Multiple Tandem Mass Spectrometry Research Articles

In-depth characterization of a cysteine-linked ADC disitamab vedotin by multiple LC-MS analysis methods and cutting-edge imaged capillary isoelectric focusing coupled with native mass spectrometry

The characterization of cysteine-linked antibody‒drug conjugates (ADCs) can be more challenging than that of monoclonal antibodies (mAbs) and lysine-linked ADCs because the interchain disulfide bonds are reduced for payload conjugation, and the chains are noncovalently bonded to each other. Furthermore, payload conjugation and disulfide bond reduction/scrambling may introduce additional charge heterogeneity to biomolecules. This study illustrates an innovative workflow employing multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin, a recent-generation cysteine-linked ADC, including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF). RPLC was employed for reduced chains analysis, subunit analysis and peptide mapping. IEX and icIEF were used for charge heterogeneity analysis. The innovation of the integrated methodology emphasizes the importance of cutting-edge icIEF-MS online coupling under near-native conditions to reveal the heterogeneity of disitamab vedotin.

Sequencing Sialic Acid Positioning in Gangliosides by High-Resolution Cyclic Ion Mobility Separations Coupled with Multiple Collision-Induced Dissociation-Based Tandem Mass Spectrometry Strategies.

Gangliosides, a diverse class of glycosphingolipids, are highly abundant in neural tissue and have been implicated in numerous aging-related diseases. Their characterization with methods such as liquid chromatography-tandem mass spectrometry is often precluded by their structural complexity, isomeric heterogeneity, and lack of commercially available authentic standards. In this work, we coupled high-resolution cyclic ion mobility spectrometry with multiple collision-induced dissociation-based tandem mass spectrometry strategies to sequence the sialic acid positions in various ganglioside isomers. Initially, as a proof-of-concept demonstration, we were able to characterize the sialic acid positions in several GD1 and GT1 species. From there, we extended our approach to identify the location of N-glycolylneuraminic acid (NeuGc) residues in previously uncharacterized GD1 and GQ1 isomers. Our results highlight the potential of this presented methodology for the de novo characterization of gangliosides within complex biological matrices without the need for authentic standards.

Profiling of Oxylipins as Markers of Oxidative Stress in Biological Samples.

Oxylipins are oxidized metabolites of polyunsaturated fatty acids (PUFAs). They represent a class of risk markers and/or therapeutic targets for diseases associated with inflammation, including cardiovascular disease and brain disorders. Because the biological activities of free PUFAs and oxylipins depend on their chemical structures and concentrations, monitoring PUFAs and oxylipin levels in biological systems is critical for understanding their roles in health and disease. Traditionally, accurate quantification of free PUFAs and oxylipins in biological samples was performed separately, as PUFAs are often 1000-fold more abundant than the derived oxidized fatty acids (oxylipins). This article describes a liquid chromatography multiple reaction monitoring tandem mass spectrometry method for the quantitative analysis of five free PUFAs and 88 oxylipins in various biological fluids, including plasma, platelet supernatants, and tissues. The same approach can also be used in conjunction with an alkaline hydrolysis step to quantify total oxylipins in fish oils. We observed that in some samples, linoleic acid levels in plasma and eicosapentaenoic acid and arachidonic acid levels in brain tissue were above the upper limit of quantification. To address this issue, we developed a data analysis method to obtain PUFA and oxylipin concentrations in these samples without additional sample preparation, thus significantly saving time and labor. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantification of polyunsaturated fatty acids (PUFAs) and oxylipins using liquid chromatography multiple reaction monitoring tandem mass spectrometry Support Protocol 1: Preparation of internal standard mixed working solution Support Protocol 2: Preparation of standard mixed stock solution Support Protocol 3: Preparation of standard mixed working solution Alternate Protocol 1: Extraction and quantitation of free PUFAs and oxylipins from mouse brain tissue Alternate Protocol 2: Extraction and quantitation of total PUFAs and oxylipins from fish oil.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

A High-Throughput Absolute Quantification of Protein-Bound Sulfur Amino Acids from Model and Crop Plant Seeds.

In this procedure, we describe a high-throughput absolute quantification protocol for the protein-bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC-MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL-Ser-d3 and L-Met-d3 as internal standards to enable the quantification of the absolute levels of the protein-bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound cysteine and methionine from seeds.

Rationally designed synthetic peptide as versatile calibrant to improve the accuracy of protein sequence analysis using MALDI mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) plays an indispensable role in analyzing protein covalent structures. The reliable identification of amino acid residues and modifications relies on the mass accuracy, which is highly dependent on calibration. However, the accuracy provided by the currently available calibrants still needs further improvement in terms of compatibility with multiple tandem MS modes or ion polarity modes, calibratable range, and minimizing suppression of and interference with analyte signals. Here aiming at developing a versatile calibrant to solve these problem, we designed a synthetic peptide format of calibrant Rx(GDPn)m (referred to as “Gly-Asp-Pro, GDP”) according to the chemical natures of amino acids and polypeptide fragmentation rules in tandem MS. With four types of amino acid residues selected and arranged through rational designs, a GDP peptide produces highly regulated fragments that give rise to evenly spaced signals in each tandem MS mode and is compatible with both positive and negative ion modes. In internal calibration, its regulated fragmentation pattern minimizes interference with analyte signals, and using a single peptide as the input minimizes suppression of the analyte signals. As demonstrated by analyses of proteins including monoclonal antibody and Aβ-42, these features allowed significant increase of the mass accuracy and precision, which improved sequence coverage and sequence resolution in sequence analyses (including de novo sequencing). This rational design strategy may also inspire further development of synthetic calibrants that benefit structural analysis of biomolecules.

CFTR protein quantification as a cystic fibrosis diagnostic biomarker in dried blood spots using multiple reaction monitoring tandem mass spectrometry

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel found on the apical surface of epithelial cells in the airway and gastrointestinal tract. A mutation in the CFTR protein is responsible for developing cystic fibrosis (CF) disease. Therefore, circulating CFTR protein could be a promising biomarker of CF disease.Multiple methodological challenges are associated with CF's available diagnostic and screening methods, such as low specificity and potential false discovery rate, mainly for ethnic groups whose CFTR mutations are not covered in the mutation panels.Herein, we have developed an absolute quantification (AQUA) method based on two CFTR signature peptides (SPs). A liquid chromatography-tandem spectrometry (LC-MS/MS) method in multiple reaction monitoring (MRM) mode (MRM transitions 1168.90 > 85.929 and 707.19 > 85.93 of SP1 and SP2, respectively) enabled the accurate quantification of CFTR protein in a dried blood spot (DBS). The method was validated successfully based on international guidelines in terms of signal linearity, precision (within-run CV 3.37–8.54%; between-run CV 5.15–11.06% for the selected SPs), and accuracy (within-run 93.4–105.59%; between-run 97.45–103.28% for the selected SPs). The level of soluble CFTR protein was evaluated as a potential biomarker for CF using patients (n = 39) and healthy controls (n = 30), were found to be in CF patients lower than controls. For instant, the level of signature peptide 1 (SP1) was 2.09 ± 0.55 nM, 68.77 ± 1.40 nM in CF patients compared to Ctrl, respectively; p < 0.0001.This study is the first to report CFTR levels in DBS using signature peptides by LC-MS/MS as a diagnostic marker for CF. The receiver operating characteristic (ROC) for CFTR SP1 and SP2 showed a significant area under the curves (AUC) 0.7714 (99% CI, p < 0.0001), and 0.8234 (99% CI, p < 0.0001), respectively. The presented MRM method provides a highly specific and sensitive approach to CFTR quantification in a DBS and could be applied in CF screening.

DecoID improves identification rates in metabolomics through database-assisted MS/MS deconvolution.

Chimeric MS/MS spectra contain fragments from multiple precursor ions and therefore hinder compound identification in metabolomics. Historically, deconvolution of these chimeric spectra has been challenging and relied upon specific experimental methods that introduce variation in the ratios of precursor ions between multiple tandem mass spectrometry (MS/MS) scans. DecoID provides a complementary, method-independent approach where database spectra are computationally mixed to match an experimentally acquired spectrum by using LASSO regression. We validated that DecoID increases the number of identified metabolites in MS/MS datasets from both data-independent and data-dependent acquisition without increasing the false discovery rate. We applied DecoID to publicly available data from the MetaboLights repository and to data from human plasma, where DecoID increased the number of identified metabolites from data-dependent acquisition data by over 30% compared to direct spectral matching. DecoID is compatible with any user-defined MS/MS database and provides automated searching for some of the largest MS/MS databases currently available.

16α-Hydroxyestrone: Mass Spectrometry-Based Methodologies for the Identification of Covalent Adducts Formed with Blood Proteins.

Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of a "bottom up" proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement, yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted in the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodology might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1 but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.

Nontargeted Analysis Strategy for the Identification of Phenolic Compounds in Complex Technical Lignin Samples.

Lignin is the second most abundant biopolymer in nature and a promising renewable resource for aromatic chemicals. For the understanding of different lignin isolation and conversion processes, the identification of phenolic compounds is of importance. However, given the vast number of possible chemical transformations, the prediction of produced phenolic structures is challenging and a nontargeted analysis method is therefore needed. In this study, a nontargeted analysis method has been developed for the identification of phenolic compounds by using an ultrahigh‐performance supercritical fluid chromatography–high‐resolution multiple stage tandem mass spectrometry method, combined with a Kendrick mass defect‐based classification model. The method is applied to a Lignoboost Kraft lignin (LKL), a sodium lignosulfonate lignin (SLS), and a depolymerized Kraft lignin (DKL) sample. In total, 260 tentative phenolic compounds are identified in the LKL sample, 50 in the SLS sample, and 77 in the DKL sample.

