The 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. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.
Elevated 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. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five modification reactions of amino acids. The positive effects of temperature dominated in the 30 min long gradients since the column operated at 90 °C provided the largest number of identified peptides and proteins. However, the adverse effects of temperature on peptide integrity in longer liquid chromatography-mass spectrometry (LC-MS) analyses required its reduction to obtain optimum results. The largest number of peptides was identified using the column maintained at 75 °C in 60 min long gradients, at 60 °C in 120 min long gradients, and at 45 °C in 240 min long gradients. Our results indicate that no universal column temperature exists for bottom-up LC-MS analyses. Quite the contrary, the temperature setting must be selected rationally to exploit the full capabilities of the state-of-the-art mass spectrometers in proteomic LC-MS analyses, with the gradient time being a critical factor.
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. We found out that the peptide formylation was associated with dissolving the peptides in a solvent containing mere 0.1% formic acid. Similar conclusions were drawn after analyzing publicly available proteomic data. We further demonstrated that these unwanted modifications can be averted via handling the samples at a low temperature or, obviously, via replacing formic acid in the solvent with trifluoroacetic acid. These simple countermeasures can contribute to a reduction in the part of the MS/MS spectra that remain unassigned to a peptide sequence.
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
1 svazek : ilustrace, tabulky ; 30 cm
Předčasný odtok plodové vody (PPROM) je příčinou podstatné části předčasných porodů a výrazně zvyšuje perinatální morbiditu a mortalitu. Hlavním důvodem je kauzální spojitost s intraamniální infekcí (IAI), k níž dochází u 40-60% PROM pacientek. V nejtěžších případech může IAI vyústit v zánětlivou odpověď plodu (FIRS) a způsobit trvalé zdravotní následky pro novorozence nebo skončit smrtí plodu. FIRS má velmi často subklinický průběh, a proto většinou zůstává neodhalen. V klinické praxi doposud nebyl etablován žádný nástroj pro jeho exaktní prenatální diagnostiku. Projekt je zaměřen na charakterizaci diagnostického potenciálu nativních polypeptidů plodové vody směrem k možnosti odhalení probíhající IAI, potažmo FIRS.; Preterm premature rupture of membranes (PPROM) is a principal cause of preterm births and considerably increases perinatal morbidity and mortality. The main reason is the causal link with intraamniotic infection (IAI), which occurs in 40-60% of PPROM patients. The most serious cases of IAI may result in fetal inflammatory response (FIRS) and cause permanent health consequences for the newborn or end up by the death of the fetus. FIRS is very often subclinical and thus frequently remains undetected. Currently, there is no tool for its precise prenatal diagnostics available. The project is focused on characterization of the diagnostic potential of native amniotic fluid polypeptides with regard to detection of IAI and FIRS.
- MeSH
- amnion mikrobiologie MeSH
- bakteriální nálož MeSH
- chorioamnionitida diagnóza MeSH
- chromatografie kapalinová MeSH
- hmotnostní spektrometrie MeSH
- infekční komplikace v těhotenství MeSH
- peptidy analýza MeSH
- předčasná porodní činnost MeSH
- předčasný odtok plodové vody MeSH
- předčasný porod MeSH
- proteomika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- třetí trimestr těhotenství MeSH
- výzkumný projekt MeSH
- zánět MeSH
- ženy MeSH
- Konspekt
- Gynekologie. Porodnictví
- NLK Obory
- gynekologie a porodnictví
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
Due 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., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.
- MeSH
- buněčný cyklus účinky léků MeSH
- doxorubicin toxicita MeSH
- fenotyp MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- myokard cytologie metabolismus MeSH
- proteom analýza MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data.
- MeSH
- delece genu MeSH
- faktory virulence genetika metabolismus MeSH
- fenotyp MeSH
- Francisella tularensis genetika metabolismus patogenita MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- protein hostitelského faktoru 1 genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- tularemie mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.
- MeSH
- analýza hlavních komponent MeSH
- bakteriální proteiny metabolismus MeSH
- chromatografie kapalinová MeSH
- faktory virulence metabolismus MeSH
- Francisella tularensis klasifikace metabolismus růst a vývoj MeSH
- proteomika metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- teplota MeSH
- Publikační typ
- práce podpořená grantem MeSH
