Tryptic Cleavage Research Articles

Mass spectrometry-complemented molecular modeling predicts the interaction interface for a camelid single-domain antibody targeting the Plasmodium falciparum circumsporozoite protein’s C-terminal domain

BackgroundBioanalytical methods that enable rapid and high-detail characterization of binding specificities and strengths of protein complexes with low sample consumption are highly desired. The interaction between a camelid single domain antibody (sdAbCSP1) and its target antigen (PfCSP-Cext) was selected as a model system to provide proof-of-principle for the here described methodology. Research design and methodsThe structure of the sdAbCSP1 – PfCSP-Cext complex was modeled using AlphaFold2. The recombinantly expressed proteins, sdAbCSP1, PfCSP-Cext, and the sdAbCSP1 – PfCSP-Cext complex, were subjected to limited proteolysis and mass spectrometric peptide analysis. ITEM MS (Intact Transition Epitope Mapping Mass Spectrometry) and ITC (Isothermal Titration Calorimetry) were applied to determine stoichiometry and binding strength. ResultsThe paratope of sdAbCSP1 mainly consists of its CDR3 (aa100–118). PfCSP-Cext’s epitope is assembled from its α-helix (aa40–52) and opposing loop (aa83–90). PfCSP-Cext’s GluC cleavage sites E46 and E58 were shielded by complex formation, confirming the predicted epitope. Likewise, sdAbCSP1’s tryptic cleavage sites R105 and R108 were shielded by complex formation, confirming the predicted paratope. ITEM MS determined the 1:1 stoichiometry and the high complex binding strength, exemplified by the gas phase dissociation reaction enthalpy of 50.2 kJ/mol. The in-solution complex dissociation constant is 5 × 10-10 M. ConclusionsCombining AlphaFold2 modeling with mass spectrometry/limited proteolysis generated a trustworthy model for the sdAbCSP1 – PfCSP-Cext complex interaction interface.

Proteotypic peptides of hairs for the identification of common European domestic and wild animal species revealed by in-sample protein digestion and mass spectrometry analysis.

The aim of this work is to offer an alternative or complementary analytical tool to the time consuming and expensive methods commonly used for the recognition of animal species according to their hairs. The paper introduces a simple and fast way for species differentiation of animal hairs called in-sample digestion. A total of 10 European animal species, including cat, cow, common degu, dog, fallow deer, goat, horse, sika deer, rabbit, roe deer, and 17 different breeds of dogs were examined using specific tryptic cleavage directly in hair followed by matrix-assisted laser desorption/ionization - time of flight mass spectrometry and liquid chromatography electrospray ionization quadrupole time of flight. Principal component analysis was used for the subsequent mass spectrometric data evaluation. This novel approach demonstrates the ability to distinguish among individual animal species, which is supported by finding characteristic m/z values obtained by the mass spectrometry for each animal species. The approach was successfully tested on two "blind" samples. On the other hand, the attempt to distinguish among hairs of different dog breeds has not been successful due to the very similar protein composition and their amino acid sequences. This article is protected by copyright. All rights reserved.

Effect of glycation derived from α-dicarbonyl compounds on the in vitro digestibility of ovalbumin: Tracing of advanced glycation end-products and immuno-active peptides

Currently, the biological consequences of advanced glycation end-products (AGEs) and their link to the antigenicity of food allergens are largely unknown due to the uncertainty in their digestive fates within the body. In this study, the influence of glycation derived from α-dicarbonyl compounds (α-DCs), precursors of AGEs, on digestive behaviors of ovalbumin (OVA) was investigated in a two-step simulated gastrointestinal (GI) model. Methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone were selected as typical α-DCs to obtain glycated OVA with different AGE-modifications (AGE-Ms). It was unveiled that α-DC-glycation reduced the digestibility of OVA via blocking tryptic cleavage sites and inducing steric hindrance, especially seen in the GO- and MGO-OVA groups. The formed AGE-Ms, depending on the precursor type, showed masking effects on the epitopes of OVA, which counteracted the negative effects of reduced digestibility on its antigenicity. Substantial changes in the peptide release patterns were also noted in glycated OVA, including alterations in the sequences and structures of several known protease-resistant epitopes of OVA. This study provides new insights into the nutritional and healthy effects of MRPs in heat-processed foods, as well as their potential connection to the modulation of egg allergy.

Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

Optimization of proteomics sample preparation for forensic analysis of skin samples

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SignificanceGenetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β.

