Human Serum Albumin Peptides Research Articles

Unraveling the binding behavior of the antifouling biocide 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one with human serum albumin: Multi-spectroscopic, atomic force microscope, computational simulation, and esterase activity

As a marine antifouling biocide, 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) exhibited high toxicity to marine organisms. This study investigated the interaction between DCOIT and human serum albumin (HSA) using several spectroscopic techniques combined with computer prediction methods. The UV–vis absorption spectra, Stern-Volmer constant (KSV) and fluorescence resonance energy transfer (FRET) results indicated that DCOIT caused static quenching of HSA fluorescence. The ΔG°, ΔH° and ΔS° values were −31.03 ± 0.17 kJ·mol−1, −133.54 ± 0.88 kJ·mol−1 and −348.46 ± 2.86 J.mol−1·K−1, respectively, suggesting that van der Waals forces and hydrogen bonds governed the spontaneous formation of the complex. Synchronous fluorescence and circular dichroism (CD) spectroscopy observed the burial of Trp residues within HSA and the unfolding of HSA secondary structure induced by DCOIT. Three-dimensional (3D) fluorescence and Atomic Force Microscopy (AFM) further detected DCOIT-induced loosening of HSA peptide chain structure. Site displacement experiments indicated that DCOIT binding at site I of HSA. Computational predictions indicated that hydrophobic interactions were also essential in the complex. The increased RMSD, Rg, SASA, and RMSF confirmed that DCOIT weakened the stability and compactness of HSA, rendering residues more flexible. Lastly, esterase activity assays demonstrated that DCOIT inhibited esterase activity and interfered with the human detoxification process.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Elevated Level of Glycated KQTALVELVK Peptide of Albumin Is Associated with the Risk of Diabetic Nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal disease. Hence, early detection of diabetic nephropathy is essential to mitigate the disease burden. Microalbuminuria, the currently used diagnostic marker of diabetic nephropathy, is not efficient in detecting it at an early stage. Therefore, we explored the utility of glycated human serum albumin (HSA) peptides for risk prediction of diabetic nephropathy. Three glycation-sensitive HSA peptides, namely, FKDLGEENFK, KQTALVELVK, and KVPQVSTPTLVEVSR, with deoxyfructosyllysine (DFL) modification were quantified by targeted mass spectrometry (MS) in a study population comprising healthy and type II diabetes subjects with and without nephropathy. Mass spectrometry, receiver operating characteristic (ROC) curve, and correlation analysis revealed that the DFL-modified KQTALVELVK peptide was better than other glycated HSA peptides and HbA1c for identifying diabetic nephropathy. DFL-modified KQTALVELVK could be a potential marker for risk prediction of diabetic nephropathy.

Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis

The posttranslational modifications (PTM) of human serum albumin (HSA) can result in the development of isoforms that have been identified as potential biomarkers for advanced hepatic diseases. However, previous approaches using top-down (TD) analysis to identify isoforms based on molecular weight may have resulted in misidentifications. The nature of the identified isoforms has never been confirmed in previous works. Here, we aimed to critically evaluate TD for the characterization and determination of HSA isoforms in patients and make an inventory of HSA-PTM.Serum samples from control subjects and patients with liver dysfunctions were analyzed using both top-down (TD) and bottom-up (BU) approaches. TD analysis involved using a LC-TOF-MS system to obtain a multicharged spectrum of HSA, which was deconvoluted to identify isoforms. Spectra were then used for relative quantitation analysis of albumin isoform abundances based on trapezoidal integration. For BU analysis, serums were reduced +/- alkylated, digested with trypsin and analyzed in the Q-TOF, data-dependent acquisition (DDA) mode to generate a SWATH-MS high-resolution mass spectral library of all HSA peptides. Tryptic digests of another set of serum samples were then analyzed using data-independent acquisition (DIA) mode to confirm the presence of HSA isoforms and their modification sites.TD detected 15 isoforms corresponding to various modifications, including glycation, cysteinylation, nitrosylation, and oxidation (di- and tri-). In BU, the spectral library containing 127 peptides allowed for the characterization of the important isoforms with their modified sites, including some modifications that were only characterized in BU (carbamylation, deamidation, and amino-acid substitution). The method used for determining isoforms offered acceptable reproducibility (intra-/inter-assay CVs < 15%) for all isoforms present at relative abundances higher than 2%.Overall, the study found that several isoforms could be missed or misidentified by TD. However, all HSA isoforms identified by TD and reported to be relevant in liver dysfunctions were confirmed by BU. This critical evaluation of TD approach helped design an adequate and reliable method for the characterization of HSA isoforms in patients and offers the possibility to estimate isoform abundances within 3 min. These findings have significant implications for the diagnosis and treatment of liver dysfunctions.