Rapid, proteomic urine assay for monitoring progressive organ disease in Fabry disease

BackgroundFabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker...

Sensitive determination of perfluoroalkane sulfonamides in water and urine samples by multiple monolithic fiber solid-phase microextraction and liquid chromatography tandem mass spectrometry

To extract perfluoroalkane sulfonamides (PFASAs) in water and urine samples effectively, a new adsorbent based on poly (1H,1H,2H,2H-nonafluorohexyl acrylate/vinyboronic anhydride pyridine complex-co-ethylenedimethacrylate) monolith (FBE) was synthesized and used as the extraction phase of multiple monolithic fiber solid-phase microextraction (MMF-SPME). Because there are abundant fluorinated (F-) alkyl chains and boron atoms in the adsorbent, the FBE/MMF-SPME displays satisfactory extraction performance for PFASAs by means of fluorophilic and B-N coordination interactions. Under the most favorable conditions, the FBE/MMF-SPME was combined with HPLC-MS/MS for the sensitive monitoring of ultra-trace PFASAs in environmental water and human urine samples. The limits of detection and limits of quantification achieved for target analytes were in the range of 0.13–1.45 ng/L and 0.44–4.80 ng/L, respectively. The developed FBE/MMF-SPME-HPLC-MS/MS method was successfully applied to quantify the level of PFASAs in water and human urine samples, and ultra-trace target PFASAs were detected in the real samples. The recoveries at different fortified concentrations ranged from 80.3% to 119% with RSD in the range of 0.9–11%. Compared with reported methods, the proposed method exhibits some merits such as high sensitivity, good method precision, low consumption of sample and environmental friendliness.

Screening and analysis of potentially active components in Shenxiong glucose injection using UHPLC coupled with photodiode array detection and MS/MS.

Shenxiong glucose injection, a pharmaceutical preparation containing a water extract of the roots of Salvia miltiorrhizae and ligustrazine hydrochloride, is widely used in clinical to treat cardiovascular diseases in China. The chemical components of the water extract have been reported and the cardioprotective effects of the injection have been evaluated. However, the chemical constituents of the injection and their correlations with its pharmacological effects have not been established. In this study, 13 chemical constituents of the injection have been identified or characterized by ultra-high performance liquid chromatography with diode array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Besides, the potentially active compounds of this preparation that directly act on cardiac cells have been screened by cell extraction and ultra high performance liquid chromatography targeted multiple reaction monitoring. As a result, eight potentially active compounds, danshensu (1), ligustrazine hydrochloride (4), salvianolic acid I/H (7), lithospermic acid (8), salvianolic acid D (9), rosmarinic acid (10), salvianolic acid B (12), and salvianolic acid C (13), were obtained and structurally characterized from the 11 target compounds used for screening. The liquid chromatography with quadrupole time-of-flight mass spectrometry and liquid chromatography with multiple reaction monitoring tandem mass spectrometry combination method has demonstrated its potency for the screening, detection, and structural identification of bioactive compounds in a complex matrix.

Quantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75–110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B2a and Biological Amines.

Aflatoxin B2a has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B2a amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using 1H, 13C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B2a dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B2a-lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and 1H NMR spectrum that match aflatoxin B1-lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B2a as well as possible interferences with known aflatoxin B1 exposure biomarkers.

Quantitative analysis of the major linkage region tetrasaccharides in heparin

Heparin is a polysaccharide based anticoagulant drug composed of a complex mixture of glycosaminoglycan chains and peptidoglycosaminoglycan chains. In an effort to better characterize this important polysaccharide based drug, we examined the peptide components of the minor peptidoglycosaminoglycan chains comprising heparin. Three different the glycan-peptide linkage regions tetrasaccharide fragments were isolated from pharmaceutical heparin using heparin lyase II and characterized the structure of these tetrasaccharides using nuclear magnetic resonance spectroscopy and mass spectrometry. A sensitive and quantitative assay was developed for these linkage regions using multiple reaction-monitoring tandem mass spectrometry. These three different linkage regions were found in heparins coming from porcine intestine and bovine lung. Two of these were also present in the low molecular weight heparin, enoxaparin.

