Resistance of 3'-phosphoglycolate DNA ends to digestion by mammalian DNase III

An essential step in the repair of free radical-mediated DNA strand breaks is the removal of sugar fragments such as phosphoglycolate from the 3' termini. While the abasic endonuclease Ape1 can remove phosphoglycolate from single-strand breaks in double-stranded DNA, an enzyme capable of removing it from 3' overhangs of double-strand breaks has yet to be identified. We therefore tested DNase III, the predominant 3' --> 5' exonuclease in mammalian cell extracts, for possible 3'-phosphoglycolate-removing activity. However, all 3'-phosphoglycolate substrates, as well as a 3'-phosphate substrate, were resistant to DNase III under conditions in which the analogous 3'-hydroxyl substrates were extensively degraded. The DNA end-binding protein Ku (an equimolar mixture of Ku70, now known as G22P1, and Ku86, now known as XRCC5) did not alter the resistance of the 3'-phosphoglycolate substrates, but the protein modulated the susceptibility of 3'-hydroxyl substrates, allowing DNase III to remove a 3' overhang but inhibiting digestion of the double-stranded portion of the substrate.     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using their fully 15N-labeled analogues as internal standards

Oxidatively induced DNA damage is implicated in disease, unless it is repaired by DNA repair. Defects in DNA repair capacity may be a risk factor for various disease processes. Thus, DNA repair proteins may be used as early detection and therapeutic biomarkers in cancer and other diseases. For this purpose, the measurement of the expression level of these proteins in vivo will be necessary. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-hydroxyguanine-DNA glycosylase (hOGG1) and Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of oxidatively induced DNA damage. We overproduced and purified (15)N-labeled analogues of these proteins to be used as suitable internal standards to ensure the accuracy of quantification. Unlabeled and (15)N-labeled proteins were digested with trypsin and analyzed by LC-MS/MS. Numerous tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their (15)N-labeled analogues were analyzed by selected-reaction monitoring on the basis of product ions. The results obtained suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases.     

Base sequence damage in DNA from X-irradiated monkey CV-1 cells

Two kinds of 3'-ends were detected in DNA scission fragments of highly repetitive primate component alpha DNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3'-ends were characterized by 5'-32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3'-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3'-phosphoryl and 3'-phosphoglycolate, and that they are converted to slower migrating fragments with 3'-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3'-phosphoryl and 3'-phosphoglycolate ends was dose-dependent over a range 100-900 Gy. In CV-1 cells the frequency of single-strand breaks in alpha DNA was 8.6 x 10(-7) breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1.6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.     

Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments

Polypeptide release factor one from Thermus thermophilus, ttRF1, was purified and subjected to crystallization. Thin crystalline needles were obtained but their quality was not satisfactory for X-ray diffraction. Stable fragments of ttRF1 suitable for crystallization were screened by limited proteolysis. Three major fragments were produced by thermolysinolysis and analyzed by N-terminal sequencing and electrospray mass spectrometry. They were N-terminal fragments generated by proteolysis at amino acid positions 211, 231 and 292. The corresponding recombinant polypeptides, ttRF1(211), ttRF1(231) and ttRF1(292), were overproduced and subjected to crystallization. Of these polypeptides, ttRF1(292) gave rise to crystals that belong to P3(1) (or P3(2)) space group with unit cell parameters a = b = 64. 5 A, c = 86.6 A and diffract up to 7 A resolution.     

Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides

Differentiated osteoblastic cell line, MC3T3-E1 expresses transglutaminase 2 (TG2) and Factor XIII (FXIII). In previous studies, we identified isozyme-specific and highly reactive glutamine-donor substrate peptides (pepF11KA and pepT26) for each isozyme. Using these peptides, we compared the reaction products with lysine-donor substrates for each isozyme in differentiating MC3T3-E1 cells. By this analysis, distinct substrates for the activated TG2 and FXIII were detected in cultured cellular extract. Possible substrates that incorporated biotin-labeled peptides were further purified using streptavidin-affinity chromatography. Several isozyme-specific substrates were identified by mass spectrometry analysis of the purified fractions. These analyses also indicate the benefit of the substrate peptides for obtaining distinct substrates in a reaction mixture where two isozymes co-exist.     

A new approach for plant proteomics: characterization of chloroplast proteins of Arabidopsis thaliana by top-down mass spectrometry

A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate "bottom-up" mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (+/-1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation.     

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.     

3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage

In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.     

Plasma desorption mass spectrometry of haemoglobin tryptic peptides for the characterization of a Hungarian alpha-chain variant.

S-Aminoethylated-alpha A and -beta A globin tryptic peptides separated by reversed-phase high-performance liquid chromatography have been analysed by plasma desorption mass spectrometry. Almost all the expected alpha A and beta A tryptic fragments were tentatively assigned relative to the known globin chain sequences based on the molecular weight obtained by plasma desorption mass spectrometric analysis of the purified peptides. The application of plasma desorption mass spectrometry for structure elucidation of a haemoglobin alpha-chain variant revealed the first case of Hb Hasharon in Hungary.     

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.     

Human HeLa cell enzymes that remove phosphoglycolate 3''-end groups from DNA.

We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate.     

Detection of new amino acid sequences of alamethicins F30 by nonaqueous capillary electrophoresis-mass spectrometry.