Идентификация фосфонилированных пептидов с применением МАЛДИ-мишени, функционализированной стеаратом лантана

As markers of intoxication, adducts of blood proteins with organophosphorus compounds (OPs) allow establishing the fact of poisoning and, furthermore, enable identification of the OPs by the attached residue. This study aimed to develop a method of specific and selective extraction of blood protein adducts carrying OPs on the surface of a matrix-assisted laser desorption/ionization (MALDI) target functionalized with multimolecular structures based on lanthanum stearate using metal affinity chromatography. We have shown the ability of adsorbent to retain both full-size and dealkylated adducts of blood proteins with OPs. The developed method allowed extraction and identification of peptides of human serum albumin and human butyrylcholinesterase modified with various OPs (after incubation of human blood plasma with OPs in concentrations from 1 to 100 ng/mL), which makes this approach applicable for the purposes of OPs identification in the context of investigation of real cases of intoxication.

Serum albumin cysteine trioxidation is a potential oxidative stress biomarker of type 2 diabetes mellitus

Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.

Identification of S419 on human serum albumin as a novel biomarker for sarin and cyclosarin exposure.

Organophosphorus nerve agents are highly toxic because they inhibit acetylcholinesterase activity, thereby causing a series of symptomatic poisoning. Upon entering the body, nerve agents bind active amino acid residues to form phosphonylated adducts. A potentially beneficial method for specific verification of exposure of nerve agents is based on albumin adducts, which have a half-life of 18 days. This appears to be more effective than the fluoride reactivation method, based on acetylcholinesterase. After the exposure of human serum albumin to nine nerve agents, human serum albumin was denatured, reduced, alkylated and digested with trypsin according to standard mass spectrometry-based proteomics procedures. The phosphonylated peptides of human serum albumin were identified using positive ion electrospray ionization with a quadrupole orbitrap mass spectrometer. The peptide KVPQVSTPTLVESR showed a good mass spectrometric response to the nine nerve agents. The tendency of sarin and cyclosarin was to bind to S419 on the peptide, while the other nerve agents (tabun, soman and V-type nerve agents) were shown to bind more readily to K414 on the peptide. This research revealed a new site, S419, of the tryptic peptide KVPQVSTPTLVEVSR on human albumin to be a valuable biomarker for sarin/cyclosarin exposure, helping to further distinguish sarin and cyclosarin poisoning from that of other nerve agents and providing an important tool for the identification of sarin or cyclosarin in terrorist attacks.

Inhibition effect of pyridoxamine on lipid hydroperoxide-derived modifications to human serum albumin.

Pyridoxamine (PM) is a promising drug candidate for treating various chronic conditions/diseases in which oxidative stress and carbonyl compounds are important factors affecting pathogenicity. These abilities of PM are mainly attributed to its inhibition of advanced glycation and lipoxidation end product formation, by scavenging reactive carbonyl species. PM might therefore prevent protein damage from lipid hydroperoxide-derived aldehydes such as 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) by trapping them. It was previously reported that PM reacts with ONE to produce pyrrolo-1,3-oxazine (PO8) through the formation of pyrido-1,3-oxazine (PO1/PO2). In this study, we found that ONE and HNE yield an identical product containing a pyrrole ring (PO7, PH2) upon reaction with PM. The structure of PO7/PH2 was shown by LC-MS and NMR analyses to be 1-(2-hydroxy-6-hydroxymethyl-3-methylpyridin-4-ylmethyl)-2-pentylpyrrole. PO1, PO7/PH2, and PO8 were the main stable PM-ONE/HNE adducts. In the incubation of human serum albumin (HSA) with ONE or HNE, Lys residues provided the most favorable modification sites for both aldehydes, and the number of HNE-modified sites was higher than that of ONE-modified sites. When HSA was allowed to react with a linoleic acid hydroperoxide in the presence of ascorbic acid, ONE modified more residues (10 Lys, 3 His, 2 Arg) than did HNE (8 His, 2 Lys), indicating the relative reactivity of aldehydes towards amino acid residues. Upon treatment with increasing concentrations of PM, the concentrations of ONE-modified HSA peptides, but not of HNE-modified peptides, were reduced significantly and dose-dependently. Concomitantly, the formation of PM-ONE adducts increased in a dose-dependent manner. The inhibition effect of PM was also confirmed in the cell system subjected to oxidative stress. Our results demonstrate that PM can inhibit lipid hydroperoxide-derived damage to proteins by trapping ONE preferentially, and the resulting PM-ONE adducts can be used as a dosimeter for ONE production to determine the levels of lipid peroxidation.