Probing the interactions between cisplatin and essential amino acids using electrospray ionization mass spectrometry

Cisplatin (cis-[PtCl2(NH3)2]) is an important platinum-containing anticancer drug for treatment of solid malignancies. Probing the interactions between cisplatin and free amino acids are beneficial for investigation of its pharmacology and side effects. In this study, the interactions of twenty amino acids with cisplatin in water, acidic, neutral and basic solutions were studied using electrospray ionization mass spectrometry (ESI MS), respectively. Adducts of cisplatin and amino acids were recognized. It had been found that the reactions of cisplatin with amino acids depended on solution media and essential amino acids. Multiple tandem mass spectrometry was employed to probe the affinities of nucleophilic functional groups, such as side chains, α-NH2 groups and α-COOH groups on amino acids for cisplatin in water. The results indicated that the chelated complexes formed were stable in aqueous solution. For free Cys, Met and His, their side chains had higher affinities than α-NH2 groups. For free Ser, Thr, Asp, Glu, Asn and Gln amino acids, their α-NH2 groups had higher affinities than the side chains and α-COOH groups. For Lys, Arg, Phe, Tyr and Trp, platinum may coordinate to their aromatic rings of the side chains or α-NH2 groups. For other amino acids Gly, Ala, Val, Leu, Ile and Pro, their α-amino groups had higher affinities for cisplatin than α-COOH groups. These results provide the insights for understanding of the mechanism and side effects of cisplatin.

Quantitative Proteomic Analysis of Peripheral Blood Mononuclear Cells in Ankylosing Spondylitis by iTRAQ.

This study was designed to identify and quantify the different proteins expression levels in ankylosing spondylitis (AS) and to explore the pathogenesis of AS. We performed isobaric tags for relative and absolute quantitation (iTRAQ) coupled with multiple chromatographic fractionation and tandem mass spectrometry to detect the proteins profiling in peripheral blood mononuclear cells (PBMCs) from AS patients and healthy controls. Mascot software and the International Protein Index and the Gene Ontology (GO) database were used to conduct the bioinformatics analysis. The differentially expressed proteins were validated by enzyme-linked immunosorbent assay (ELISA). A total of 1,232 proteins were identified by iTRAQ, of which 183 showed differential expression and 18 differentially expressed proteins were acute phase reactants. Upon mapping of the differentially expressed proteins to GO database, we found four differentially expressed proteins involved in the biological process of cell killing, including up-regulated cathepsin G (CTSG), neutrophil defensin3 (DEFA3), protein tyrosine phosphatase receptor type C (PTPRC), and down-regulated peroxiredoxin-1(PRDX1),which were consistent with the verified results of ELISA. Our proteomic analyses suggested that the proteins involved in the biological process of cell killing might play an important role in the pathogenesis of AS.

Confirmation of extensive natural distribution of azaspiracids in the tissue compartments of mussels (Mytilus edulis)

Liquid chromatography multiple tandem mass spectrometry (LC–MS/MS) has been applied to demonstrate that azaspiracid (AZA1), its isomer, AZA6, and its methyl- and demethyl-analogues, AZA2 and AZA3 respectively, are distributed throughout mussel (Mytilus edulis) tissue compartments. Large differences in both the toxin content and the toxin profiles were observed in 20 individual mussels from the same batch. The toxin levels found in the hepatopancreas of mussels were, AZA1 (0.02–5.0 μg/g); AZA2 (0.12–1.9 μg/g), AZA3 (0.06–0.88 μg/g) and AZA6 (0.05–2.0 μg/g). This study also examined the toxin content in mussel tissue compartments and it was found that the gills contributed significantly to the overall level of toxins in mussels. Although polyether toxins are usually concentrated in the hepatopancreas of shellfish this is not always the situation with azaspiracids. The mean distribution of azaspiracids in mussels was; hepatopancreas (60.6%), gills (12.0%) and adductor muscle (27.4%).

Comparative proteomic analysis of renal tissue in IgA nephropathy with iTRAQ quantitative proteomics.

Immunoglobulin (Ig) A nephropathy (IgAN) is the most common form of glomerulonephritis. In clinical practice, it is difficult to monitor the repeating relapse in patients suffering from IgAN, which usually occurs within 10 years of end-stage renal disease. In order to identify and quantify the total protein content in the renal tissue of patients with IgAN, isobaric tags for relative and absolute quantification (iTRAQ) technology was performed. iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry was used to analyze the total protein of normal renal tissue in IgAN and healthy patients. The individual proteins were identified by the Mascot search engine and any that were differentially expressed were monitored. A total of 574 different proteins were identified, and 287 proteins were up- or downregulated by >1 fold alteration in levels. The results showed that iTRAQ-based quantitative proteomic technology for the identification and relative quantitation of the renal tissue proteome is efficiently applicable. The differential expression of the proteome profiles for IgAN patients was determined. Further studies using large cohorts of patient samples with long-term clinical follow-up data should be conducted to evaluate the usefulness of the pathogenesis and novel biomarker candidates of IgAN, which may develop a novel technique for the diagnosis of IgAN.