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

Coenzyme-Mediated Electro-RAFT Polymerization for Amplified Electrochemical Interrogation of Trypsin Activity.

Trypsin is a key proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. Taking advantage of the coenzyme-mediated electrografting of ferrocenyl polymers as a novel strategy for signal amplification, herein, a signal-on cleavage-based electrochemical biosensor is reported for the highly selective interrogation of trypsin activity at ultralow levels. The construction of the trypsin biosensor involves (i) the immobilization of peptide substrates (without free carboxyl groups) via the N-terminus, (ii) the tryptic cleavage of peptide substrates, (iii) the site-specific labeling of the reversible addition-fragmentation chain transfer (RAFT) agents, and (iv) the grafting of ferrocenyl polymers through the electro-RAFT (eRAFT) polymerization, which is mediated by potentiostatic reduction of nicotinamide adenine dinucleotide (NAD+) coenzymes. Through the NAD+-mediated eRAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA), the presence of a few tryptic cleavage events can eventually result in the recruitment of a considerable amount of ferrocene redox tags. Obviously, the NAD+-eRAFT polymerization is low-cost and easy to operate as a highly efficient strategy for signal amplification. As expected, the as-constructed biosensor is highly selective and sensitive toward the signal-on interrogation of trypsin activity. Under optimal conditions, the detection limit can be as low as 18.2 μU/mL (∼72.8 pg/mL). The results also demonstrate that the as-constructed electrochemical trypsin biosensor is applicable to inhibitor screening and the interrogation of enzyme activity in the presence of complex sample matrices. Moreover, it is low-cost, less susceptible to false-positive results, and relatively easy to fabricate, thus holding great potential in diagnostic and therapeutic applications.

Investigation and Highly Accurate Prediction of Missed Tryptic Cleavages by Deep Learning.

Trypsin is one of the most important and widely used proteolytic enzymes in mass spectrometry (MS)-based proteomic research. It exclusively cleaves peptide bonds at the C-terminus of lysine and arginine. However, the cleavage is also affected by several factors, including specific surrounding amino acids, resulting in frequent incomplete proteolysis and subsequent issues in peptide identification and quantification. The accurate annotations on missed cleavages are crucial to database searching in MS analysis. Here, we present deep-learning predicting missed cleavages (dpMC), a novel algorithm for the prediction of missed trypsin cleavage sites. This algorithm provides a very high accuracy for predicting missed cleavages with area under the curves (AUCs) of cross-validation and holdout testing above 0.99, along with the mean F1 score and the Matthews correlation coefficient (MCC) of 0.9677 and 0.9349, respectively. We tested our algorithm on data sets from different species and different experimental conditions, and its performance outperforms other currently available prediction methods. In addition, the method also provides a better insight into the detailed rules of trypsin cleavages coupled with propensity and motif analysis. Moreover, our method can be integrated into database searching in the MS analysis to identify and quantify mass spectra effectively and efficiently.

Evaluation of Filter, Paramagnetic, and STAGETips Aided Workflows for Proteome Profiling of Symbiodiniaceae Dinoflagellate

The integrity of coral reef ecosystems worldwide rests on a fine-tuned symbiotic interaction between an invertebrate and a dinoflagellate microalga from the family Symbiodiniaceae. Recent advances in bottom-up shotgun proteomic approaches and the availability of vast amounts of genetic information about Symbiodiniaceae have provided a unique opportunity to better understand the molecular mechanisms underpinning the interactions of coral-Symbiodiniaceae. However, the resilience of this dinoflagellate cell wall, as well as the presence of polyanionic and phenolics cell wall components, requires the optimization of sample preparation techniques for successful implementation of bottom-up proteomics. Therefore, in this study we compare three different workflows—filter-aided sample preparation (FASP), single-pot solid-phase-enhanced sample preparation (SP3), and stop-and-go-extraction tips (STAGETips, ST)—to develop a high-throughput proteotyping protocol for Symbiodiniaceae algal research. We used the model isolate Symbiodinium tridacnidorum. We show that SP3 outperformed ST and FASP with regard to robustness, digestion efficiency, and contaminant removal, which led to the highest number of total (3799) and unique proteins detected from 23,593 peptides. Most of these proteins were detected with ≥2 unique peptides (73%), zero missed tryptic peptide cleavages (91%), and hydrophilic peptides (>70%). To demonstrate the functionality of this optimized SP3 sample preparation workflow, we examined the proteome of S. tridacnidorum to better understand the molecular mechanism of peridinin-chlorophyll-protein complex (PCP, light harvesting protein) accumulation under low light (LL, 30 μmol photon m−2 s−1). Cells exposed to LL for 7 days upregulated various light harvesting complex (LHCs) proteins through the mevalonate-independent pathway; proteins of this pathway were at 2- to 6-fold higher levels than the control of 120 μmol photon m−2 s−1. Potentially, LHCs which were maintained in an active phosphorylated state by serine/threonine-protein kinase were also upregulated to 10-fold over control. Collectively, our results show that the SP3 method is an efficient high-throughput proteotyping tool for Symbiodiniaceae algal research.

Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library.

This work presents methods for identifying and then creating a mass spectral library for disulfide-linked peptides originating from the NISTmAb, a reference material of the humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced and non-reduced samples under neutral and weakly basic conditions followed by nanoflow liquid chromatography tandem mass spectrometry (LC–MS/MS). Spectra of peptides containing disulfide bonds are identified by both MS1 ion and MS2 fragment ion data in order to completely map all the disulfide linkages in the NISTmAb. This led to the detection of 383 distinct disulfide-linked peptide ions, arising from fully tryptic cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation mixtures, and metal adducts. Fragmentation features of disulfide bonds under low-energy collision dissociation were examined. These include (1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide bond cleavage, often leading to extensive fragmentation; and (3) double cleavage products resulting from breakages of two peptide bonds or both peptide and disulfide bonds. Automated annotation of various complex MS/MS fragments enabled the identification of disulfide-linked peptides with high confidence. Peptides containing each of the nine native disulfide bonds were identified along with 86 additional disulfide linkages arising from disulfide bond shuffling. The presence of shuffled disulfides was nearly completely abrogated by refining digest conditions. A curated spectral library of 702 disulfide-linked peptide spectra was created from this analysis and is publicly available for free download. Since all IgG1 antibodies have the same constant regions, the resulting library can be used as a tool for facile identification of “hard-to-find” disulfide-bonded peptides. Moreover, we show that one may identify such peptides originating from IgG1 proteins in human serum, thereby serving as a means of monitoring the completeness of protein reduction in proteomics studies. Data are available via ProteomeXchange with identifier PXD023358.

Capillary electrophoretic profiling of in-bone tryptic digests of proteins as a potential tool for the detection of inflammatory states in oral surgery.

The commonly used histological assessment of pathological states of alveolar bone tissues in oral surgery needs laborious and time-consuming processing by an experienced histologist. Therefore, a simpler and faster methodology is required in this field. Following this demand, this paper reports a straightforward approach using the tryptic cleavage of proteins directly in bone without its demineralization, followed by the capillary electrophoresis-ultraviolet detection profiling of the yielded protein digest. Cleavage-derived peptides were separated by capillary electrophoresis in acidic background electrolytes, pH 2.01-2.54. The best resolution of peptide fragments with the highest peak capacity was achieved in the background electrolyte composed of 55mM H3 PO4 , 14mM tris(hydroxymethyl)aminomethan, pH 2.01. The differences in the obtained capillary electrophoresis-ultraviolet detection profiles with characteristic patterns for particular bone samples were subsequently discriminated by linear discriminant analysis over principal components. This approach was first verified on porcine bone tissues as model samples; jawbone and calf bone tissues could be discriminated with an accuracy of 100%. Subsequently, the method was capable of differentiating unequivocally between human healthy and inflammatory alveolar bone tissues obtained from oral surgery. This procedure seems to be promising as complement or even an alternative to the traditional histological discrimination between healthy and inflammatory bone tissues in oral surgery.

Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin.

