Characterization of changes in the proteome in different regions of 3D multicell tumor spheroids.

Three dimensional multicell tumor spheroids (MCTS) provide an experimental model where the influence of microenvironmental conditions on protein expression can be determined. Sequential trypsin digestion of HT29 colon carcinoma MCTS enabled segregation into four populations comprising proliferating cells from the surface (SL), an intermediate region (IR), nonproliferating hypoxic cells from the perinecrotic region (PN), and a necrotic core (NC). Total protein was extracted from each population and subjected to iTRAQ-based quantitative proteomics analysis. From a total of 887 proteins identified, 209 were observed to be up-regulated and 114 were down-regulated in the PN and NC regions relative to the SL. Among the up-regulated proteins, components of glycolysis, TCA cycle, lipid metabolism, and steroid biosynthesis increased progressively toward the PN and NC regions. Western blotting, immunohistochemistry, and enzyme assays confirmed that significant changes in the expression of proteins involved in cellular metabolism occur in the nonproliferating fraction of cells within the viable rim. The presence of full length, functional proteins within the NC was unexpected, and further analysis demonstrated that this region contains cells that are undergoing autophagy. This study has identified possible targets that may be suitable for therapeutic intervention, and further studies to validate these are required.

Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus.

The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.

The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae.

The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.

Peptide mass fingerprinting of chaperonin-containing TCP-1 (CCT) and copurifying proteins.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research, CCT isolated from mouse testis lysate sediments at 20S in a sucrose gradient and accounts for about 70% of the total protein in this fraction. We intend to identify all the other proteins that copurify with CCT and to compile a reference profile for future studies. Their identification can be accelerated by a combination of protease digestion, matrix-assisted laser desorption-mass spectrometry, and database matching known as peptide mass fingerprinting. We applied this strategy to 32 polypeptides resolved by 2-dimensional gel electrophoresis, and 23 known proteins and 6 novel proteins were identified. We analyzed isoelectric variants of the CCT subunits and differences in the peptide mass spectra of two CCT theta isoforms indicated a novel posttranslational modification of this subunit.

Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.

Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry.

A rapid and sensitive method for sequencing oligosaccharides has been developed, using matrix-assisted laser desorption mass spectrometry to monitor the digestion of glycopeptides by specific exoglycosidases. Recombinant human tissue inhibitor of metalloproteinases (TIMP), which has two glycosylation sites, has been characterized to illustrate this new approach for obtaining site-specific information. Glycopeptides which span residues Asn30 and Asn78 were generated by tryptic digestion of 1 nmol of TIMP and separated by reverse-phase high-performance liquid chromatography. The oligosaccharide composition of the glycoforms was inferred from the observed mass shifts following digestion by peptide-N-glycosidase F. Composition and sequence were then elucidated by digestion with specific exoglycosidases, using a total of 200 pmol of each glycopeptide. Glycopeptides from well-characterized proteins, fetuin, alpha 1-acid glycoprotein, and tissue plasminogen activator were also analyzed to confirm exoglycosidase specificity for glycopeptides and establish the quantitative significance of the relative intensities of peaks in the mass spectra. Both TIMP glycosylation sites exhibited extensive heterogeneity comprising mainly fucosylated complex oligosaccharides, but in different proportions. The Asn78 site also contained 4.4% nonfucosylated mannose (Man4) oligosaccharide. The merits and limitations of this approach as a universal method for oligosaccharide analysis are discussed.

The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Sample immobilization protocols for matrix-assisted laser-desorption mass spectrometry.

Methods are described for the purification of proteins prior to analysis by matrix-assisted laser-desorption mass spectrometry. Contaminated protein samples were immobilized onto the surfaces of sample targets and rinsed. In general, a layer of electrosprayed nitrocellulose gave better results than the roughened gold surface of an untreated target. Using this approach, spectra could be obtained from low picomole quantities of protein in the presence of contaminants which did not inhibit the binding of the protein to the substrate.

Characterisation of two forms of phosphoribulokinase isolated from the green alga, Scenedesmus obliquus.

Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42,000-Mr subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of Mr 470,000 and apparent subunit composition X8Y4. The subunits X and Y are of Mr 39,000 and 42,000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD. Depolymerisation of the latent phosphoribulokinase to give the low-Mr form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol. The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-Mr latent form of the enzyme.

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes.

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts--fructose-1,6-bis-phosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase), and phosphoribulokinase (PRK)--were partially purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin system, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5'-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.