Sol‐Gel Materials Used in Phosphoproteomics and Glycoproteomics Applications

Glycosylation and phosphorylation are two of the most commonly seen and important post‐translational modifications (PTMs) of proteins. Both play key role in many vital biological processes. Mass spectrometry is the most favored technique used for monitoring the dynamics of PTMs. Mass spectrometric analysis of phosphopeptide and glycopeptide is a crucial step in phosphoproteomics and glycoproteomics to understand the role of these modifications in the cellular pathways. Enrichment and purification of (phospho‐ and glyco‐) peptides and glycans are recommended prior to mass spectrometric analysis because of the lower amount of modified peptides in a peptide mixture. Herein, we present titanium/silica and zirconium/silica sol‐gel materials for the enrichment of (phospho‐ and glyco‐) peptides and glycans to enhance MALDI‐MS analysis performance. Enrichment of (phospho‐ and glyco‐) peptides was successfully applied using standard proteins including β‐casein, fetuin, and IgG as well as some complex medium. The sol‐gel materials were compared with commercial metal oxides regarding their modified peptide enrichment performances

Model Sentetik Polimerlerin Ve Komplekslerinin Gaz Fazı Karakteristiklerinin İncelenmesi

Mass spectrometry is the most convenient technique for obtaining useful information about the formation parameters of noncovalent complexes in both solution and gas phases with high accuracy and sensitivity. Ion mobility-mass spectrometry (IM-MS) additionally gives conformational data about the analyzed molecules. From the drift time data obtained from the IM-MS analyses, the collision cross sections (CCS) of ions can be determined, which is a size parameter related to the averaged momentum transfer impact area of the ions. At the beginning of the studies, poly-L-lysine (PLL) and polystyrene sulfonic acid (PSS) were analyzed using MALDI tandem time-of-flight (ToF/ToF) mass spectrometer. The end groups of their chains could be determined by evaluating data obtained from MALDI mass spectra. Mixtures of PLL and PSS were prepared in various molar ratios and the signals of their noncovalent complexes could be detected in the negative ion mode MALDI-MS analysis. The MALDI-MS/MS spectra confirmed the proposed stoichiometries of the observed noncovalent complexes of PLL and PSS. Noncovalent complexes of PLL and PSS could also be detected in both positive and negative ion mode using ESI-Q/ToF-MS with ion mobility capabilities. Complexes with various charge states and stoichiometries could be identified easily utilizing ion mobility separation. Most of the noncovalent complex ions obtained in positive and negative ion modes were fragmented upon collisionally activated dissociation in the transfer cell unit of the instrument. ESI-MS/MS spectra obtained for both positive and negative ion modes correlated well with the stoichiometry of the complexes. Another polyanion, polyacrylic acid (PAA) sample was analyzed by itself in detail and six different types of PAA series with various end groups were detected in the sample. It was determined that some of the series must have been present in the PAA sample solution while the other series were probably generated in gas phase during the MS experiment. CCS of PLL-PAA complex ions with different PAA series were derived separately for comparison of their compactness. The results prove that the sizes of end groups of each species affect the conformation of the noncovalent complex in the gas phase. When the poly-L-glutamic acid (PGA) was analyzed, it was determined that its sample contains linear and cyclic forms of PGA. A mixture of PLL and PGA was also analyzed in order to monitor the complexes formed between these polyelectrolytes. The CCS of polyelectrolyte ions were derived individually for comparing their compactness. It was obtained that the gas-phase conformations of the polyelectrolytes depend on the nature of functional groups located in their repeating unit. The CCS values of polyelectrolyte complex ions were also derived. Comparison of the CCS values of the complexes indicated that differences in complex ion formation may be caused by reducing repulsion forces between charged groups or forming more extended structures. Noncovalent complexes of lysozyme formed with PSS oligomers were also examined in the dissertation to determine how the CCS of protein changed with the stoichiometry of the PSS chains in the complex.ÖZET i ABSTRACT iii TEŞEKKÜR v İÇİNDEKİLER vi ŞEKİLLER viii SİMGELER VE KISALTMALAR xii 1. GİRİŞ 1 2. GENEL BİLGİLER 3 2.1. Sentetik Polimerler 3 2.2. Polimer Karakterizasyonu 4 2.3. Kütle Spektrometrik Polimer Analizi 7 2.3.1. İyonlaştırma Yöntemleri 8 2.3.1.1. Matriks-Yardımlı Lazer Desorpsiyon/İyonlaştırma (MALDI) 9 2.3.1.2. Elektrosprey İyonlaştırma (ESI) 12 2.3.2. Kütle Ayırıcıları 17 2.3.2.1. Dört Kutuplu Kütle Ayırıcısı 18 2.3.2.2. Uçuş Zamanlı Kütle Ayırıcısı 19 2.3.2.3. Dört Kutuplu/Uçuş Zamanlı Kütle Ayırıcısı (Q/TOF) 22 2.3.3. Dedektörler 22 2.4. Tandem Kütle Spektrometrisi 23 2.5. İyon Hareketliliği-Kütle Spektrometrisi (IM-MS) 27 2.5.1. Kaydırmalı Dalga İyon Hareketliliği Spektrometrisi (Traveling Wave Ion Mobility Spectrometry, TWIMS) 29 2.5.2. Etkin Çarpışma Kesiti (Collision Cross Section, CCS) 31 2.5.3. IM-MS ile Polimer Karakterizasyonu 34 2.6. Kovalent Olmayan Kompleksler 37 2.7. Polielektrolitler 41 2.7.1. Polielektrolit Çoklu Tabakaları 42 2.7.2. Polielektrolit Kompleksleri 45 3. DENEYSEL ÇALIŞMALAR 49 3.1. Kullanılan Kimyasallar 49 3.2. MALDI-MS Analizleri 49 3.3. İyon Hareketliliği Kütle Spektrometrisi (IM-MS) Analizleri 51 3.4. Moleküler Modelleme ve Simülasyon 52 4. SONUÇLAR VE TARTIŞMA 54 4.1. Standart Bir Polikatyon-Polianyon Çiftinin MALDI-MS ile Analizi 54 4.1.1. Standart Bir Polikatyon-Polianyon Çiftinin Komplekslerinin MALDI-MS Analizleri 58 4.2. İyon Hareketlilik Kütle Spektrometrisi ile Polielektrolitlerin ve Komplekslerinin Analizleri 63 4.2.1. PLL-PSS Komplekslerinin IM-MS Analizleri 68 4.2.2. Polikatyon-Polianyon Komplekslerinin Etkin Çarpışma Kesitlerinin Hesaplanması 81 4.2.3. Bir Polielektrolitin Farklı Zincir Sonu Gruplarına Sahip Oligomerlerinin Analizi 86 4.2.4. Polielektrolitlerin Zincir Sonu Gruplarının Komplekslerin Etkin Çarpışma Kesitlerine Etkilerinin İncelenmesi 92 4.2.5. Halkalı Yapıdaki Polielektrolitin Komplekslerinin İncelenmesi 93 4.2.6. Polielektrolit Komplekslerinin CCS Değişimlerinin Kıyaslanması 99 4.3. Protein-Polielektrolit Etkileşiminin İyon Hareketliliği-Kütle Spektrometrisi ile İzlenmesi 101 5. SONUÇLAR 107 6. KAYNAKLAR 113 EKLER 131 ÖZGEÇMİŞ 140Kütle spektrometrisi, kovalent olmayan komplekslerin çözelti ve gaz fazlarındaki oluşum parametreleri ile ilgili faydalı bilginin yüksek doğruluk ve hassasiyetle elde edilmesi için en uygun tekniktir. İyon hareketliliği-kütle spektrometrisi (IM-MS) ise bunlara ek olarak analiz edilen moleküllerin konformasyonları ile ilgili detaylı veriler de sağlamaktadır. IM-MS analizlerinden elde edilen sürüklenme zamanı verilerinden iyonların ortalama momentum transfer etki alanları ile ilişkili bir boyut parametresi olan etkin çarpışma kesitleri (CCS) belirlenebilmektedir. Çalışmaların başlangıcında, poli- L-lizin (PLL) ve polistiren sülfonik asit (PSS) örnekleri, MALDI tandem uçuş zamanlı (ToF/ToF) kütle spektrometresi ile analiz edilmiştir. Bu türlerin zincir sonu grupları, MALDI kütle spektrumlarından alınan veriler değerlendirilerek belirlenebilmiştir. PLL ve PSS karışımları, farklı molar oranlarda hazırlanmış ve bunların kovalent olmayan komplekslerine ait sinyaller, negatif moddaki MALDI-MS analizi ile gözlenebilmiştir. Kovalent olmayan PLL-PSS kompleksleri için önerilen stokiyometri oranları, alınan MALDI-MS/MS spektrumları ile doğrulanmıştır. Kovalent olmayan PLL-PSS kompleksleri, iyon hareketliliği analizi özelliğine sahip ESI-Q/ToF-MS cihazı ile pozitif ve negatif iyon modunda gözlenebilmiştir. İyon hareketliliğine göre ayırma özelliğinden yararlanılarak farklı yük ve stokiyometrilere sahip kompleksler kolaylıkla tayin edilebilmiştir. Pozitif ve negatif iyon modunda gözlenen kovalent olmayan kompleks iyonlarının çoğu, cihazın transfer hücresinde gerçekleştirilen çarpışmayla aktifleştirilmiş parçalama ile daha küçük iyon birimlerine ayrılmıştır. Pozitif ve negatif iyon modlarında alınan ESI-MS/MS spektrumlarının kompleks stokiyometrileri ile uyumlu oldukları görülmüştür. Bir başka polianyon olan poli(akrilik asit) (PAA) örneği, tek başına analiz edilmiş ve bu örneğin farklı zincir sonu gruplarına sahip altı adet PAA iyon serisi içerdiği gözlenmiştir. Bazı serilerin PAA çözeltisinde kesin olarak bulunduğu, diğerlerinin ise muhtemelen kütle spektrometrik analiz sırasında gaz fazında oluştukları belirlenmiştir. Farklı PAA serileri içeren PLL-PAA kompleks iyonlarının sıkılıklarının kıyaslanması için CCS değerleri ayrı ayrı hesaplanmıştır. Alınan sonuçlar, her türün zincir sonu grubu büyüklüklerinin kovalent olmayan komplekslerinin gaz fazındaki konformasyonlarını etkilediklerini göstermiştir. Poli(L-glutamik asit) (PGA) polianyonu analiz edildiğinde örnek içerisinde doğrusal ve halkalı formlarının bulunduğu belirlenmiştir. PLL-PGA karışımı da bu polielektrolitlerin komplekslerinin gözlenmesi için analiz edilmiştir. Polielektrolit iyonlarının sıkılıklarının kıyaslanması için her birinin CCS değerleri ayrı ayrı hesaplanmıştır. Alınan verilere göre polielektrolitlerin gaz fazındaki konformasyonlarının tekrar eden birimleri üzerindeki fonksiyonel gruplara bağlı olduğu belirlenmiştir. Polielektrolit kompleks iyonlarının CCS değerleri de hesaplanmıştır. Komplekslerin CCS değerlerinin kıyaslanması, yüklü grupların itme kuvvetlerini azaltması veya daha geniş yapıların oluşması gibi durumların kompleks iyonlarının oluşumlarındaki farklılıkları meydana getirebildiklerini göstermiştir. Lizozim proteinin CSS değerinin komplekse katılan PSS zinciri sayısına göre değişimini belirlemek için lizozimin PSS oligomerleri ile oluşturdukları kovalent olmayan kompleksler de bu tez çalışmasında incelenmiştir

