Matrix-assisted Laser Desorption Time-of-flight Research Articles

Your horse is a donkey! Identifying domesticated equids from Western Iberia using collagen fingerprinting

Skeletal remains of two equid species, Equus caballus (horse) and Equus asinus (donkey), have been found in archaeological contexts throughout Iberia since the Palaeolithic and Chalcolithic periods, respectively. These two species play different economic and cultural roles, and therefore it is important to be able to distinguish between the two species to better understand their relative importance in the past human societies. The most reliable morphological features for distinguishing between the two domesticated equids are based on cranial measurements and tooth enamel folds, leading to only a small percentage of archaeological remains that can be identified to species. Ancient DNA (aDNA) analysis can be used to reliably distinguish the two equids, but it can be cost prohibitive to apply to large assemblages, and aDNA preservation of non-cranial elements is often low. Collagen peptide mass fingerprinting by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a minimally destructive and cost-effective alternative to aDNA analysis for taxonomic determination. However, current ZooMS markers lack resolution below the genus level Equus. In this paper, we report a novel ZooMS peptide marker that reliably distinguishes between horses and donkeys using the enzyme chymotrypsin. We apply this peptide marker to taxonomically identify bones from the Iberian Peninsula ranging from the Iron Age to the Late Modern Period. The peptide biomarker has the potential to facilitate the collection of morphological data for zooarchaeological studies of equids in Iberia and throughout Eurasia and Africa.

P025 A study on candiduria in neonates and infants from a tertiary care center, North India.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjective Candida albicans is the major cause of fungal UTI in neonates and infants but nowadays incidence of other species of Candida is also increasing, and these are mostly multidrug resistant. It is, therefore, important to determine the causative Candida species in fungal UTI for appropriate management. This study was undertaken to determine the Candida species distribution in UTI along with susceptibility patterns and outcomes in infants and neonates admitted to various wards and intensive care units (ICUs) of our hospital. The incidence rate of candiduria in ICUs was also assessed.MethodUrine samples were collected from infants and neonates presented in pediatric and neonatal ICUs and clinical wards with a clinical suspicion of candiduria. Infants at risk of invasive candidiasis were also included in the study. Identification of Candida species was done by Gram's staining, germ tube test, chlamydospore formation on corn meal agar, color of colonies on CHROMagar, and confirmed by Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF). Antifungal susceptibility was performed by using the Broth microdilution method as per the latest CLSI guidelines (M27-A3/M27-S4).ResultUrine samples were received from 219 infants, and Candida was isolated from samples from 52 infants (isolation rate 23.75%), of which 30 were admitted to pediatric or neonatal ICU and 22 in the wards. The incidence rate of candiduria in ICU was 3.25%. Candida albicans was the most frequently isolated species from the samples of infants in the wards (13/22 ie, 59%), while Candida tropicalis was most frequently isolated from samples of infants in the ICUs (13/30 ie, 43.34%). Candida glabrata was the least commonly isolated species and was only encountered in the ICU. The species distribution of isolates is given in Table 1. There was no discrepancy between the results of conventional methods of identification and MALDI-TOF.Antifungal susceptibility was performed for 18 randomly selected isolates. All were found to be susceptible to caspofungin, micafungin, itraconazole, voriconazole, fluconazole, and amphotericin B. MIC distribution for various isolates is given in Table 2.ConclusionHigh index of suspicion of candiduria is necessary for early diagnosis of fungal UTI and initiation of antifungal treatment, especially in critically ill infants requiring intensive care. Species other than C. albicans are also encountered more frequently nowadays and these species often have higher MICs for commonly used antifungal drugs, which may lead to delayed or failed response to routine antifungal therapy and necessitate prolonged use and or higher doses of antifungals. Identification of Candida isolates at species level along with analysis of the susceptibility patterns is therefore important for successful outcomes in candiduria in neonates and infants.

