Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.     

Alkaline degradation of oligosaccharides coupled with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry: a method for sequencing oligosaccharides

A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the "peeling" reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by beta-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.     

Comparison of fragmentation modes for the structural determination of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry

Fragment ions from underivatized N-linked oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry were obtained by spontaneous fragmentation on a magnetic sector mass spectrometer, by post-source decay (PSD) on a reflectron time-of-flight (TOF) instrument and by collision-induced dissociation on a magnetic sector instrument fitted with an orthagonal-TOF analyser. Spontaneous fragmentation on the magnetic sector instrument produced ions mainly by glycosidic cleavage together with two abundant ions formed by cross-ring cleavage of the reducing-terminal residue. The PSD spectra were similar, the majority of ions being formed by glycosidic cleavage. Internal fragment ions were abundant. High-energy collision-induced dissociation spectra recorded with the orthagonal-TOF analyser, differed considerably from the other types of spectra, particularly in the appearance of major fragment ions produced by cross-ring cleavages of most of the constituent monosaccharide residues. These ions allowed much sequence and branching information to be obtained from the oligosaccharide.     

Evaluation of IgG glycation levels by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been employed for the evaluation of the glycation level of IgG from healthy subjects and also from well- and badly-controlled diabetic patients. The measurements have been performed on untreated plasma protein fractions. The data obtained have shown that a clear mass increase, originating from non-enzymatic glycation processes, is observed in the case of diabetic patients: for well-controlled ones it is in the range 512-1565 Da, while it becomes 827-4270 Da for badly-controlled diabetic patients. This approach indicates that MALDI mass spectrometry is a highly specific tool that can be employed in the metabolic control of diabetic patients and in studies relating the IgG glycation level to possible immunological impairment.     

Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of partially methyl-esterified enzymatic pectin digests is described. The sensitivities of several matrices, positive and negative ion modes and desalting techniques for these acidic oligosaccharides were compared. The most favorable results were obtained with a thin-layer preparation of a mixture of 2,4,6-trihydroxyacetophenone and nitrocellulose in the negative ion mode. Results are presented demonstrating the sensitive characterization of separated and unseparated high-ester pectin digests obtained after complete digestion using Aspergillus niger pectin lyase and the analysis of digests after chemical modification. In the case of unseparated digests, the analysis of methylation patterns is demonstrated. Oligomers with a degree of polymerization up to 40 were detected after enrichment of large oligomers by propan-2-ol precipitation.     

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.     

Accurate peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Partial 18O-labeling of peptides has been applied to post-source decay experiments in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The ions which originate from the carboxyl terminus of a peptide partially retain 18O atoms which have readily been incorporated into the C-terminal carboxyl groups during enzymatic hydrolysis in a buffer containing 40 atom percent H218O. The isotopic resolution of singly charged precursor ions and their product ions obtained for peptides up to ca. 2800 Da has been achieved using the delayed extraction method, which permits the rapid identification and assignment of the 18O-labeled and non-labeled ion species in the PSD spectra. The results obtained from several 18O-labeled peptides, derived from an enzymatic digest, demonstrate the accuracy and reproducibility of the present method, which will be in widespread use for protein identification via database searching or for investigations of totally unknown proteins.     

Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry

During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

The hydrolytic degradation of poly(DL-lactide) (PLA50) material was investigated in order to elucidate the effects of temperature and acidity of the external medium on the degradation characteristics. It was observed that at 60 degrees C and at pH = 7.4, degradation was extremely rapid as compared with degradation at 37 degrees C. After only 2 days, heterogeneous degradation was observed due to larger internal autocatalysis. On the other hand, in the case of degradation at 37 degrees C in an acidic medium with pH = 3.7, the heterogeneous degradation was preceded by a much longer lag time. Water absorption was found to be pH dependent. At pH = 7.4, water absorption was increased due to the osmotic pressure driving the buffer solution into the polymer matrix to neutralize acidic endgroups, which was not the case for degradation at pH = 3.7. In both cases, the oligomeric stereocomplex was obtained as degradation residue at the end of the degradation period.     

Off-line coupling of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An automated fraction collection interface is used in conjunction with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry to analyze material isolated by capillary electrophoresis (CE). CE fractions are deposited directly on the MALDI probes so that individual peaks from the electropherogram are associated with a single sample spot on the probe. MALDI matrices with high acid concentrations afford enhanced tolerance of electrophoresis buffers. The utility of this hybrid instrument is demonstrated by separation and mass analysis of a tryptic digest of cytochrome c and synthetic mixtures of four proteins. Mass assignments corresponding to the protonated molecular ions are in good agreement with those predicted from molecular structure. Miniaturization of the interface affords enhanced sensitivity, with good-quality spectra from separations of as little as 25 fmol of protein.     

Polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry for tRNA mutant analysis

In analogy to two-dimensional analysis, the mobility shift in native polyacrylamide gel electrophoresis (PAGE) due to a nucleotide substitution of a single-stranded transfer ribonucleic acid (tRNA) fragment serves as the first dimension for tRNA mutation analysis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), as the second dimension, allows precise determination of the mass of the tRNA fragments resolved by native PAGE. Off-line combination of native PAGE with MALDI-MS is demonstrated for high-resolution analysis of tRNAval and its mutants, including a three-nucleotide deletion and 12 single-base substitutions. Three approaches, including direct extraction of tRNAs from gel into buffer solution, dissolution of membrane in the matrix solution, and direct desorption of tRNAs from the membrane, are studied for coupling native PAGE with MALDI-MS. The membrane dissolution method is simple, and the resulting mixture is amenable to MALDI-MS analysis. In the membrane dissolution method, as little as 1 microg or 40 pmol of tRNA sample is loaded on a native gel, separated, capillary eluted onto a nitrocellulose membrane, and recovered using the matrix solution of 2,4,6-trihydroxyacetophenone in acetone.     

Exploring infrared wavelength matrix-assisted laser desorption/ionization of proteins with delayed-extraction time-of-flight mass spectrometry

We report a study of the application of delayed extraction (DE) to infrared-wavelength matrix-assisted time-of-flight mass spectrometry (IR-MALDI-TOF-MS) of proteins. The shapes of the spectral peaks obtained with DE-IR-MALDI-MS are compared with those obtained from the same samples and matrix using continuous extraction (CE) IR-MALDI-MS. Application of DE results in significant improvements in the peak resolution, revealing spectral features (in proteins with molecular masses < 12 kDa) that were not resolved in the corresponding CE-IR-Maldi mass spectra. Particularly significant is a series of peaks on the high mass side of the protonated protein peaks that arise through replacement of protons by adventitious sodium ions in the sample. We deduced that these sodium replacement species are a significant contributor to the broad tails (and resulting peak asymmetries) that are a feature of the DE-IR-MALDI mass spectra of proteins with molecular masses > or = 17 kDa. The peak width reduction observed in IR-MALDI by DE suggests that, as in UV-MALDI, the initial velocity distribution for ions produced in the MALDI process contributes to the peak broadness in the CE mass spectra. In a systematic comparison between DE UV-MALDI and DE IR-MALDI, we determined that photochemical matrix adduction is present in UV-MALDI but absent in IR-MALDI. In addition, we find that protein ions produced by IR irradiation are less internally excited (i.e., cooler), exhibiting less fragmentation, more Na+ replacement and/or unspecified noncovalent adduction, and more heme adduction with apomyoglobin. Thus, IR-MALDI appears to be a softer means for producing gas-phase protein ions than is UV-MALDI. It will be of considerable practical interest to determine whether large protein ions produced by IR-MALDI are sufficiently cool to survive transport through reflecting TOF mass spectrometers (without loss of small neutral species such as H2O, NH3, and CO2) and the extended time periods required for detection by quadrupole ion trap and Fourier transform ion cyclotron resonance mass analyzers.     

Structure analysis of trivalent glycoclusters by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

Post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectra were found to be useful for the structural elucidation of a series of tris [2-(glycosylthiourylene)ethyl]amines. The reported fragmentation behaviours of [M + H]+, [M + Na]+ and [M - H]- ions differ from each other significantly; however, they can be compared to tree pruning in every case. Whereas detailed structural information on unprotected glycoclusters is obtained from all PSD experiments, only the positive-ion mode can be used to gain relevant information about the acetylated glycoclusters.     

Studies on the selection of new matrices for ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new group of compounds has been successfully tested as matrices for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS). Several new matrices for UV-MALDI MS have been found by choosing, as potential matrices, compounds that perform an intramolecular proton transfer along an intramolecular H-bond under UV irradiation. Compounds of this type are, for example, salicylamide, salicylanilide, several ortho-hydroxyacetophenones and ortho-hydroxybenzophenones. The matrix activity of these compounds is compared to the corresponding meta- and para-isomers and to the matrix activity of such well known matrices as 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone. It was found that meta- and para-substituted hydroxycarbonyl compounds show either a significantly lower or no matrix activity compared with the ortho isomers.     

Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A methodological approach

It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin, oriP, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the oriP region. Second, the DNA sequence of oriP alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in oriP. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in oriP at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.     

Characterization of transthyretin mutants from serum using immunoprecipitation, HPLC/electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry

A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitive diagnosis of ATTR relies on the detection and identification of TTR variants. The approach presented here is based on isolation of serum TTR using immunoprecipitation. The detection of the variant is achieved by mass measurement of the intact protein with electrospray ionization mass spectrometry (ESIMS). The liquid chromatography/ESIMS analysis of the tryptic digest of the protein followed by subsequent matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and MALDI postsource decay of the relevant recovered chromatographic fraction containing the variant peptide allows the identification of unknown variants. The method was successfully tested using serum from ATTR patients with known variants (Val30-->Met and Val122-->Ile). A new TTR variant, Ser23-->Asn, was detected and identified using the above method where isoelectric focusing and restriction enzyme analysis failed to identify the nature of the variant.     

Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans.