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Development of Methods for High-Throughput Enrichment of Glycoproteins and Glycopeptides Employing Multiple Lectin Affinity Chromatography/Tandem Mass Spectrometry
Maděra, Milan ; Pacáková, Věra (vedoucí práce) ; Feltl, Ladislav (oponent) ; Foret, František (oponent) ; Kučerová, Zdenka (oponent)
5 CONCLUSIONS This thesis surrrnrarizes the development of a multimethodological analýical approach employing microcolumn lectin affi1t1,9fuom1tography coupled to highir".otu,to; separation and dete9tion techniques, in order to facilitate the enrichmení of glyco-proteins aná giycopeptioes originating from small quantities of real samples. This is u"toauy a sipificant-ítep towara satis$'ing the demands of contemporary glycoproteomics, which rely orirurt *a small scale analyses, allowing the identification of low abundant protein components' The overď conclusions and contribution to the contemporaÍy scierrce are drawn in tbree followine chaoters. 5.1 combining Lectin Microcolumns with High-Resolution separation Techniques for Enrichment of Glycoproteins and Glycopeptides This section describes coupling small-scďe lectin aÍfinity chromatograpby on-line to high- resolution separation and detection techniques, with the utiliý of a microcolumn loaded with a lectin immobilized onto macroporous silica. Experimental results, involving optimization of coupling procedure, fabrication of lectin microcolumns, verification of tteir Uin&ng efficiency and their possibility of interfacing on-line with nano LC-MS/]víS, me summarizžd into thé following conclusions; o optimized coupling procedure involved resuspending only 125...
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Stanovení perzistentních organohalogenových polutantů metodou GC/MS/MS
Grabic, Roman ; Pacáková, Věra (vedoucí práce) ; Ševčík, Jiří (oponent) ; Matisová, Eva (oponent)
Conclusions o MSiMS methods for analysis of PCDD/Fs, PCBs, OCPs, and PBDES were develoPed o GC separations were optimized for MS/MS detection methods o GC/MS/MS optimized methods were validated. o Limits of quantification at level 0.1 - I pg/pl were achieved. o Linear response over four orders of magnitude was achieved except for some OCPs. o The selectivity of methods is better or comparable with a reference HRMS method' o Cost of GC/MS/MS analysis is significantly lower comPared to GC/HRMS o Developed methods were successfully applied for o Human blood, milk and adipose tissue analysis o Studies of de novo synthesis and destruďion of halogenated POP o In vitro study of PCB accumulation in human cells o List of application is enclosed Page 12
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Hodnocení cizorodých látek pro toxikologické a farmakologické účely
Bořek-Dohalský, Václav ; Feltl, Ladislav (vedoucí práce) ; Pacáková, Věra (oponent) ; Fischer, Jan (oponent) ; Majstřík, Viktor (oponent)
L INTRODUCTION Processes in the nature and also in the human organism are affected by conditions of its running. People are notably affecting their living con- ditions both positively and unfortunately negatively. The environment is exposed to countless human interventions - social impacts, influence of physical factors and chemical compounds. The chemical products belong into many conquests of the modern civilization, which are inherent part of our life. Except their benefit they could also have the negative effect in many cases. Acquiring data for new chemical compounds that are extraneous to organisms (xenobiotics) is difficult, but it is much more difficult to describe the effect of xenobiot- ics onto the living organisms. Toxicology deals with the adverse effects of these compounds; pharmacology is the practice of searching for their bene- ficial effects. My PhD thesis looks at both of these branches. I was en- gaged in possibilities of alternative testing of the acute toxicity of binary mixtures and possibilities of predicting the acute toxicity using the phys- icochemical descriptors of these mixtures in terms of toxicology. In the section covering pharmacology I worked on the development and valida- tion of analytical methods for the determination of drugs in a biological matrix. I' IL lI lfr' ffi...
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High-performance chromatographic methods for determination of proteins
Vařilová, Tereza ; Pacáková, Věra (vedoucí práce) ; Feltl, Ladislav (oponent) ; Coufal, Pavel (oponent) ; Kučerová, Zdenka (oponent)
High-performance chromatographic methods for determination of proteins Ab strakt disertačnípráce Praha,2006 Autor: Mgr. Tereza Vařilová Školitelé: Prof. RNDr.Věra Pacáková, CSc. (Katedra analýické chemie, Karlova Univerzita, Praha) Prof. Dr. Hans-Gerd Janssen (Unilever Research & Development, Nizozemí) Ana|ýza biologicky aktivních látek je rychle se rozvíjejícíobor, kteý tvoří důležitou část aplikací v analýické chemii. Mezi látkami jako jsou peptidy, hormony, léky a drogy tvoří proteiny důleŽitou skupinu bíologicky aktivních látek. Naročným úkolem je izo|ace, separace a identifikace iednotlivých složek složitýchsměsí (např. krevní sénrm). Pro t1to f{9|1z p2ií vysokoúčinnéchromatografické metody svénezastupitelné místo. Proteomika je dynamicky se rozvijejícía dtúežitávědní disciplína. Jejípokrok by nebyl možný bez r{rvoje v metodách analytických a preparativních separací. Komplexnost biologický.ch systémůklade velképoŽadavky na pouŽité metody. V současnosti se běžné vytlŽivá šest ruzných módů HPLC pro ana|ýzu proteinů: iontově qiměrurá chromatografie (IEC)' vylučovací(SEC), afinitní (ALC), chromatografie s hydrofobní interakcí (HIC), chronratograÍie na normální fazi (i.IPLC) a chromatografie na reverzni fázi (RPLC). Dva z těchto módů, konkrétně afinitní chromatograťte a vylučovacíchromatografie, byly...