P400 A study of the ecology, evolution and resistance mechanism of Candida auris at a tertiary care center in North India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM AimTo study the ecology, evolution, and resistance mechanism of Candida auris, using samples from patients, healthcare workers, hospital and environmental niches, using amplified fragment length polymorphism (AFLP) and antifungal susceptibility testing (AFST).MethodsA total of 720 samples were screened for C. auris, including clinical samples from patients (tissue, body fluids), surveillance samples from patients (axillar/groin swabs), swabs and water samples from environmental locations and objects, surface swabs from hospital locations, and screening samples from healthcare personnel for hand carriage of C. auris. Samples were cultured on Sabouraud Dextrose agar (SDA) and CHROMagar. Colonies morphologically suggestive of C. auris were identified by Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF) and isolates were subjected to antifungal susceptibility testing (AFST) by broth micro-dilution method. DNA was extracted for analysis by amplified fragment length polymorphism (AFLP) and cluster analysis. The amplicons were subjected to capillary electrophoresis and fluorescent amplified length polymorphism (FALP) for the generation of a heat map and dendrogram to evaluate single nucleotide polymorphisms and single nucleotide variations (SNPs and SNVs).ResultsOut of 720 samples, C. auris was isolated and identified by MALDI-TOF from 50, including 37 from routine patient samples, 12/674 axillar/groin surveillance swabs, and 1/66 samples from hands of healthcare workers. C. auris was not isolated from any environmental samples or hospital surfaces.AFST revealed high overall rates of resistance to three important antifungal drugs−93.22%, 38.98%, and 52.54% of isolates were resistant to fluconazole, voriconazole, and amphotericin B respectively. Resistance to echinocandins was lower−1.81% of isolates were resistant to caspofungin, and micafungin. Additionally, 18 isolates showed only intermediate sensitivity to both voriconazole and caspofungin.The highest rates of resistance to amphotericin B, and azoles were observed in isolates from blood (62.5% of isolates) and axillar/groin swabs (44.5% of isolates) respectively. Resistance to caspofungin was seen in 14.28% of isolates from both groups.AFLP and capillary electrophoresis of extracted DNA revealed 188 variations in the range of 300-662 nucleotides. A total of 10 samples had no change in the nucleotides and were labeled as ‘constant’. The dendrograms generated by bioinformatic analysis of FALP results yielded two different clusters provisionally designated as cluster I and cluster II. Cluster I could be further distinguished into sub-cluster Ia and sub-cluster Ib, indicating further variations.Conclusions Candida auris is a pathogen of emerging importance in our center, with significant levels of resistance to several important antifungal drugs. Incidence of both the pathogen and antifungal drug resistance was observed in samples collected from patients, but not from the hospital or environment, and minimally from healthcare personnel. This suggests that the source of most C. auris infections is colonizers from the patient rather than environmental sources or healthcare workers, and infection control measures should be tailored accordingly.

The Fruit Proteome Response to the Ripening Stages in Three Tomato Genotypes.

The tomato is a horticultural crop that appears in various colors as it ripens. Differences in the proteome expression abundance of a tomato depend on its genotype and ripening stage. Thus, this study aimed to confirm the differences in changes in the proteome according to four ripening stages (green, breaker, turning, and mature) of three tomato genotypes, i.e., yellow, black, and red tomatoes, using a gel-based proteomic technique. The number of protein spots shown as two-dimensional electrophoresis (2-DE) gels differed according to tomato genotype and ripening stage. A total of 286 variant proteins were determined using matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, confirming 233 identified protein functions. In three tomato genotypes in each ripening stage, grouping according to the Munich Information Center for Protein Sequences (MIPS) functional categories confirmed the variant proteins involved in the following: energy processes (21%); metabolism (20%); protein fate (15%); protein synthesis (10%); a protein with a binding function or cofactor requirement (8%); cell rescue, defense, and virulence (8%); cellular transport, transport facilitation, and transport routes (6%); the biogenesis of cellular components (5%); cell cycle and DNA processing (2%); others (5%). Among the identified protein spots in the function category, two proteins related to metabolism, four related to energy, four related to protein synthesis, and two related to interaction with the cellular environment showed significantly different changes according to the fruit color by the ripening stage. This study reveals the physiological changes in different types of tomatoes according to their ripening stage and provides information on the proteome for further improvement.

Development of an inexpensive matrix-assisted laser desorption-time of flight mass spectrometry method for the identification of endophytes and rhizobacteria cultured from the microbiome associated with maize.