Thermosensitive Metal Chelation Dual-Template Epitope Imprinting Polymer Using Distillation-Precipitation Polymerization for Simultaneous Recognition of Human Serum Albumin and Transferrin.

A new type of thermosensitive dual-template epitope molecular imprinting polymer was prepared and coated on magnetic carbon nanotubes (MCNTs@D-EMIP) for simultaneous recognition of human serum albumin (HSA) and transferrin (Trf) via the strategies of dual-template epitope imprinting, metal chelation imprinting, and distillation-precipitation polymerization (DPP). C-terminal peptides of HSA and C-terminal peptides of Trf were selected as templates, zinc acrylate and N-isopropylacrylamide were used as functional monomers, and MCNTs@D-EMIP was prepared by the method of DPP. The two types of template epitopes were immobilized by metal chelation and six-membered ring formed with zinc acylate. MCNTs@D-EMIP was prepared in only 30 min, which was much shorter than other polymerization methods. The resultant MCNTs@D-EMIP showed excellent specific recognition ability toward HSA and Trf. The adsorption amounts of MCNTs@D-EMIP for HSA and Trf were 103.67 and 68.48 mg g-1 and the imprinting factors were 2.57 and 2.17, respectively. In addition, MCNTs@D-EMIP displayed a thermosensitive property to realize temperature-controlled recognition and release of target proteins. Furthermore, the results of high-performance liquid chromatography analysis proved that MCNTs@D-EMIP could be applied to specifically recognize two types of targets simultaneously in the biosample. The proposed strategy provided a preparation method for the thermosensitive dual-template epitope imprinting polymer via dual-template imprinting, metal chelation imprinting, and DPP.

Targeted Quantification of the Glycated Peptides of Human Serum Albumin.

Glycated human serum albumin (HSA) serves as an important marker for monitoring the glycemic status. Developing methods for unambiguous identification and quantification of glycated peptides of HSA using high-throughput technologies such as mass spectrometry has a great clinical significance. The following protocol describes the construction of reference spectral libraries for Amadori-modified lysine (AML), N(ε)-(carboxymethyl) lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of synthetically modified HSA using high-resolution mass spectrometers. The protocol also describes work flows, for unambiguous identification and quantification of glycated modified peptides of HSA in clinical plasma using standard spectral libraries by various mass spectrometry approaches such as parallel reaction monitoring(PRM), sequential window acquisition of all theoretical fragment ion spectra (SWATH), and MSE.

Profiling the Serum Albumin Cys34 Adductome of Solid Fuel Users in Xuanwei and Fuyuan, China.

