Pierre Thibault

Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte‐myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.

Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector and a protein digestion bed on a monolithic substrate. A large channel, 800 microm wide, 150 microm deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 microm diameter beads. Separation was performed in channels etched 10 microm deep, 30 microm wide and about 45 mm long, feeding into a capillary, attached to the chip with a low dead volume coupling, that was 30 mm in length, with a 50 microm i.d. and 180 microm o.d. Sample was pumped through the reactor bed at flow rates between 0.5 and 60 microL/min. The application of this device for rapid digestion, separation and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA). The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 microL/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for cytochrome c to 71% for BSA, with some missed cleavages observed. Melittin was consumed within 5 s. In contrast, a similar extent of digestion of melittin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ratio of trypsin to sample. This chip design thus provides a convenient platform for automated sample processing in proteomics applications.

Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry☆☆☆

The application of capillary electrophoresis-mass spectrometry (CE-MS) to the analysis of compounds of concern to the aquaculture industry is reported. Two different approaches to coupling the CE column to an IonSpray atmospheric pressure ionization (API) interface, viz., a liquid-junction and a coaxial arrangement, are describe and compared with regard to ruggedness, ease of use, sensitivity and electrophoretic performance. The different injection modes used in three commercial capillary electrophoresis systems were also evaluated for their applicability to CE-MS. The use of CE-MS for the analysis of a variety of classes of antibiotics used in the fish aquaculture industry, such as the sulfonamides and their potentiators (e.g., trimethoprim), is demonstrated and was used to confirm the presence of these components in shellfish extracts at the low ppm level. CE-MS was also applied to the analysis of marine toxins such as saxitoxin and its analogues which are associated with paralytic shellfish poisoning, and also the toxins responsible for amnesic and diarrheic shellfish poisoning. Tandem mass spectrometry (MS-MS) was used to provide structural information on these analytes, and the ability to distinguish isomeric compounds based on their different migration and fragmentation characteristics using CE-MS-MS is demonstrated.

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins

Flagellin from Campylobacter coli VC167 is post‐translationally modified at ≥ 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7‐diacetamido‐3,5,7,9 ‐ tetradeoxy ‐ l ‐ glycero ‐ l ‐ manno ‐ nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81‐176. VC167 lacked two modi‐fications present in 81‐176 and instead had two unique modifications of masses 431 and 432u2003Da. Flagellins from both C. jejuni 81‐176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81‐176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS‐PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315u2003Da and 431u2003Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81‐176 pseA gene in trans resulted in flagellins containing both 315 and 431u2003Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.

Identification of differentially expressed proteins in human glioblastoma cell lines and tumors

An in‐frame deletion of 801 bp in exons 2–7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGΔEGFR) that confers high malignancy to tumor cells. Two‐dimensional gel electrophoresis followed by in‐gel digestion of separated spots and protein identification by LC‐MS‐MS and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGΔEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT‐PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low‐grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors. GLIA 42:194–208, 2003.

Mass spectral analyses of microcystins from toxic cyanobacteria using on-line chromatographic and electrophoretic separations

The application of capillary electrophoresis and of reversed-phase liquid chromatography coupled to electrospray mass spectrometry is presented for the analysis of microcystins isolated from toxic strains of Microcystis aeruginosa. The separation performance of these two techniques is compared in terms of both sensitivity and of resolution of closely related microcystins. Quantitation of microcystin-LR present at low micrograms/ml concentrations in cell extracts is demonstrated using both techniques. A marked advantage of capillary electrophoresis over liquid chromatography was its ability to resolve different desmethyl microcystin-LR analogues. Identification of these positional isomers was facilitated using capillary electrophoresis combined with tandem mass spectrometry (MS-MS). Rationalization of fragment ions observed in MS-MS spectra of microcystins was made possible through comparison with 15N labelled microcystins obtained from stable isotope feeding experiments. The potential of tandem mass spectrometry in providing selective detection of microcystins in cell extracts, and in structural characterization of novel microcystins, was also investigated.

Development of electrophoretic conditions for the characterization of protein glycoforms by capillary electrophoresis—electrospray mass spectrometry☆

A capillary electrophoresis (CE) method using acidic buffers and capillaries coated with Polybrene, a cationic polymer has been developed for the separation of glycoproteins and glycopeptides. Electrophoretic conditions have been optimized to provide resolution of individual glycoforms observed for different glycoprotein preparations. These conditions were found to be entirely compatible with the operation of electrospray mass spectrometry (ESMS), which facilitated the assignments of possible carbohydrate compositions of glycopeptides arising from digests of glycoproteins. By using operating conditions enhanced the formation of oxonium fragment ions prior to mass spectral analysis, selective identification of glycopeptides was achieved for complex samples such as those from proteolytic digests or chemical cleavages. Examples of applications are presented for ribonuclease B, ovalbumin, horseradish peroxidase, and a lectin from Erithrina corallodendron using both CE-ESMS and CE with ultraviolet detection (CE-UV).

Analysis of paralytic shellfish poisons by capillary electrophoresis

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.

Glycopeptide specificity of the secretory protein folding sensor UDP–glucose glycoprotein:glucosyltransferase

Secretory and membrane N‐linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N‐linked Man9‐GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9‐GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino‐acid residues close to N‐linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.

Jadomycin, a novel 8H-benz[b]oxazolo[3,2-f]phenanthridine antibiotic from from streptomyces venezuelae ISP5230.☆

In a galactose-isoleucine medium at 37 °C, Streptomyces venezuelae ISP5230 produces jadomycin (1), the first 8H-benz[b]oxazolo[3,2]phenanthridine derivative to be isolated from natural sources. The structure of 1 was assigned by interpretation of the spectral data.