Many endophytes and rhizobacteria associated with plants support the growth and health of their hosts. The vast majority of these potentially beneficial bacteria have yet to be characterized, in part because of the cost of identifying bacterial isolates. Matrix-assisted laser desorption-time of flight (MALDI-TOF) has enabled culturomic studies of host-associated microbiomes but analysis of mass spectra generated from plant-associated bacteria requires optimization. In this study, we aligned mass spectra generated from endophytes and rhizobacteria isolated from heritage and sweet varieties of Zea mays. Multiple iterations of alignment attempts identified a set of parameters that sorted 114 isolates into 60 coherent MALDI-TOF taxonomic units (MTUs). These MTUs corresponded to strains with practically identical (>99%) 16S rRNA gene sequences. Mass spectra were used to train a machine learning algorithm that classified 100% of the isolates into 60 MTUs. These MTUs provided >70% coverage of aerobic, heterotrophic bacteria readily cultured with nutrient rich media from the maize microbiome and allowed prediction of the total diversity recoverable with that particular cultivation method. Acidovorax sp., Pseudomonas sp. and Cellulosimicrobium sp. dominated the library generated from the rhizoplane. Relative to the sweet variety, the heritage variety c ontained a high number of MTUs. The ability to detect these differences in libraries, suggests a rapid and inexpensive method of describing the diversity of bacteria cultured from the endosphere and rhizosphere of maize.

Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)x.

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ β-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Metabolic profiling of zebrafish (Danio rerio) embryos by NMR spectroscopy reveals multifaceted toxicity of \u03b2-methylamino-L-alanine (BMAA)

β-methylamino-L-alanine (BMAA) has been linked to several interrelated neurodegenerative diseases. Despite considerable research, specific contributions of BMAA toxicity to neurodegenerative diseases remain to be fully resolved. In the present study, we utilized state-of-the-art high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), applied to intact zebrafish (Danio rerio) embryos, as a model of vertebrate development, to elucidate changes in metabolic profiles associated with BMAA exposure. Complemented by several alternative analytical approaches (i.e., in vivo visualization and in vitro assay), HRMAS NMR identified robust and dose-dependent effect of BMAA on several relevant metabolic pathways suggesting a multifaceted toxicity of BMAA including: (1) localized production of reactive oxygen species (ROS), in the developing brain, consistent with excitotoxicity; (2) decreased protective capacity against excitotoxicity and oxidative stress including reduced taurine and glutathione; (3) inhibition of several developmentally stereotypical energetic and metabolic transitions, i.e., metabolic reprogramming; and (4) inhibition of lipid biosynthetic pathways. Matrix-assisted laser desorption time-of-flight (MALDI-ToF) mass spectrometry further identified specific effects on phospholipids linked to both neural development and neurodegeneration. Taken together, a unified model of the neurodevelopmental toxicity of BMAA in the zebrafish embryo is presented in relation to the potential contribution of BMAA to neurodegenerative disease.

Chemical detection of cysteine-rich circular petides in selected tropical Violaceae and Moringaceae families using modified G-250 and mass spectrometry

Cysteine-rich circular peptides (CRCs) comprise a large family of gene encoded and low molecular weight polypeptides that has recently engaged the attention of scientists. This class of peptides exhibit a continuous circular configuration and a cystine knot backbone, which defines their resilient nature-directed structural design. Many CRCs have been reported in medicinal plants the first of which is kalata B1 cyclotide from the traditional African plant Oldenlandia affinis. Their detection and isolation can be very challenging and evasive. Only about 1% of plant species have so far been reportedly screened. A modified preliminary chemo-microscopic/macroscopic method involving the use of G-250 stain was applied followed by thin layer chromatographic protosite reaction for plant selection. This was followed by the Matrix Assisted Laser Desorption Time of Flight (MALDI-TOF) Mass spectrometry guided experiment for cyclotide discovery. The blue colouration produced upon sample reaction with modified G-250 led to the selection of potential circular peptide containing plant samples. A further MALDI-TOF MS-guided screening resulted in the detection of circular peptides and cyclotides in Moringa oleifera, Rinorea dentata, R. oblongifolia and R. brachypatela. Viola odorata and Viola tricolor (positive controls) indicated the presence of cyclotides. Results from this study can serve as proof-of-concept for plant selection based on preliminary cysteine-rich circular peptide detection in plants especially with the use of G-250 stain.Keywords: Cysteine-rich Cyclotides, Violaceae, Moringaceae, TLC, Microscopy, MALDI-TOF MS