The microheterogeneous alamethicin F30 (ALM F30) isolated from the fermentation of Trichoderma viride strain NRRL 3199 was analyzed by nonaqueous capillary electrophoresis coupled to electrospray ion-trap mass spectrometry (ESI-IT-MS) and electrospray time-of-flight mass spectrometry (ESI-TOF-MS). Tandem ESI-IT-MS was used for elucidation of the amino acid sequence based on the fragmentation pattern of selected parent ions. The MS/MS spectra using the [M + 3H](3+) or [M + 2H](2+) ions as precursor ions displayed the respective b- and the y-type fragments resulting from cleavage of the particularly labile Aib-Pro bond. The MS(3) of these fragments generated the b acylium ion series, as well as internal fragment ion series. Eleven amino acid sequences were identified, characterized by the exchange of Ala to Aib in position 6, Gln to Glu in positions 7 or 19 as well as the loss of the C-terminal amino alcohol. In addition, two truncated pyroglutamyl peptaibols were found. Overall, seven new sequences are reported compared to earlier LC-MS studies. The composition of the components was confirmed by on-line ESI-TOF-MS detection. Mass accuracy well below 5 ppm was observed. Quantification of the individual components was achieved by a combination of UV and TOF-MS detection.     

Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI

The self-complementing dodecamer 5'-CGCGAATTCGCG-3' and its complexes with the antibiotic netropsin and the restriction endonuclease EcoRI provide substrates of known three-dimensional structure to study the stereochemistry and mechanism of the artificial nuclease of 1,10-phenanthroline-copper ion [(OP)2Cu+]. Analysis of the reaction products with the 5'-32P dodecamer on 20% sequencing gels has demonstrated the presence of 3'-phosphoglycolate ends in addition to 3'-phosphomonoester ends expected from previous studies. A reaction intermediate, which is a precursor to 3'-phosphomonoester termini, has been trapped; in contrast, no comparable species for the 5'-phosphomonoester termini can be detected when 3'-labeled DNAs are utilized as substrates. The reactive oxidative species formed by the coreactants (OP)2Cu+ and hydrogen peroxide is distinguishable in its chemistry from the hydroxyl radicals produced by cobalt-60 gamma-irradiation. The freely diffusible hydroxyl radicals generated by cobalt-60 irradiation produce equivalent amounts of 3'-phosphomonoester and 3'-phosphoglycolate termini whereas the 3'-phosphomonoesters are the preferred product of (OP)2Cu+ and H2O2. On the basis of the structures of the products obtained, the principal site of attack of the coordination complex is on the C-1 of the deoxyribose within the minor groove. This conclusion is supported by the footprinting of netropsin binding to the dodecamer. Crystallographic results have demonstrated that netropsin binds to the minor groove at the central AATT residue. A clear protection of attack by the coordination complex at the deoxyriboses associated with A-5, T-6, T-7, and C-9 is fully consistent with attack from the minor groove without intercalation during the course of the cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

Mass spectrometric analysis of long-chain esters of diols

Homologous series of synthetic long-chain monoesters and diesters of 1,2-ethanediol were analyzed by mass spectrometry, and the patterns of fragmentation were studied. Under electron impact saturated ethanediol monoesters yielded prominent ions characteristic of the short-chain diol, such as the rearranged ion formed by 2,3-cleavage (m/e 104) and the ion caused by 3,4-cleavage (m/e 117). Fragments characteristic of the constituent long-chain moieties were the acylium ions [RCO](+), [RCO - 1](+), and the ions [RC(OH)(2)](+). The mass spectra of ethanediol diesters exhibited very intense peaks due to the ions formed by loss of the acyloxy group, [M - RCOO](+), or one carboxylic acid, [M - RCOOH](+). These ions are distinctive for diol diesters. Precise mass measurements by high resolution mass spectrometry verified the composition of the ion fragments. Spectral studies of some monoesters and diesters of 1,3-propanediol, 1,4-butanediol, 2,3-butanediol, and also of some monounsaturated homologues, demonstrated that mass spectrometry is very suitable for the identification, distinction, and analysis of diol lipids.     

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: Structural characterization and identification as histidine-rich glycoprotein

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.     

Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

This paper reviews methods for the analysis of N-linked glycans by mass spectrometry with emphasis on studies conducted at the Oxford Glycobiology Institute. Topics covered are the release of glycans from sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, their purification for analysis by mass spectrometry, methods based on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization for producing fragment ions, and details of their fragmentation. MALDI mass spectrometry provided a rapid method for profiling neutral N-linked glycans as their [M + Na](+) ions which could be fragmented by collision-induced decomposition to give spectra containing both glycosidic and cross-ring fragments. Electrospray ionization mass spectrometry was more versatile in that it was relatively easy to change the type of ion that was formed and, furthermore, unlike MALDI, electrospray did not cause extensive loss of sialic acids from sialylated glycans. Negative ions formed by addition of anions such as chloride and, particularly, nitrate, to the electrospray solvent were stable and enabled singly charged ions to be obtained from larger glycans than was possible in positive ion mode. Fragmentation of negative ions followed specific pathways that defined structural details of the glycans that were difficult to obtain by classical methods such as exoglycosidase digestion.     

重组人胰高血糖素样肽-1的表达、纯化及其生物学活性

为获得重组人胰高血糖素样肽 1[recombinanthumanglucagon likepeptide 1(7～ 37) ,rhGLP 1]并研究其生物学活性 ,采用亚磷酸二酯法合成hGLP 1cDNA的 6个寡核苷酸片段 ,拼接成完整的hGLP 1cDNA ,构建重组质粒pGEX hGLP 1,转化大肠杆菌BL2 1(DE3)获得表达菌株 .高密度发酵培养的菌体超声破碎后 ,裂解液用Glutathione Sepharose 4B亲和层析纯化得到GST融合蛋白 .经CNBr裂解、QAE SepharoseFF柱层析和脱盐 ,得到纯度大于 90 %的rhGLP 1,质谱测定分子量结果与理论值一致 .生物学活性分析表明 ,rhGLP 1具有明显的降血糖活性 .     

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.