emphasis>-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)

Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to α1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. Conclusion: Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.     

Evaluation of Chemical Derivatisation Methods for Protein Identification using MALDI MS/MS

In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra.*Australian Peptide Conference Issue.**This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program.     

A reference map and identification of porcine testis proteins using 2-DE and MS

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Arbutin derivatives from the buds of Vaccinium dunalianum and their MS/MS spectrometric fragmentations

Four arbutin derivatives were isolated from the buds of Vaccinium dunalianum in which 4-hydroxyphenyl-6′-(3''-O-β-D-glucopyranosyl-4''-hydroxycinnamoyl)-O-β-D-glucopyranoside (1) was a new compound. The structure of the new compound was determined on the basis of NMR and HR-ESI-MS data. All the arbutin derivatives were subjected to the MS/MS analyses from which the MS/MS spectrometric fragmentations were summarized.     

MALDI‐TOF MS identification of microbiota associated with pest insect Diabrotica speciosa

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Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

Structure elucidation of ostreocin-A and ostreocin-E1, novel palytoxin analogs produced by the dinoflagellate Ostreopsis siamensis,using LC/Q-TOF MS

Palytoxin analogs are marine toxins with large complex polyol structures. A benthic dinoflagellate Ostreopsis siamensis produces more than ten palytoxins (ostreocins, OSTs). The limited sample availability of minor OSTs restricts the definition of their chemical structures. The present investigation characterizes structures of two minor OSTs, i.e., ostreocin-A (OSTA) and ostreocin-E1 (OSTE1), using ostreocin-D (OSTD) as a reference compound, by liquid chromatography/quadrupole-time-of-flight mass spectrometry. The molecular formulas of OSTA and OSTE1 were C₁₂₇H₂₁₉N₃O₅₄ and C₁₂₇H₂₁₇N₃O₅₂, respectively. Compared to OSTD, OSTA has an extra oxygen atom whereas OSTE1 lacks one oxygen atom and two hydrogen atoms. The MS/MS experiments (precursor ions: [M + H]⁺ and [M-H]^?) suggested a hydroxyl substitution at C82 in OSTA and alteration(s) between C53 and C100 in OSTE1. Further analysis of structural details in OSTE1 was performed through a pseudo-MS³ experiment (precursor ion: m/z 1432.748). Accordingly, the planar structures of OSTA and OSTE1 were assigned to 42,82-dihydroxy-3,26-didemethyl-19,44-dideoxypalytoxin and 42-hydroxy-3,26-didemethyl-19,44,73-trideoxypalytoxin-72-ene, respectively.

Abbreviations:CID: collision induced dissociation; HR-LC/MS/MS: high-resolution liquid chromatography/tandem mass spectrometry; LC/ESI/Q-TOF MS: liquid chromatography/quadrupole time-of-flight mass spectrometry equipped with an electrospray ionization source; NMR: nuclear magnetic resonance; OSTs: ostreocins; OSTA: ostreocin-A; OSTB: ostreocin-B; OSTD: ostreocin-D; OSTE1: ostreocin-E1; OVTX-a: ovatoxin-a; OVTXs: ovatoxins; PLTX: palytoxin     

Serum N-glycan and O-glycan analysis by mass spectrometry for diagnosis of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDGs) are caused by defects in genes that participate in biosynthetic glycosylation pathways. To date, 19 different genetic defects in N-glycosylation, 17 in O-glycosylation, and 21 in multiple glycosylation are known. Current diagnostic testing of CDGs largely relies on indirect analysis of glycosylation of serum transferrin. Such analysis alone is insufficient to diagnose many of the known glycosylation disorders. To improve the diagnosis of these groups of CDGs, we have developed serum or plasma N- and O-glycan profiling using a combination of MALDI–TOF/MS and LC–MS/MS technologies. Using this approach, we analyzed samples from nine patients with different known multiple glycosylation disorders, including three with COG deficiencies, one with TMEM165-CDG, two with PGM1-CDG, and three with SLC35A2-CDG, and one patient with combined type I and type II of unknown molecular etiology. Measurement of the relative quantities of various N- and O-glycan species clearly differentiates patients and controls. Our study demonstrates that structural analysis and quantitation of combined N- and O-glycan profiles are reliable diagnostic tools for CDGs.     

Discrimination of intact mycobacteria at the strain level: a combined MALDI-TOF MS and biostatistical analysis

New methodologies for surveillance and identification of Mycobacterium tuberculosis are required to stem the spread of disease worldwide. In addition, the ability to discriminate mycobacteria at the strain level may be important to contact or source case investigations. To this end, we are developing MALDI-TOF MS methods for the identification of M. tuberculosis in culture. In this report, we describe the application of MALDI-TOF MS, as well as statistical analysis including linear discriminant and random forest analysis, to 16 medically relevant strains from four species of mycobacteria, M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. Although species discrimination can be accomplished on the basis of unique m/z values observed in the MS fingerprint spectrum, discrimination at the strain level is predicted on the relative abundance of shared m/z values among strains within a species. For the 16 mycobacterial strains investigated in the present study, it is possible to unambiguously identify strains within a species on the basis of MALDI-TOF MS data. The error rate for classification of individual strains using linear discriminant analysis was 0.053 using 37 m/z variables, whereas the error rate for classification of individual strains using random forest analysis was 0.023 using only 18 m/z variables. In addition, using random forest analysis of MALDI-TOF MS data, it was possible to correctly classify bacterial strains as either M. tuberculosis or non-tuberculous with 100% accuracy.     

Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.