Xuanwei and Fuyuan counties in China have the highest lung cancer rates in the world due to household air pollution from combustion of smoky coal for cooking and heating. To discover potential biomarkers of indoor combustion products, we profiled adducts at the Cys34 locus of human serum albumin (HSA) in 29 nonsmoking Xuanwei and Fuyuan females who used smoky coal, smokeless coal, or wood and 10 local controls who used electricity or gas fuel. Our untargeted "adductomics" method detected 50 tryptic peptides of HSA, containing Cys34 and prominent post-translational modifications. Putative adducts included Cys34 oxidation products, mixed disulfides, rearrangements, and truncations. The most significant differences in adduct levels across fuel types were observed for S-glutathione (S-GSH) and S-γ-glutamylcysteine (S-γ-GluCys), both of which were present at lower levels in subjects exposed to combustion products than in controls. After adjustment for age and personal measurements of airborne benzo(a)pyrene, the largest reductions in levels of S-GSH and S-γ-GluCys relative to controls were observed for users of smoky coal, compared to users of smokeless coal and wood. These results point to possible depletion of GSH, an essential antioxidant, and its precursor γ-GluCys in nonsmoking females exposed to indoor-combustion products in Xuanwei and Fuyuan, China.

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes.

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity.2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma.3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated.4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.

Antibody Enrichment and Mass Spectrometry of Albumin-Cys34 Adducts

Untargeted analyses of tryptic peptides of human serum albumin (HSA) have been used to investigate unknown exposures to reactive electrophiles (adductomics). To reduce the complexity of the analytical matrix and thereby enhance identification of adducts by liquid chromatography-high-resolution mass spectrometry (LC-HRMS), a polyclonal anti-T3 antibody was designed to capture Cys34 adducts in tryptic digests of HSA (T3 is the third largest tryptic peptide). Epitopes were selected from sequences at both C- and N-termini based on the three-dimensional structure of the T3 peptide to minimize the influence of modified Cys34 residues. The assay was simplified by attaching magnetic beads to the anti-T3 antibody. When applied to commercial HSA and to plasma samples from healthy humans and analyzed by LC-HRMS, antibody treatment greatly reduced the background of non-T3 peptides in the sample matrix. Although other lipophilic HSA peptides were still present, presumably due to nonspecific binding to the antibody-magnetic-bead surfaces, their concentrations in antibody-treated samples were reduced about 6-fold compared to the same samples that had not been treated with the antibody. Analysis of antibody-enriched HSA digests from human plasma samples revealed 10 modified T3 peptides of which 8 were identified from accurate masses. Identified peptides included Cys34 oxidation and cysteinylation products and modifications representing losses of water and Lys and transpeptidation of Arg.

Glutathione-based zwitterionic stationary phase for hydrophilic interaction/cation-exchange mixed-mode chromatography

As a naturally hydrophilic peptide, glutathione was facilely immobilized onto silica surface to obtain a novel hydrophilic interaction/cation-exchange mixed-mode chromatographic stationary phase (Click TE-GSH) via copper-free “thiol-ene” click chemistry. The resulting material was characterized by solid state 13C/CP MAS NMR and elemental analysis. The measurement of ζ-potential indicated the cation-exchange characteristics and adjustable surface charge density of Click TE-GSH material. The influence of acetonitrile content and pH value on the retention of ionic compounds was investigated for understanding the chromatographic behaviors. The results demonstrated that Click TE-GSH column could provide both hydrophilic and cation-exchange interaction. Taking advantage of the good hydrophilicity and inherent cation-exchange characteristics of Click TE-GSH material, the resolution of neutral fructosan with high degree of polymerization (DP), basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides was successfully realized in hydrophilic interaction chromatography (HILIC), hydrophilic interaction/cation-exchange mixed-mode chromatography (HILIC/CEX), cation-exchange chromatography (CEX) and electrostatic repulsion/hydrophilic interaction chromatography (ERLIC). On the other hand, the separation of standard peptides varying in hydrophobicity/hydrophilicity and charge was achieved in both CEX and HILIC/CEX mode with high efficiency and distinct selectivity. To further demonstrate the versatility and applicability of Click TE-GSH stationary phase, the separation of a human serum albumin (HSA) tryptic digest was performed in HILIC/CEX mode. Peptides were adequately resolved and up to 86 HSA peptides were identified with sequence coverage of 85%. The results indicated the good potential of Click TE-GSH material in glycomics and proteomics.