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Polymethakrylátové monolitické kolony v kapilární kapalinové chromatografii
Grafnetter, Jan ; Coufal, Pavel (vedoucí práce) ; Pacáková, Věra (oponent) ; Ventura, Karel (oponent) ; Sýkora, David (oponent)
3 Conclusions This Thesis gives chromatographic and structural characteristics of the butyl methacrylate monolithic columns prepared for the capillary liquid chromatography. Results of this Thesis clearly demonstrate the possibility of preparation of capillary monolithic columns with a good repeatability of the principal chromatographic parameters. A completely new technique for preparation of the monolithic columns for use in the on-column detection mode was introduced. Different photometric detection modes were applied to monolithic capillary columns in CLC. The advantages and disadvantages of the on-column detection mode in comparison to the additional detection capillary were studied and described. It was shown that the best separation and the most sensitive quantification in the short analysis time could be reached by using the monolithic columns with detection window placed directry on the separation capillary, just after the stationary phase or within the monolith. The attempts leading to preparation of chiral monolithic stationary phases, which would allow enantioseparation of selected chiral analýes' were not totally successful. No time was left to find the suitable chromatographic conditions for enantioseparations. \ I
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Analysa pigmentů a pojiv v barevné vrstvě uměleckých děl
Kučková, Štěpánka ; Pacáková, Věra (vedoucí práce) ; Štulík, Karel (oponent) ; Kopecká, Ivana (oponent) ; Novotná, Miroslava (oponent)
4. Conclusions 1. The method of gas chromatography was successfully optimised for a reliabie oeteňation of individual components in coiophony and Mexioan coPai' ificant 2. of arl the stu<iied pigments, verdigris exhibits the most stgn effect on the composition of fresh corophony and Mexican copal' 3. The degradative processes caused by aftif,rcial ageing of diterpenoids (pimaradiene tolttul"' and abietadiene acids) in colophony and triterpenoids (ct- and B-amyrin) in Mexican copal were detected' ___________ i I -. l i3 c:oriern oť u]tramícrotom. ,,.^'::..:. distonion can be solved by the use oť compare tt," int.u.i A more convenienl reÍlection ...nn,o,..o spectra ", ;;;": J:ffi: ";J-",,;,J.5. The detection iimit corour rayers ,.. Jt ".t infrared spectroscopy for the non-aged moder (*zw) forprussian :',t'' n (w/w) for c6pp1 rimits were rouna Tand0'3-1 "@;;;;: phthalocvanine' 1-3 %o phtharocyanin " r, n'l'.the artinciatl;' ;JTil:t:Jffi:: ó. MALDI.T.F MS is w/w) and indigo (3.l0 % wlw). in fresh and artificial able to ídentify indigo ar are 0.01 o/o lv/wÍ.b. i".: aged model cil;, copper phthalocyanine 7. MALDI-T.. ,, ,o,tt*o and 0'03 n *,* ^i,""' The detection limits their morecurar ions. :ntifies carmine, tu..ui. ropper phthalocyanine' associatedproreins. In addition' t"*;;:1""::"::ff:;;'Jl 8. The technique...
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Studium interakcí podílejících se na mechanizmu separace v chromatografických separačních systémech
Kalíková, Květa ; Tesařová, Eva (vedoucí práce) ; Pacáková, Věra (oponent) ; Ševčík, Juraj (oponent)
Univerzita Karlova v Praze Přírodovědecká fakulta Katedra fyzikální a makromolekulární chemie Studium interakcí podílejících se na mechanizmu separace v chromatografických separačních systémech Autoreferát dizertační práce Květa Kalíková Praha 2009 Dizertační práce shrnuje výsledky získané během let 2005-2009 na Katedře fyzikální a makromolekulární chemie, PřF UK v Praze. Školitel: Doc. RNDr. Eva Tesařová, CSc. 2 OBSAH 1. Úvod 4 2. Cíle práce 4 3. Výsledky a diskuze 5 3.1 Charakterizace separačních systémů metodou LFER (publikace I) 5 3.2 Popis systémových píků v RP HPLC s vodnými mobilními fázemi (publikace II) 6 3.3 Příklady nalezení a využití optimalizovaných separačních systémů 9 3.3.1 Achirální systémy 9 3.3.1.1 Separace estrogenů (publikace III) 9 3.3.1.2 Separace 1,4-benzodiazepinů (připravovaná publikace) 9 3.3.2 Chirální systémy (publikace IV) 11 3.3.2.1 Optimalizace a validace HPLC metody pro enantioseparaci (±)-cloprostenolu (publikace V) 11 4. Závěry 12 5. Literatura 13 6. Seznam publikovaných prací 14 7. Příspěvky na konferencích 15 8. Životopis 17 3 1. Úvod Vysokoúčinná kapalinová chromatografie (HPLC) se řadí mezi nejčastěji používané separační metody. Vyniká vysokou spolehlivostí, dobrou opakovatelností a robustností. Výhodou této metody je možnost ovlivňovat separaci nejen volbou...
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