Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometer Research Articles

Fibrinogen alpha chain precursor and apolipoprotein A-I in urine as biomarkers for noninvasive diagnosis of calcium oxalate nephrolithiasis: a proteomics study.

Calcium oxalate nephrolithiasis is the most common urological disease, but noninvasive and convenient methods of diagnosis are rarely available. Objective. The present study aimed to identify potential urine biomarkers for noninvasive diagnosis of CaOx nephrolithiasis. Methodology. Urine samples from 72 patients with CaOx nephrolithiasis and 30 healthy controls were collected and proteomics analysis was performed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS). Results. Thirteen proteins/peptides displayed statistically significant differences. The peptides of m/z 1207.23 and 2773.86 were selected by the genetic algorithm (GA) to build a possible diagnostic model. The area under the curve of m/z 1207.23 and 2773.86 was 0.936 and 0.987, respectively. The diagnostic model in distinguishing patients and healthy subjects showed 100% sensitivity and specificity. The peak at m/z 2773.86 was identified as fibrinogen alpha chain (FGA) with the sequence G.EGDFLAEGGGVR.G, and the peak at m/z 2773.86 was identified as apolipoprotein A-I (apoA-I) with the sequence L.PVLESFKVSFLSALEEYTKKLNTQ. Conclusion. The study results strongly suggested that urinary FGA and apoA-I are highly sensitive and specific biomarkers for noninvasive diagnosis of CaOx nephrolithiasis.

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover

In stolon of white clover (Trifolium repens L.), the 17.3kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS–PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28days of water-deficit. Water deficit led to decrease protein concentration. SDS–PAGE revealed that two major proteins of 17.3 and 16kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.

Genetic Variants of Bank1 Gene in Autoimmune Thyroid Diseases: A Case-Control Association Study

To investigate BANK1 gene variation, and its association with autoimmune thyroid disease and clinical features. We genotyped 3 single nucleotide polymorphisms (SNPs) rs10516487, rs3733197 and rs4522865 of BANK1 gene in 667 patients with autoimmune thyroid diseases (417 with Graves' disease and 250 with Hashimoto's thyroiditis) and 301 healthy controls. The Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) Platform was used to detect the 3 SNPs. There was a significant association in rs3733197 A allele and AITD patients (P=0.043). SNP rs3733197 A allele was found in 29.63% chromosome of AITD patients who are 18 years old or below, compared with 19.90% chromosome in those 19 years or above (P=0.017). Also SNP rs3733197 A allele showed a significant association in HT patients when compared with controls (P=0.031, OR=0.73 and 95% CI=0.55-0.97). Stratification for specific autoantibodies in AITD patients TGAb positive when compared with SE (shared epitope) positive showed a significant association in rs3733197 SNP (P=0.010, OR=0.68 and 95%=CI 0.51-0.91). However, no significant association was found between 3 SNPs and GD. Our findings suggest the existence of association between BANK1 gene and AITD thus adding BANK1 gene to the list of the predisposing genes to AITD.

Effect of Stabilizers on Bioactivity of Peptide-24 in PLGA Microspheres

In the present study, Poly (D,L-lactide-co-glycolide) microspheres (PLGA MSs) were prepared for delivering a novel oligopeptide derived from rhBMP-2 (Peptide-24). Hydroxypropyl-β-cyclodextrin (HP-β-CD) and Bovine serum albumin (BSA) were used as stabilizers for retaining bioactivity of the oligopeptide. The morphology, diameter, drugloading rates and encapsulation rates of the PLGA MSs were detected and compared. The PLGA MSs were incubated for 3 and 30 days respectively to obtain the release supernatant containing Peptide-24. The structure and bioactivity of released Peptide-24 from PLGA MSs were evaluated through physicochemical detections and cell culture. The structure integrity of the Peptide-24 was confirmed by Far-UV circular dichroism and matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometer (MALDI-TOF-MS) analysis. The interaction between PLGA matrix and loaded Peptide- 24 was verified through Raman. The results showed that the diameter of PLGA MSs was from 8.62 to 15.34 μm, the loading rate was 0.7-0.8%, and the encapsulation rate was 69.3-85.3%. The released Peptide-24 from PLGA MSs was proved to retain original bioactivity by the cellular activity and alkaline phosphatase (ALP) test. HP-β-CD is a kind of excellent stabilizer for retaining the bioactivity of Peptide-24 in PLGA MSs.