Proteomics Study on Cross-Linking Reaction of Formaldehyde and HSA

Possible clinical diagnosis biomarkers of diabetes were proposed using proteomics and bioinformatics methods through the preliminary study on the formaldehyde-human serum albumin crosslinkage. Using LC/MS techniques, the experiment on in vivo cross-linking reaction of formaldehyde with pure protein or plasma protein revealed that the cross-linking reaction of human serum albumin and forrmaldehyde would occur and the concentrations of some normal peptides would change. A group of significant peptides of human serum albumin were selected. As the results of in vivo cross-linking reaction, Non-enzymatic glycosylation modification of HSA were observed in diabetic. The same change of the concentrations of some peptides happened. The above-mentioned results were demonstrated with the hematoplasmas of normal people and diabetics.

Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses.

T84-Intestinal Epithelial Exosomes Bear MHC Class II/Peptide Complexes Potentiating Antigen Presentation by Dendritic Cells

Intestinal epithelial cells release antigen-presenting vesicles (exosomes) bearing major histocompatibility complex class II/peptide complexes stimulating specific immune responses in vivo. To characterize further the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, bind immune target cells, and induce T-cell activation was analyzed in vitro. The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide (3)H-HSA 64-76 and to activate a HLA-DR4-restricted T-cell hybridoma was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of fluorescein isothiocyanate-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. T84-derived exosomes, enriched in CD9, CD81, CD82, and A33 antigen, were capable of binding specifically human serum albumin (HSA) 64-76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T-cell hybridoma directly but induced a productive T-cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (x10(-3)). Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to major histocompatibility complex class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces.

Correlations between air levels of hexahydrophthalic anhydride (HHPA) and HHPA-adducted albumin tryptic peptides in nasal lavage fluid from experimentally exposed volunteers.

Organic acid anhydrides (OAAs) are low molecular weight, reactive compounds extensively used in industry. Exposure to these compounds may lead to allergic symptoms such as rhinitis and asthma. It is important to develop better and more informative methods for assessment of exposure to OAAs. The aim of this study was to develop a method for analysis of specific hexahydrophthalic anhydride (HHPA)-adducted tryptic peptides of human serum albumin (HSA) in nasal lavage (NAL). Furthermore, these peptides were evaluated as biomarkers of exposure. The proteins in the NAL samples were reduced, alkylated and digested with trypsin and the obtained peptides were analyzed using liquid chromatography/tandem mass spectrometry. The total amount of hydrolyzable HHPA in an HHPA-HSA conjugate was used for calibration. A deuterium-labeled HHPA-HSA conjugate was used as internal standard. Five volunteers were exposed to 10, 40 and 80 microg/m3 of HHPA in an exposure chamber and NAL samples were collected before and after exposure. Acceptable precisions of the assay at 13-14% were found for three adducted peptides. The mean levels of these three peptides for the five subjects ranged between 5-22, 15-75 and 33-125 pmol/mL NAL for the exposures at 10, 40 and 80 microg/m3, respectively. High correlations between air levels and the measured peptides were found on an individual basis but there were large inter-individual differences ranging between 63 and 110% for the three peptides. The large differences remained after protein adjustments. It was possible to detect exposures below 10 microg/m3 with the method. Thus, these adducted peptides may be used as biomarkers of exposure, which may better estimate the risk than previous biomarkers developed for OAAs.

Peptide sequence motif analysis of tandem MS data with the SALSA algorithm.

We have developed a pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) for the detection of specific features in tandem MS (MS-MS) spectra. Application of the SALSA algorithm to the detection of peptide MS-MS ion series enables identification of MS-MS spectra displaying characteristics of specific peptide sequences. SALSA analysis scores MS-MS spectra based on correspondence between theoretical ion series for peptide sequence motifs and actual MS-MS product ion series, regardless of their absolute positions on the m/z axis. Analyses of tryptic digests of bovine serum albumin (BSA) by LC-MS-MS followed by SALSA analysis detected MS-MS spectra for both unmodified and multiple modified forms of several BSA tryptic peptides. SALSA analysis of MS-MS data from mixtures of BSA and human serum albumin (HSA) tryptic digests indicated that ion series searches with BSA peptide sequence motifs identified MS-MS spectra for both BSA and closely related HSA peptides. Optimal discrimination between MS-MS spectra of variant peptide forms is achieved when the SALSA search criteria are optimized to the target peptide. Application of SALSA to LC-MS-MS proteome analysis will facilitate the characterization of modified and sequence variant proteins.