Recombinant human erythropoietin (rhEPO) is an important biopharmaceutical for which glycosylation is a critical quality attribute. Therefore, robust analytical methods are needed for the in-depth characterization of rhEPO glycosylation. Currently, the protease GluC is widely established for the site-specific glycosylation analysis of rhEPO. However, this enzyme shows disadvantages, such as its specificity and the characteristics of the resulting (glyco)peptides. The use of trypsin, the gold standard protease in proteomics, as the sole protease for rhEPO is compromised, as no natural tryptic cleavage site is located between the glycosylation sites Asn24 and Asn38. Here, cysteine aminoethylation using 2-bromoethylamine was applied as an alternative alkylation strategy to introduce artificial tryptic cleavage sites at Cys29 and Cys33 in rhEPO. The (glyco)peptides resulting from a subsequent digestion using trypsin were analyzed by reverse-phase liquid chromatography-mass spectrometry. The new trypsin-based workflow was easily implemented by adapting the alkylation step in a conventional workflow and was directly compared to an established approach using GluC. The new method shows an improved specificity, a significantly reduced chromatogram complexity, allows for shorter analysis times, and simplifies data evaluation. Furthermore, the method allows for the monitoring of additional attributes, such as oxidation and deamidation at specific sites in parallel to the site-specific glycosylation analysis of rhEPO.

Direct tryptic cleavage in bone tissue followed by LC-MS/MS as a first step towards routine characterization of proteins embedded in alveolar bones

While methods like radiology or histology are routinely used in oral surgery, a simple and effective method for characterization of alveolar bones at the molecular level is missing. This is primarily due to the fact that proteomic analysis of hard tissue represents a serious analytical challenge. In this study, we developed a simple technique for the proteomic characterization of bone tissue in the field of oral surgery. After a prewashing step with different solvents, specific digestion with trypsin was performed directly in porcine alveolar bone tissues without the prior reduction and alkylation of disulfide bonds. The released peptides were then analyzed using liquid chromatography-tandem mass spectrometry. When 2-propanol was used in the pre-incubation step, this approach identified 487 proteins in the maxillary (upper jaw) bone and 443 proteins in the mandibular (lower jaw) bone, 40 of each being specifically ascribed to bone tissue. This suggests that routine use of our technique can provide deeper insights into the field of oral surgery at the molecular level. Furthermore, it could also have diagnostic potential in differentiating between the proteomic patterns of healthy and pathological alveolar bone tissue. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD018836.

Dual-Probe Approach for Mass Spectrometric Quantification of MUC1-Specific Terminal Gal/GalNAc In Situ.

Protein glycosylation is a prevalent post-translational modification that mediates a variety of cellular processes. For membrane proteins, glycosylation at their terminal motif is usually more functional. Among the various glycosylation types found in membrane proteins, O-glycosylation is the most common and is closely correlated with a variety of cancer types, including breast cancer. Slightly aberrant expression of certain O-glycans can significantly affect cancer progression, especially at the cancer-related membrane protein level. To collect biological information on protein-specific glycosylation and further explore clinical applications, quantitative detection of glycosylation is essential. However, few assays have been reported for the in situ detection of protein-specific glycosylation to date. Herein, we developed a dual-probe approach for mass spectrometric quantification of protein-specific glycosylation using the terminal galactose/N-acetylgalactosamine (Gal/GalNAc) of MUC1 as a model. The dual-probe (i.e., protein probe and glycan probe) system was first designed and built. The protein probe contained an aptamer for MUC1 protein recognition and a capture DNA sequence. Correspondingly, the glycan probe had a DNA sequence complementary to that of the capture DNA, a substrate peptide containing a reporter peptide, and a tryptic cleavage site, and could be covalently linked with the terminal Gal/GalNAc. Exonuclease III enabled recycling of the hybridization-dehybridization process in a restricted space. Finally, the reporter peptide was tryptically released and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass response of the reporter peptide represented the amount of MUC1-specific terminal Gal/GalNAc. This dual-probe approach was applied for in situ detection of MUC1-specific terminal Gal/GalNAc in three human breast cancer cell lines and 32 pairs of matched breast cancer tissue samples. The relationship between MUC1-specific terminal Gal/GalNAc expression and breast cancer diagnosis/prognosis was also assessed.

Electrochemically controlled grafting of polymers for ultrasensitive electrochemical assay of trypsin activity