A marine phytoplankton (Prymnesium parvum) up-regulates ABC transporters and several other proteins to acclimatize with Fe-limitation

Iron (Fe) is one of the vital limiting factors for phytoplankton in vast regions of the contemporary oceans, notably the high nutrient low chlorophyll regions. Therefore, it is apparent to be acquainted with the Fe uptake strategy of marine phytoplankton under Fe-limited condition. In the present study, marine phytoplankton Prymnesium parvum was grown under Fe-deplete (0.0025μM) and Fe-rich (0.05μM) conditions, and proteomic responses of the organism to Fe conditions were compared. In sodium dodecyl sulfate (SDS) gel electrophoresis, 7 proteins (16, 18, 32, 34, 75, 82, and 116kDa) were highly expressed under Fe-deplete condition, while one protein (23kDa) was highly expressed under Fe-rich condition. These proteins were subjected to 2-dimensional gel electrophoresis (2-D DIGE) to differentiate individual proteins, and were identified by matrix-assisted laser desorption–ionization-time of flight-mass spectrometer (MALDI-TOF-MS) analysis. The results showed that under Fe-deplete condition P. parvum increases the biosynthesis of ATP binding cassette (ABC) transporters, flagellar associated protein (FAP), and Phosphoribosylaminoimidazole-succinocarboxamide synthase. These proteins are assumed to be involved in a number of cellular biochemical processes that facilitate Fe acquisition in phytoplankton. Under Fe-deplete condition, P. parvum increases the synthesis of ribulose biphosphate carboxylase (RuBisCo), malate dehydrogenase, and two Fe-independent oxidative stress response proteins, manganese superoxide dismutase (MnSOD) and Serine threonine kinase (STK). Thus, marine phytoplankton may change their Fe acquisition strategy by altering the biosynthesis of several proteins in order to cope with Fe-limitation.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant K a was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high-resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high-performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI-Orbitrap or the MALDI-TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI-Orbitrap mass spectrometer enabled differentiation between two possible RNA-based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In-source decay (ISD) analysis using a MALDI-TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix. The ESI-Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on-line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI-TOF mass spectrometer, in combination with 2,4-DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds.

Direct detection of peptides and proteins on a microfluidic platform with MALDI mass spectrometry

The ability to detect and quantify proteins of individual cells in high throughput is of enormous biological and clinical relevance. Most methods currently in use either require the measurement of large cell populations or are limited to the investigation of few cells at a time. In this report, we present the combination of a polydimethylsiloxane-based microfluidic device to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) that allows the detection of as few as 300 molecules at the peptide level and ~10(6) to 10(7) molecules at the protein level. Moreover, we performed an immunoassay with subsequent MALDI-TOF-MS to capture and detect insulin immobilized on a surface (~0.05 mm(2)) in this device with a detection limit of 10(6) insulin molecules. This microfluidic-based approach therefore begins to approach the sample handling and sensitivity requirements for MS-based single-cell analysis of proteins and peptides and holds the potential for easy parallelization of immunoassays and other highly sensitive protein analyses.

Preparation of novel hyperbranched flame‐retardant polymer and its application into natural rubber systems

AbstractFlame‐retardant hyperbranched pentaerythritol (FR‐HPER) was prepared by condensation polymerization between pentaerythritol (PER), the monomer, and phosphate acid we prepared. It was then characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, thermogravimetric analysis, and matrix‐assisted laser desorption ionization time of flight mass spectrometer (MALDI‐TOF‐MS). This FR‐HPER was used in the preparation of natural rubber (NR)/FR‐HPER composites. Different types of NR/FR‐HPER composites were prepared with different amounts of FR‐HPER, and compared with the composites directly incorporated with PER. Then, the tensile, wear resistant, thermal, and FR properties were researched and compared. Results showed that some mechanical and FR properties of NR/FR‐HPER composites showed obvious improvements compared with that of NR and NR/PER systems. Schemes of toughening, wear resistant, and FR mechanism were proposed. The hyperbranched polymer may reduce the amount and size of voids in NR matrix, and thus increase the tensile properties. Meanwhile, the synergistic effect between nitrogen in PER and phosphorus in phosphate acid can increase the amount of carbonaceous layers, and thus may inhibit the pyrolysis degree of NR matrix during burning. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Association between thyroid stimulating hormone receptor gene intron polymorphisms and autoimmune thyroid disease in a Chinese Han population