Somatic DNA Mutation Analysis.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

Comparative Proteomic Analysis of Rapamycin Versus Cyclosporine Combination Treatment in Mouse Podocytes

BackgroundThe mechanism of podocyte injury observed with the use of rapamycin (RPM) remains unclear. The conversion from calcineurin inhibitors (CNIs) to RPM in kidney transplant recipients has been associated with a higher incidence of proteinuria and renal injury. In this study, we performed proteomic analyses to investigate the alteration of protein expression in mouse podocytes treated with RPM in comparison with CNI/RPM combination. MethodsImmortalized mouse podocytes were treated with 20 nmol/L RPM or 20 nmol/L RPM + 1 μg/mL cyclosporine. Podocyte proteins were separated by 2-dimensional polyacrylamide gel electrophoresis (2DE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Selected proteins were analyzed by means of Western blot assay. ResultsWe identified 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes. There are 3 distinct patterns of protein expression: (1) potentiated down- or upregulation of proteins by CNI/RPM treatment compared with isolated RPM treatment (n = 4); (2) partial offset of down-regulation by CNI/RPM in comparison with RPM treatment (n = 25); (3) no difference in down-regulation between RPM and CNI/RPM treatment (n = 5). We found a significant interplay between RPM and CNI on the expression of the selected proteins in mouse podocytes. This might explain the higher incidence of proteinuria by CNI/RPM combination in clinical settings. ConclusionsFurther study is required to elucidate the target protein associated with RPM-induced podocyte injury.

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693: a promising biocatalyst for the efficient degradation of natural and functionalized mcl-PHAs.

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % β-sheet, 19.4 % β-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/β hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings.Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC–MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli.

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.

Depletion of PKM2 leads to impaired glycolysis and cell death in 2-demethoxy-2,3-ethylenediamino hypocrellin B-photoinduced A549 cells

2-Demethoxy-2,3-ethylenediamino hypocrellin B (EDAHB) is an efficient photosensitizer that mediates cancer cell apoptosis. In order to better understand the molecular mechanisms involved in its antitumour activity, we used proteomics technology to identify candidate targets in A549 cells using EDAHB-mediated photodynamic therapy (EDAHB-PDT). The protein profile changes between untreated and PDT-treated A549 cells were analysed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Differentially expressed protein spots were identified using matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometry; and 15 differentially expressed proteins (over 2-fold, p<0.05) were identified in PDT-treated A549 cells compared with untreated cells. Among them, the expression of pyruvate kinase M2 (PKM2), a key enzyme involved in glycolysis, was found to be significantly decreased in A549 cells following EDAHB-PDT. Transient ectopic over-expression of PKM2 attenuated death of EDAHB-PDT-treated A549 cells, whereas knockdown of PKM2 expression by RNA interference increased the photocytotoxicity of EDAHB. Moreover, a decrease in lactate production was detected in PDT-treated A549 cells. These observations suggest that PKM2 plays an important role in the antitumour action of EDAHB-PDT; thus, it may be a potential molecular target to increase the efficacy of PDT in cancer therapy.

A Platform for Characterizing Therapeutic Monoclonal Antibody Breakdown Products by 2D Chromatography and Top-Down Mass Spectrometry

With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization. Described here is the implementation of existing advanced analytical technologies, including high-performance mass spectrometry (LTQ-Orbitrap XL-ETD) and a chip-based nanoelectrospray autosampling robot (TriVersa NanoMate), for the thorough identification and characterization of breakdown products derived from a force-degraded monoclonal antibody. Many anticipated breakdown products were identified, including Fab fragment (48,325 Da) and heavy chain polypeptide hydrolysis product (15,521 Da). Using high-resolution collisionally induced and electron transfer dissociation methods, additional identifications were made with specific localization of unpredicted modifications. As examples, a modified Fab fragment (N- and C-terminal cyclization, 47,902 Da) and a hydrolyzed free light chain impurity components (23,191 Da) were identified with a high degree of confidence (E value, <1e-5). This work describes the approach for top-down characterization of breakdown products and is readily applicable to additional monoclonal antibodies (mAb) characterization experiments, including charge isoform characterization and aggregate analysis, for a more thorough understanding of therapeutic mAb drug products.