Synthesis and characterization of novel ABA type poly(Ester-ether) triblock copolymers

ATAKAY, MEHMET/0000-0002-7102-2974WOS: 000466029400003Oligomers of poly(3-hydroxypropionate) (P3HP) were synthesized via hydrogen transfer polymerization of acrylic acid. Olefinic end-group of the oligomers were modified through epoxidation and bromination to obtain activated oligomers with epoxy and bromide end-groups, respectively. Terminally hydroxyl oligomeric poly(ethylene glycol) (PEG-1450) was also converted to sodium alkoxide of PEG-1450 through reaction with sodium hydride. Novel ABA type P3HP-b-PEG-b-P3HP triblock copolymers were successfully obtained through simple nucleophilic addition reactions between alkoxy and epoxy/alkyl bromide. Water uptake measurements of the triblock copolymers were calculated. Characterization of the modified oligomers and the triblock copolymers was performed by using FT-IR, H-1-NMR and MALDI-MS analyses. Thermal transitions and degradation features of the copolymers were investigated by using DSC and TGA methods. Spectroscopic and thermal analyses revealed that both end-group modifications and coupling reactions were successfully achieved.Ordu University Scientific Research Fund [ODU-BAP HD-1606]This work was supported by Ordu University Scientific Research Fund (grand number: ODU-BAP HD-1606)

Synthesis and characterization of the ABA-type poly(ester-ether-ester) block copolymers

WOS: 000523671800001Polyethylene glycol with average molar mass of 1,450 Da (PEG-1450) was treated with equivalent amount of sodium hydride to convert hydroxy groups to sodium alkoxide. Assuming the PEG oligomer with alkoxy end-groups as a macroinitiator, anionic ring-opening polymerization of some lactones (beta-propiolactone, alpha-methyl-beta-propiolactone, delta-valerolactone and epsilon-caprolactone) with different ring sizes were performed to obtain the ABA-type poly(ester-ether-ester) block copolymers. Copolymers with various compositions were obtained with high yields. Spectroscopic and thermal characterization of the copolymers were performed by using DSC, TGA, FT-IR, and H-1-NMR spectroscopy. Formation of the block copolymers and their mass distributions were determined by MALDI mass spectrometry. Structural analyses of the copolymers revealed that chain extensions increased in order of alpha-methyl-beta-propiolactone, beta-propiolactone, delta-valerolactone and epsilon-caprolactone