Autoimmune thyroid disease (AITD) is a multifactorial disease with a genetic susceptibility and environmental factors. The thyroid stimulating hormone receptor gene (TSHR) which is expressed on the surface of the thyroid epithelial cell is thought to be the main auto-antigen and a significant candidate for genetic susceptibility to AITD. This case-control study aimed at evaluating the association between single nucleotide polymorphisms (SNP) of TSHR and AITD in a Chinese Han population. We recruited 404 patients with Graves' disease (GD), 230 patients with Hashimoto's thyroiditis (HT) and 242 healthy controls. The Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) Platform was used to detect five SNPs (rs179247, rs12101255, rs2268475, rs1990595, and rs3783938) in TSHR gene. The frequencies of allele T and TT genotype of rs12101255 in GD patients were significantly increased compared with those of the controls (P=0.004/0.015, OR=1.408/1.446). The allele A frequency of rs3783938 was greater in HT patients than in the controls (P=0.025, OR=1.427). The AT haplotype (rs179247-rs12101255) was associated with an increased risk of GD (P=0.010, OR=1.368). The allele A of rs179247 was associated with ophthalmopathy in GD patients. These data suggest that the polymorphisms of rs12101255 and rs3783938 are associated with GD and HT, respectively.

Dermatophyte Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

The performance of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, ≥1.7; species, ≥2.0) and adjusted cutoff values established by this study (genus, ≥1.5; species, ≥1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P = 0.005)- and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.

MALDI-TOF mass spectrometry: the quantum leap

With this special issue on the matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, our aim is that infectious diseases specialists and microbiologists not yet familiar with this new tool will appreciate the importance of this ‘magical laser’ for microbial identification. We hope that even specialists will appreciate this overview on this new technology and its applications, since MALDI-TOF mass spectrometry really represents a quantum leap in diagnostic microbiology. In their review article, Emonet et al. thoroughly explain how MALDI-TOF mass spectrometry allows identification of bacterial species available in pure culture. They also compare the different mass spectrometer specifically adapted to routine microbiology laboratories and discuss their respective advantages and disadvantages. Then, they nicely present two other recent mass spectrometry-based tools, i.e. the MALDI-resequencing (MALDI-RE) and the association of a new broad-range PCR approach coupled with the analysis of generated amplicons by electrospray ionization mass spectrometry (PCR-ESI-MS), that allows better discrimination of strains and may be performed directly on clinical samples without a culture-based amplification step. In the second article of this special issue, Bizzini and I present the performance of MALDI-TOF mass spectrometry in routine setting. Although current protocols allow accurate identification of most strains routinely isolated in clinical microbiology laboratories, some clades will repeatedly be misidentified due to poor quality of databases or due to the close relatedness of different species (i.e. within the Streptococcus genus; or between E. coli and Shigella species). Despite these limitations, MALDI-TOF mass spectrometry really appears as a quantum leap in diagnostic microbiology that significantly reduce the time to results and costs of microbial identification. The benefit of protein extraction is also discussed in this review as well as some future possible applications such as the search for virulence factors and/or for some antibiotic resistance determinants. In the article by M. Drancourt, the usefulness of MALDI-TOF mass spectrometry for direct bacterial identification starting from positive blood culture bottles is discussed. This approach was successful in various laboratories using different protocols. In the near future, it will be important to further improve the identification, especially of Gram positive cocci and to precise the clinical impact of such rapid identification of the etiological agents of bacteremia. Then, in the last article of this special issue, P. Murray presents an overview of the usefulness of MALDI-TOF mass spectrometry for taxonomy and epidemiology. Based on the literature currently available, it appears that MALDI-TOF mass spectrometry may be used for microbial identification at the subspecies level and might even prove to be as discriminative then pulse-field gel electrophoresis (PFGE) for some selected species. However, protein extraction and precise protein calibration seems important to obtain reliable results at subspecies, type and subtypes levels. This ‘magic laser’ technology will also have applications in the future in the taxonomy of obligate intracellular bacteria such as members of the Rickettsiales and Chlamydiales order, for which no or little phenotypic traits are available, given their strict intracellular growth. Although current protocols allows accurate identification of most strains routinely isolated in clinical microbiology laboratories, there are still much place for improvement, especially in the quality and completeness of databases and in the quality control of the process. Thus, as an example, it may be important to check the MALDI-TOF microplate for the absence of residual trace of proteins on cleaned microplates. The performance of the extraction step and of the MALDI-TOF mass spectrometer needs although to be daily checked by routinely testing a few selected bacterial ATCC strains with and without a specific extraction step. Thank to this special issue on MALDI-TOF mass spectrometry, you will surf from technical aspects to the application of this quantum leap technology allowing accurate, easy and cheap microbial identification and, to some extent, straightforward typing. The author has no conflicts of interest to declare.