As one of the most important proteolytic enzymes, trypsin is useful as a reliable and specific biomarker for the diagnosis of pancreatitis and other pathological conditions. In this paper, a novel signal-on electrochemical biosensor based on the use of electrochemically controlled grafting of polymers as an amplification strategy is described for the ultrasensitive assay of trypsin activity. The carboxyl-group-free peptide, serving as the substrate for the recognition of trypsin, is first immobilized via its N-terminus. The tryptic cleavage of peptide substrate can generate a free carboxyl group at the C-terminus of the truncated peptide, to which through the carboxylate-Zr(IV)-carboxylate linkage the carboxyl-group-containing initiator for atom transfer radical polymerization (ATRP) can be conjugated. The subsequent surface-initiated grafting of polymers (SI-GOP) based on electrochemically controlled ATRP (eATRP), with ferrocenylmethyl methacrylate (FcMMA) as the monomer, can bring a large amount of Fc tags to electrode surface, resulting in the generation of a very high detection signal. The eATRP-based SI-GOP is easy to operate and low-cost as an amplification strategy. Under optimal conditions, the detection limit for trypsin activity can be down to 0.016 mU mL−1 (~2.68 pM or ~0.064 ng mL−1). As the current signal increases with trypsin activity, this trypsin biosensor is less susceptible to false positives due to the signal-on mode. Moreover, it is highly selective and applicable to inhibitor screening and the assay of trypsin activity in the presence of complex biological matrices. Taking together, this electrochemical trypsin biosensor may hold great potential in diagnostic applications.

Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.

In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.

Thiol/disulfide exchange occurs in rotavirus structural proteins during contact with intestinal villus cell surface.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.

Duplex-Specific Nuclease-Mediated Amplification Strategy for Mass Spectrometry Quantification of MiRNA-200c in Breast Cancer Stem Cells.

MicroRNAs (miRNAs) play a significant role in numerous biological processes and are implicated in a range of cancers, including breast cancer. MiRNAs have the potential to be biomarkers in clinical practice because of their distorted and unique expression, especially with regard to their presence in cancer stem cells (CSCs) that have applications in cancer diagnosis and treatment. Thus, the absolute determination of miRNA expression levels is a prerequisite for exploring their applications. Nevertheless, currently available methods may not be adequate for the detection of miRNAs in CSCs due to the inherently low population of these cells. Therefore, we combined a duplex-specific nuclease (DSN)-mediated amplification strategy with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in this study. We designed the substrate peptide GDKAVLGVDPFR, which contains the reporter peptide AVLGVDPFR and a tryptic cleavage site (lysine at position 3) in combination with a biotinylated DNA sequence that was complementary to a target miRNA (i.e., miR-200c). Then, this newly synthesized DNA-peptide probe was hybridized with the target miRNA. Upon the introduction of the DSN, the enzyme degraded the probe into two parts where the target miRNA was integrated and thereby triggered further cleavage. The accumulated peptide fragments were ultimately digested with trypsin to release the reporter peptide for LC-MS/MS quantification. Under these circumstances, the miRNA signal was converted and amplified into a mass response of the reporter peptide. After optimization of the parameters, including temperature, hybridization/DSN time, and the amounts of DSN and streptavidin agarose beads, we demonstrated a linear detection range between 1 fM and 200 fM for miR-200c. The detection limit obtained was 3 orders of magnitude lower than those previously reported. Finally, quantification of miR-200c in breast cancer stem cells (BCSCs) and in stem cells isolated from breast tumors was performed. We also compared these data with quantitative reverse transcription PCR (qRT-PCR) results.

An amplification strategy using DNA-Peptide dendrimer probe and mass spectrometry for sensitive MicroRNA detection in breast cancer

MicroRNAs (miRNAs) are emerging as novel biomarkers for diagnosis and treatment of various cancers, including breast cancer. Because the value of biomarkers primarily depends on whether they are quantifiable, great effort has been taken to develop assays for sensitive and accurate quantification of miRNAs. However, most of current assays have high nonspecific amplification effect, which limits quantification accuracy. In this study, we circumvented copying of nucleic acid sequence and developed a signal amplification strategy based on a novel DNA-peptide dendrimer (DPD) probe coupled with mass spectrometry. The DPD probe RP8-MAP4-DNA contained three functional domains, including substrate peptides containing eight reporter peptides and tryptic cleavage sites, peptide dendrimer scaffold and DNA complementary to target miRNA. The probe was first hybridized with the target miRNA (i.e., miR-21) that was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was liberated and quantified using LC-MS/MS. The signal intensity was approximately 8 fold greater than that without signal amplification. Finally, the developed assay was applied for the quantitative analysis of miR-21 in 3 human breast cell lines and 102 matched pairs of breast tissue samples. The miR-21 expression in tissue was also evaluated depend on histopathological features, molecular subtypes and prognosis of breast cancer. The result demonstrated that combination of DPD probe and mass spectrometry is a promising strategy for quantification of miRNAs and illustration of their biomarker potential, especially those at low abundance.