Mutational analysis of PTEN/PIK3CA/AKT pathway in oral squamous cell carcinoma

The phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma (AKT) viral oncogene pathway is involved in regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes associated with the PI3K/AKT pathway including PI3K, AKT, RAS and PTEN, are infrequently found within head and neck squamous cell carcinoma and more specifically are rarely reported in oral squamous cell carcinoma (OSCC) cases. We aimed to investigate the frequency of mutations in AKT1, PTEN, PIK3CA, and RAS (K-RAS, N-RAS, H-RAS) genes in 37 cases of oral squamous cell carcinoma (OSCC). Mutational analysis of PTEN, RAS, PIK3CA and AKT genes was performed using chip-based matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and by direct sequencing. The only gene mutated in our series was the PIK3CA. Missense mutations of the PIK3CA gene were found in 4 of our cases (10.8%); no correlation has been found with oral location, stage and survival. The absence of mutations in AKT1, PTEN, and RAS genes in the present study is in accordance with previous studies confirming that these genes are rarely mutated in OSCC. Our data confirm that PIK3CA is important to OSCC tumorigenesis and can contribute to oncogene activation of the PIK3CA/AKT pathway in OSCC. The knowledge of the PIK3CA's involvement in OSCC is important because a specific kinase inhibitor could be considered as a future therapeutic option for OSCC patients with PIK3CA mutations.

The relationship between UGT1A4 polymorphism and serum concentration of lamotrigine in patients with epilepsy

Lamotrigine (LTG) which has a widespread use in epilepsy treatment as an antiepileptic agent is metabolized by UDP-glucuronosyl transferase (UGT) enzymes. In this study, single nucleotide polymorphisms, P24T and L48V, of the UGT1A4 enzyme have been investigated in a Turkish population of patients with epilepsy (n=131) by comparing serum levels of LTG of wild type and polymorphic subjects. High performance liquid chromatography (HPLC) was used to measure serum concentrations of LTG. The P24T and L48V polymorphisms of the UGT1A4 enzyme were analyzed with a matrix assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry method. The frequencies of the heterozygous alleles for L48V or P24T polymorphisms were 22.4% and 3.8%, respectively. L48V polymorphism was found to decrease the serum concentration of LTG in patients on monotherapy or polytherapy. The LTG levels of non smoking monotherapy patients were 52% lower for the L48V polymorphism than for wild type alleles. Also the LTG levels were significantly lower for non smoking or smoking polymorphic alleles than for normal. The high frequency of the L48V polymorphism detected in the Turkish population indicates that LTG dose adjustments in patients with the UGT1A4 L48V polymorphic enzyme should be taken into account.

Comparative proteomics of the mixotrophic dinoflagellate Prorocentrum micans growing in different trophic modes

Protein profiles of a common mixotrophic dinoflagellate, Prorocentrum micans, growing autotrophically and mixotrophically (fed on the cryptophyte Rhodomonas salina) were compared using two-dimensional gel electrophoresis (2DE) to determine if they vary in different trophic modes. Approximately 2.3% of the detected proteins were differentially expressed in the different trophic modes. Twelve proteins observed only in the mixotrophic condition had lower pI value ( 5). When the internal amino acid sequences of five selected proteins differentially expressed between autotrophic and mixotrophic conditions were analyzed us ing matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, two proteins that were specifically expressed in the autotrophic condition showed homology to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) and a bacterial catalase. Three mixotrophy-specific proteins showed homology to certain hypothetical proteins from an insect and bacteria. These results suggested the presence of certain gene groups that are switched on and off according to the trophic mode of P. micans.

Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.