Functional Substituted Phthalocyanines Bearing Ter-pyridine Complexes as Macromolecular Oxidation Catalysts for Bleaching Systems

The present study dealing with the oxidation catalysts using in laundry bleaching process comprises of the complexion of 4'-(N-2-phthalonitrile-N-methylamino)-2,2':6',2''-terpyridine (2) with the related MnCl2.4H(2)O and CoCl2.6H(2)O salts in ethanol to obtain 4'-(N-2-phthalonitrile-N-methylamino)-2,2':6',2''-terpyridine-Mn (II) complex (3) and 4'-(N-2-phthalonitrile-N-methylamino)-2,2':6',2''-terpyridine-Co (II) complex (4). Terpyridine metal complexes substituted Zinc phthalocyanines, Tetrakis [4'-(N-2-phthalonitrile-N-methylamino)-2, 2': 6', 2''-terpyridine-Mn(II) complex] phthalocyaninato zinc(II) (5) and Tetrakis [4'-(N-2-phthalonitrile-N-methylamino)-2,2':6',2''-terpyridine-Co(II) complex] phthalocyaninato zinc(II) (6) were also prepared by the tetramerization of the terpyridine-metal complexes (3, 4) in the presence of ZnCl2. FT-IR, H-1-NMR, C-13-NMR, UV-Vis, ESI-MS and MALDI-MS spectra were applied to characterize the prepared compounds. 4-nitrophthalonitrile and some other starting materials crystallized in the synthetic pathway. 4-nitrophthalonitrile crystalizes in different morphology and 4'-(N-2- Hydroxyehyl-N-methylamino)-2, 2' : 6', 2''- terpyridine (1) crystallizes in the monoclinic, space group. The compound 2 and 3 crystallize in the monoclinic space group. The bleach performances of the prepared terpyridine complexes (3, 4) and their phthalocyanine derivatives (5, 6) were examined by the degradation of Morin dye by using online spectrophotometric method (OSM)

Comparison of Two Methods for Purification of Enterocin B, a Bacteriocin Produced by Enterococcus faecium W3

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B

(2S,5R)-2-Isopropyl-5-Methylcyclohexanone Hydrazones

Hydrazones were obtained in 76-78% yield via condensation of (2S,5R)-2-isopropyl-5-methylcyclohexanone with 4-R-phenoxyacetic acid hydrazides in the presence of a catalytic amount of glacial acetic acid. The structure of the target compounds has been established by FTIR-ATR, Raman, H-1-NMR and C-13-NMR spectral analysis and EI/FAB/ESI mass spectrometry. Thermal properties of hydrazones 3a-3e were elucidated by differential scanning calorimetry (DSC) and their purity by HPLC coupled to mass spectrometry. Synthesized compounds were found to exist as Z/E geometrical isomers about C=N bond and cis/trans amide conformers.WoSScopu

Synthesis and characterization of amphiphilic triblock copolymers including -alanine/-methyl--alanine and ethylene glycol by "click" chemistry

WOS: 000460703600029Terminally azide poly--alanine (PBA-Az) was directly obtained by hydrogen transfer polymerization of acrylamide in the presence of sodium azide as an initiator. However, terminally azide poly(-methyl -alanine) (PmBA-Az) was synthesized by the reaction between terminally bromo poly(-methyl -alanine) and sodium azide. Dipropargyllated polyethylene glycol (PEG-di-Pr) was synthesized by using the reaction of PEGs with different molecular weights and propargyl bromide. Synthesis of poly(-alanine-b-ethylene glycol-b--alanine) and poly(-methyl -alanine-b-ethylene glycol-b--methyl -alanine) amphiphilic ABA triblock copolymers was achieved via click chemistry of PBA-Az or PmBA-Az and PEG-di-Pr with different molecular weight. Click reaction parameters such as concentration and time were assessed. Macromonomers and the amphiphilic triblock copolymers were characterized by using H-1-NMR, FT-IR, MALDI-MS, TGA, and elemental analysis techniques. The multi-instruments studies of the obtained amphiphilic triblock copolymers reveal that the copolymers easily formed as a result of click chemistry