Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography.
View Article and Find Full Text PDFWe have recently demonstrated the ability of a C stationary phase with a positively charged surface (PCS-C) to provide superior chromatographic separation of peptides using mobile phase acidified with a mere 0.01 % formic acid, significantly improving MS sensitivity. Here, we examined three columns packed with different PCS-C phases using the MS-favorable mobile phase acidified with low formic acid concentrations to establish the impact of separation performance and better MS sensitivity on peptide identifications.
View Article and Find Full Text PDFThe default choice of mobile phase acidifier for bottom-up LC-MS proteomic analyses is 0.10% formic acid because of its decent acidity, decent ion pairing ability, and low suppression of electrospray ionization. In recent years, state-of-the-art columns have been designed specifically to provide efficient separation even when using an MS-friendly mobile phase of low ionic strength.
View Article and Find Full Text PDFRadioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e.
View Article and Find Full Text PDFSample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics.
View Article and Find Full Text PDFThe performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories.
View Article and Find Full Text PDFA wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies.
View Article and Find Full Text PDFElevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses.
View Article and Find Full Text PDFPreterm prelabour rupture of membranes beyond the 34th week of gestation (late PPROM) is frequently associated with the risk of the microbial invasion of the amniotic fluid (MIAC) and histological chorioamnionitis (HCA). Hence, we employed a Tandem Mass Tag-based approach to uncover amniotic fluid proteome response to the presence of MIAC and HCA in late PPROM. Protein dysregulation was associated with only five cases in the group of 15 women with confirmed MIAC and HCA.
View Article and Find Full Text PDFFrancisella tularensis is a highly virulent intracellular bacterium and the causative agent of tularemia. The disease is characterized by the suboptimal innate immune response and consequently by the impaired adaptive immunity. The virulence of this pathogen depends on proteins encoded by a genomic island termed the Francisella Pathogenicity Island (FPI).
View Article and Find Full Text PDFThe literature reports on cationic and anionic phthalocyanines (Pcs) for photodynamic therapy suggest systematically significant differences in activity. In this work, ten different zinc(II) Pcs with carboxylate functions or quaternary nitrogens (hydrophilic anionic, hydrophilic cationic, amphiphilic anionic, and amphiphilic cationic) were investigated, with the aim of revealing reasons for such differences. assays on HeLa, MCF-7, and HCT-116 cells confirmed higher photoactivity for cationic Pcs (EC ∼ 3-50 nM) than for anionic Pcs (EC ∼ 0.
View Article and Find Full Text PDFBackground: Amniotic fluid is clinically accessible via amniocentesis and its protein composition may correspond to birth timing. Early changes in the amniotic fluid proteome could therefore be associated with the subsequent development of spontaneous preterm delivery.
Objective: The main objective of this study was to perform unbiased proteomics analysis of the association between mid-trimester amniotic fluid proteome and spontaneous preterm delivery and gestational duration, respectively.
The ability of concentrated formic acid to formylate reactive amino acid residues is known from previous reports. In contrast, solvents containing a low concentration of formic acid are generally recognized as a safe environment for proteomic applications. The primary objective of this study was to explain the excessive formylation rate in tryptic peptides that did not come into contact with concentrated formic acid.
View Article and Find Full Text PDFDue to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-μL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments.
View Article and Find Full Text PDFThe nucleoid-associated HU proteins are small abundant DNA-binding proteins in bacterial cell which play an important role in the initiation of DNA replication, cell division, SOS response, control of gene expression and recombination. HU proteins bind to double stranded DNA non-specifically, but they exhibit high affinity to abnormal DNA structures as four-way junctions, gaps or nicks, which are generated during DNA damage. In many pathogens HU proteins regulate expression of genes involved in metabolism and virulence.
View Article and Find Full Text PDFBreast cancer is the most common and molecularly well-characterized malignant cancer in women; however, its progression to metastatic cancer remains lethal for 78% of patients within 5 years of diagnosis. Identifying novel markers in high risk patients using quantitative methods is essential to overcome genetic, inter-tumor, and intra-tumor variability, and to translate novel findings into cancer diagnosis and treatment. Using untargeted proteomics, we recently identified 13 proteins associated with some key factors of breast cancer aggressiveness: estrogen receptors, tumor grade, and lymph node status.
View Article and Find Full Text PDFBiochim Biophys Acta Proteins Proteom
May 2017
Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics.
View Article and Find Full Text PDFObjectives: To confirm the initial iTRAQ-based proteomic findings related to the plasma fibronectin level and hypertrophic cardiomyopathy using an antibody-based assay.
Methods: The iTRAQ technique was used for the discovery proteomic analysis of the pooled plasma samples. The ELISA was used for verification of fibronectin plasma concentration in individual samples.
Objective: The aim of this study was to identify early proteomic biomarkers of spontaneous preterm delivery (PTD) in mid-trimester amniotic fluid from asymptomatic women.
Methods: This is a case-cohort study. Amniotic fluid from mid-trimester genetic amniocentesis (14-19 weeks of gestation) was collected from 2008 to 2011.
Unlabelled: Transgelin and transgelin-2 have been discussed as potential markers of various cancers. Here we identified increased transgelin level in lymph node positive vs. negative, low grade primary breast cancer tissues using 2-DE in the cohort of 12 patients.
View Article and Find Full Text PDFDue to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e.
View Article and Find Full Text PDFCeramides with ultralong chains (≥30 carbons), also known as acylceramides, play a critical role in the survival of mammals on dry land. An efficient and scalable synthesis of four major classes of ultralong human skin ceramides is reported. The key approach involves the use of a succinimidyl ester that acts as a protective group, helps overcome the extremely low solubility, and simultaneously activates the fatty acid for its clean and high-yielding attachment to a sphingoid base.
View Article and Find Full Text PDFSpontaneous preterm birth significantly contributes to the overall neonatal morbidity associated with preterm deliveries. Nearly 50% of cases are associated with microbial invasion of the amniotic cavity followed by an inflammatory response. Robust diagnostic tools for neonates jeopardized by infection and inflammation may thus decrease the overall neonatal morbidity substantially.
View Article and Find Full Text PDFProteins, peptides and nucleic acids are commonly isolated and purified in almost all bioscience laboratories. Methods based on molecular recognition are currently the most powerful tool in separation processes due to their selectivity and recovery. The aim of this study was to prove the versatility and the ability of an affinity carrier containing the immobilised ligand oracin (previously developed by our workgroup) to selectively bind carbonyl-reducing enzymes.
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