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1-A6), which were confirmed by Western blotting and MALDI-TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The (199)Val-Ser-Tyr-Asn(202) sequence in peptide T12 of A1 and A2 was replaced by (199)Leu-Ser-His-Glu(202) in A3 and A4 (T12') while in A5 and A6 this sequence was (199)Val-Ser-Tyr-Val(202) (T12''). Peptide T1 showed the presence of (10)Asn in the isoforms A1-A5 while in A6 this amino acid was replaced by (10)Lys (T1'). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.

Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography

In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N, N′-methylenebisacrylamide as the cross-linker, and N, N, N′, N′-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top–down proteomic analysis.

らせん状のイオン軌道を利用した飛行時間質量分析計の開発

A novel time-of-flight mass spectrometer that utilizes a spiral ion trajectory was developed. In this mass spectrometer, the ions sequentially pass through four toroidal electrostatic sectors and revolve along a figure-eight-shaped orbit on a particular projection plane. Each toroidal electrostatic sector has eight stories, and during multiple revolutions, the ion trajectory shifted perpendicular to the projection plane in every cycle, thereby generating a spiral trajectory. Two prototypes, orthogonal acceleration time-of-flight mass spectrometer (OA-TOFMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS) were made and tested. The total flight path lengths were 20 and 17 m for OA-TOFMS and MALDI-TOFMS, respectively. By adopting an ion optical system that has a flight path length more than five times longer than that of the commonly used reflectron ion optical system, the m/z-dependent mass resolving power was reduced while improving the mass accuracy of the mass measurements.

遅延引き出し法の基礎

The delayed extraction (also known as pulsed extraction) technique involves the application of a time delay between the ionization pulse and an accelerating voltage pulse for desorption/ionization from a surface. This technique can achieve energy focusing in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer and is used to improve mass resolving power. We explain the principles of delayed extraction (pulsed extraction) used in a MALDI TOF mass spectrometer.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Effects of Iron-cored Carbonballs as Matrix on Matrix-Assisted Laser Desorption Ionization-time of Flight Mass Spectrometer Analysis

Mass spectrometry is an important technique for characterizing the structures of surfaces and has several characteristics that are especially valuable in the bioanalytical application. Matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF-MS) is commonly used in preliminary protein identification. To prepare the sample for analysis, purified-proteins of cryptic digestion are carefully mixed with saturated liquid-phase matrices, classified as high-molecular-weight organic polymer, which are essential to promote analytical accuracy via mechanism of laser-energy transfer to peptides. Signals generated from matrix in the analytical process interfere with signals of small peptides in the same region of mass-to-charge ratio. In order to improve analytical results, we demonstrated that matrix in MALDI-TOF-MS can be replaced by carbon nanomaterial. We substituted iron-cored carbonballs for matrix well mixing with trypsinized bovine serum albumins (BSA) and performed mass spectrometrical analyses. Experimental results indicated that in addition to iron-cored carbonballs possessed equivalent functionality as matrix does, several improved aspects were also observed.

Rapid characterization of edible oils by direct matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis using triacylglycerols

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis of solutions of edible fats/oils yielded spectra useful for their rapid differentiation and classification. Results also reflected the individual fatty acid components and their degree of unsaturation. After dissolution in hexane, MALDI-MS analysis revealed spectra showing characteristic triacylglycerols (TAGs), the main fat/oil components, as sodium adduct ions. The Euclidean distances calculated using the mass and intensity values for 20 TAGs were used to evaluate and compare spectra. With cluster analysis, animal fats grouped together differently than vegetable oils and the individual oils grouped together by type. The ion abundances for the individual TAGs and their presumed compositions were used to approximate the overall fatty acid composition of canola, soybean, corn, olive and peanut oil, as well as lard. Using this approach the calculated fatty acid compositions and degree of unsaturation generally fell within about 4% of literature values. When the degree of saturation was compared with values calculated from the package labeling the differences were about 7%.

Analysis of NOD2-mediated Proteome Response to Muramyl Dipeptide in HEK293 Cells

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(WT) that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2(WT) and disease-associated NOD2(SNP13) variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.