Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Reexamination of the cysteine residues in glucocerebrosidase

Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure.     

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.     

Enhancement of analyte ionization in desoprtion/ionization on porous silicon (DIOS)-mass spectrometry (MS)

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a relatively new laser desorption/ionization technique for mass spectrometry without employing an organic matrix. This present study was carried to survey the experimental factors to improve the efficiency of DIOS-MS through electrochemical etching condition in structure and morphological properties of the porous silicon. The porous structure of silicon structure and its properties are crucial for the better performance of DIOS-MS and they can be controlled by the suitable selection of electrochemical conditions. The fabrication of porous silicon and ion signals on DIOS-MS were examined as a function of silicon orientation, etching time, etchant, current flux, irradiation, pore size, and pore depth. We have also examined the effect of pre- and post-etching conditions for their effect on DIOS-MS. Finally, we could optimize the electrochemical conditions for the efficient performance of DIOS-MS in the analysis of small molecule such as amino acid, drug and peptides without any unknown noise or fragmentation.     

Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and/or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying concentrations of ammonia or methylamine and monitored in time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimized. Amidation was performed best under anhydrous conditions at a concentration of 4 M ammonia or methylamine at ambient temperature. Amidation using methylamine reached almost completeness (DAm 95) without hardly any hydrolysis of methyl-esters while amidation with ammonia reached a DAm of 70 on average. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueous solutions instead off anhydrous ammonia resulted in 6–10% lower DAm values due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagent for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties.     

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.     

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.     

大鼠脑皮质表达蛋白质组学研究

文章用蛋白质组学方法初步分析大鼠脑皮质蛋白质的表达。提取大鼠脑皮质蛋白质,双向凝胶电泳分离,考马斯亮蓝染色,胰蛋白酶胶内酶解,用基质辅助激光解吸/电离飞行时间质谱对酶解后的肽段进行分析,根据肽质量指纹图谱,检索专业数据库(Swissprot),对蛋白质进行鉴定。鉴定出84个蛋白,分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白等。文章结果丰富了大鼠脑皮质蛋白质组数据库,为在大鼠模型上研究神经疾病奠定了基础。     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

Identification of Putative Biomarkers for Toluene-Degrading Burkholderia and Pseudomonads by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Peptide Mass Fingerprinting

The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.     

TRIP12 ubiquitination of glucocerebrosidase contributes to neurodegeneration in Parkinson’s disease

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Extracellular and transmembrane region of a podocalyxin-like protein 1 fragment identified from colon cancer cell lines

Regulated intramembrane proteolysis of membrane proteins has been shown to play an important role in cell differentiation and in the pathogenesis of diseases. The aim of the present study was to identify novel peptides generated by intramembrane proteolysis. The peptides were identified in serum-free cultured (SFC) media from various cell lines by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). A 2315-Da peptide found only in medium from SFC colon cancer cell lines was identified and shown to consist of a portion of both the extracellular and transmembrane regions of human podocalyxin-like 1. This protein fragment was not found in lung or pancreatic cancer cell lines by immunoprecipitation-SELDI tests using an antibody specific to this fragment, suggesting that this human podocalyxin-like protein 1 fragment may be unique to colon cancer cell lines.     

Protein profiling in respiratory disease: techniques and impact

Multifactorial diseases such as respiratory disease call for a global analysis of such disorders. Recent advances in protein profiling techniques may allow for early diagnosis of respiratory disease, which is crucial for intervention and treatment. In order to reduce false-positive rates, clinical diagnosis requires a high degree of sensitivity and specificity to be an effective screening tool. Protein profiles identified by ProteinChip^® (Ciphergen Biosystems) technology coupled with mass spectrometry affords a global analysis of clinical samples and is beginning to reach acceptable levels of sensitivity and specificity. Combining the profile with another diagnostic tool enhances the effectiveness of protein profiles to classify disease. Although current efforts have centered on serum protein profiling, the local environment of the lung may be better reflected in proteins of bronchoalveolar lavage or sputum. Identification of biomarkers of disease by protein profiling anaylses may lead to an understanding of the mechanisms of this disease and contribute to the discovery of new therapeutics for the prevention and treatment of disease. Advancing these analyses are techniques such as ProteinChip mass spectrometry, laser capture microdissection, tissue microarrays and fluorescently labeled antibody bead arrays, which enable the direct global analysis of complex mixtures. Effective high-throughput and ease of use of clinical testing will arrive with improvements in bioinformatics and decreases in instrumentation costs.     

MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery.     

Current status and future prospects in the search for protein biomarkers of immunosenescence

Complex adaptations including changes in cellular redox status, the production of high levels of pro-inflammatory cytokines and alterations in immunity occur as the result of aging of the immune system (immunosenescence). These events are thought to underlie the progression of chronic degenerative diseases of aging, such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. It is envisaged that identifying early biomarkers of immune aging would aid in identifying individuals at risk of age-related disease and would allow the discovery of novel intervention strategies. Proteomics has emerged as a rapidly expanding and innovative field, investigating protein expression, interaction and function at a global level. Several proteomic strategies, including use of mass spectrometry and non-mass spectrometry-based detection systems (including secondary antibody labeling with fluorescent tags) may be particularly advantageous in identifying biomarkers of immune health. Application of these approaches may identify factors that both contribute to (and define) age-dependent deregulation of the immune system.     

Mass spectrometric analysis of glycosylated viral proteins

Introduction: Viral diseases contribute much to human and animal suffering and enormous efforts are directed at developing appropriate vaccines for protection. Glycoproteins constitute much of the viral surfaces and are obvious targets for such vaccine development. This review describes mass spectrometric methods used for the structural determination of these compounds.

Areas covered: The review describes the structures of the N- and O-linked glycans found on glycoproteins and mass spectrometric methods for their ionization and fragmentation. The steps, such as determination of glycan attachment sites and the structures of the attached glycans following their release from the glycoproteins are described and examples are given of the uses of the various analytical methods using mainly influenza, Ebola and HIV as representative examples. Also included are tables listing work on many other viruses.

Expert commentary: Recent technological advances, such as the introduction of ion mobility techniques, have greatly improved analyses in this area and have enabled larger amounts of information to be gathered in shorter time periods on ever smaller amounts of material. Such techniques should greatly accelerate the discovery of vaccine targets and lead to the production of vaccines for diseases not currently available.