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1.
Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.  相似文献   

2.
A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 μg of pure horseradish peroxidase were successfully purified.  相似文献   

3.
Cartilage extracellular matrix molecules synthesized and maintained by chondrocytes form a strong, elastic tissue functioning to cushion and protect the subchondral bone. Osteoarthritis is characterized by degradation of cartilage extracellular matrix molecules resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. With recent interest in the degradation of cartilage extracellular matrix molecules, a need for more detailed structural information exists. Posttranslational modifications are believed to play a role in determining the susceptibility of these molecules to proteolytic degradation during the development of osteoarthritis. The purpose of this paper is to show how the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to extracellular matrix protein and proteoglycan structure will help elucidate problems in extracellular matrix biochemistry. Methodological issues relating to the high molecular weight, polydispersity, and high degree of posttranslational modification of these molecules are discussed. MALDI-TOF mass spectrometry provides an improved level of detail for extracellular matrix protein and proteoglycan structure and is useful in addressing issues surrounding the causes of degradation during osteoarthritis.  相似文献   

4.
Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]+ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]+ and [M + Na]+ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.  相似文献   

5.
6.
Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.  相似文献   

7.
Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.  相似文献   

8.
A robust high-throughput single-nucleotide polymorphism (SNP) genotyping method is reported, which applies allele-specific extension to achieve allelic discrimination and uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to measure the natural molecular weight difference of oligonucleotides for determination of the base in a single-nucleotide polymorphic location. Tenfold PCR is performed successfully by carefully designing the primers and adjusting the conditions of PCR. In addition, two ways used for PCR product purification are compared and the matrix used in mass spectrometry for high-throughput oligonucleotide analysis is evaluated. The result here shows that the method is very effective and suitable for high-throughput genotyping of SNPs.  相似文献   

9.
A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.  相似文献   

10.
Cardiolipin (CL) is an anionic tetraacylphospholipid found in mammalian tissues, inner membrane of mitochondria and in the cytoplasmic membrane of Gram-positive and -negative bacteria. Lipid A is the principal structural component responsible for the range of biological activities of lipopolysaccharides. Here we report a MALDI-MS-based method for the sensitive simultaneous analysis of CL and lipid A from Helicobacter pylori cells. The sensitivity was demonstrated by the analysis of CL and lipid A from a single bacterial colony of in vitro grown H. pylori strain NCTC 11637 (ATCC 43504). We then characterized the CL and lipid A structures in H. pylori cells grown under three different conditions, on agar-horse blood plates, in liquid culture and ex vivo. The results revealed the presence of high amounts of myristic (C14:0) and 19-carbon cyclopropane (C19:0cyc) fatty acids. Alterations in CL structure were observed in H. pylori cells cultivated on plates as compared with the bacteria grown in broth culture. Furthermore, significant changes in lipid A acylation pattern were detected in H. pylori cells during formation of coccoids. In contrast, structural analysis of CL from ex vivo H. pylori cells recovered from the stomachs of infected Mongolian gerbils demonstrated only minor changes in acyl chain combination. This is the first report of simultaneous analysis of CL and lipid A from ex vivo cells of H. pylori.  相似文献   

11.
Harvey DJ 《Proteomics》2005,5(7):1774-1786
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.  相似文献   

12.
Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.  相似文献   

13.
Fu Y  Xu S  Pan C  Ye M  Zou H  Guo B 《Nucleic acids research》2006,34(13):e94
A new matrix of 3,4-diaminobenzophenone (DABP) was demonstrated to be advantageous in the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With DABP as a matrix, intact oligonucleotide ions can be readily produced with lower laser powers, resulting in better detection limits, less fragmentation and fewer alkali metal ion adducts compared with the results obtained with conventional matrices. Importantly, minimal fragmentation and fewer alkali metal ion adducts were seen even at low concentrations of oligonucleotides. It was also found that samples prepared with DABP are highly homogenous and therefore reducing the need for finding ‘sweet’ spots in MALDI. In addition, excellent shot-to-shot reproducibility, resolution and signal-to-noise ratio were seen with DABP as the matrix.  相似文献   

14.
Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSn spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.  相似文献   

15.

Background

MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response.

Methodology/Principal Findings

A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells.

Conclusions/Significance

This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended to analyze the cell composition of tissues and the activation state of immune cells.  相似文献   

16.
17.
The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of 18O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.  相似文献   

18.
Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H2O), improving matrix crystallization. Peptide-bead cleavage with NH4OH was cheaper and safer than, yet as efficient as, NH3/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, α-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix.  相似文献   

19.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]+ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.  相似文献   

20.
The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid β-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method requires a simple sample preparation avoiding derivatization. It has high sensitivity, specificity, and throughput capability, and it requires minimal instrument maintenance. The use of chromatographic separation allows us to identify and quantify isomeric compounds that cannot be solved by appropriate multiple reaction monitoring (MRM) transitions. Urinary concentrations of the different acylglycines were determined using deuterated internal standards. The reference interval for the various metabolites was established using 120 healthy controls. The diagnostic usefulness of the method was demonstrated in three patients with propionic acidemia (PA), one patient with isovaleric acidemia (IVA), two patients with beta ketothiolase deficiency (BKTD), one patient with short branched chain amino acid deficiency (SBCAD), four patients with medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD), one patient with isobutyryl-coenzyme A dehydrogenase deficiency (IBDHD), and one patient with multiple acyl-coenzyme A dehydrogenase deficiency (MADD).  相似文献   

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