TY  - JOUR
AU  - Duvnjak, Marija
AU  - Butorac, Ana
AU  - Kljak, Kristina
AU  - Nišavić, Marija
AU  - Cindrić, Mario
AU  - Grbeša, Darko
PY  - 2022
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/10483
AB  - The starch availability and nutritional value of corn (Zea mays L.) are affected by zein proteins. The aim of the study was to see whether the proposed reduction of γ-zeins during the fermentation of silages is a result of either the enzymatic proteolytic activity or of the acidic environment, and how this reduction affects starch availability and degradability in high-moisture corn. A mass spectrometry (MS) technique was used to quantify the 16- and 27-kDa γ-zeins. Briefly, two-dimensional gel electrophoresis (2-DE) was used for γ-zein separation, followed by densitometry for protein quantification and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/TOF) for protein identification. The results show that the reduction in γ-zeins induced by the ensiling led to a more pronounced starch availability and in vitro degradation, and this reduction was dependent on the type of proteolysis. More specifically, the results indicate that the reduction of γ-zeins in the ensiled corn was primarily driven by the enzymatic proteolysis. Furthermore, we demonstrated that 2-DE followed by densitometric quantification and the mass spectrometry analysis for protein identification can be used as a state-of-the-art method for γ-zein evaluation both in fresh and fermented/ensiled corn samples.
T2  - Fermentation
T1  - The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages
VL  - 8
IS  - 10
SP  - 551
DO  - 10.3390/fermentation8100551
ER  - 

@article{
author = "Duvnjak, Marija and Butorac, Ana and Kljak, Kristina and Nišavić, Marija and Cindrić, Mario and Grbeša, Darko",
year = "2022",
abstract = "The starch availability and nutritional value of corn (Zea mays L.) are affected by zein proteins. The aim of the study was to see whether the proposed reduction of γ-zeins during the fermentation of silages is a result of either the enzymatic proteolytic activity or of the acidic environment, and how this reduction affects starch availability and degradability in high-moisture corn. A mass spectrometry (MS) technique was used to quantify the 16- and 27-kDa γ-zeins. Briefly, two-dimensional gel electrophoresis (2-DE) was used for γ-zein separation, followed by densitometry for protein quantification and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/TOF) for protein identification. The results show that the reduction in γ-zeins induced by the ensiling led to a more pronounced starch availability and in vitro degradation, and this reduction was dependent on the type of proteolysis. More specifically, the results indicate that the reduction of γ-zeins in the ensiled corn was primarily driven by the enzymatic proteolysis. Furthermore, we demonstrated that 2-DE followed by densitometric quantification and the mass spectrometry analysis for protein identification can be used as a state-of-the-art method for γ-zein evaluation both in fresh and fermented/ensiled corn samples.",
journal = "Fermentation",
title = "The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages",
volume = "8",
number = "10",
pages = "551",
doi = "10.3390/fermentation8100551"
}

Duvnjak, M., Butorac, A., Kljak, K., Nišavić, M., Cindrić, M.,& Grbeša, D.. (2022). The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages. in Fermentation, 8(10), 551.
https://doi.org/10.3390/fermentation8100551

Duvnjak M, Butorac A, Kljak K, Nišavić M, Cindrić M, Grbeša D. The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages. in Fermentation. 2022;8(10):551.
doi:10.3390/fermentation8100551 .

Duvnjak, Marija, Butorac, Ana, Kljak, Kristina, Nišavić, Marija, Cindrić, Mario, Grbeša, Darko, "The Evaluation of γ-Zein Reduction Using Mass Spectrometry—The Influence of Proteolysis Type in Relation to Starch Degradability in Silages" in Fermentation, 8, no. 10 (2022):551,
https://doi.org/10.3390/fermentation8100551 . .

TY  - JOUR
AU  - Nišavić, Marija
AU  - Hozić, Amela
AU  - Hameršak, Zdenko
AU  - Radić, Martina
AU  - Butorac, Ana
AU  - Duvnjak, Marija
AU  - Cindrić, Mario
PY  - 2017
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/1552
AB  - Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.
T2  - Analytical Chemistry
T1  - High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions
VL  - 89
IS  - 9
SP  - 4847
EP  - 4854
DO  - 10.1021/acs.analchem.6b04466
ER  - 

@article{
author = "Nišavić, Marija and Hozić, Amela and Hameršak, Zdenko and Radić, Martina and Butorac, Ana and Duvnjak, Marija and Cindrić, Mario",
year = "2017",
abstract = "Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.",
journal = "Analytical Chemistry",
title = "High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions",
volume = "89",
number = "9",
pages = "4847-4854",
doi = "10.1021/acs.analchem.6b04466"
}

Nišavić, M., Hozić, A., Hameršak, Z., Radić, M., Butorac, A., Duvnjak, M.,& Cindrić, M.. (2017). High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions. in Analytical Chemistry, 89(9), 4847-4854.
https://doi.org/10.1021/acs.analchem.6b04466

Nišavić M, Hozić A, Hameršak Z, Radić M, Butorac A, Duvnjak M, Cindrić M. High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions. in Analytical Chemistry. 2017;89(9):4847-4854.
doi:10.1021/acs.analchem.6b04466 .

Nišavić, Marija, Hozić, Amela, Hameršak, Zdenko, Radić, Martina, Butorac, Ana, Duvnjak, Marija, Cindrić, Mario, "High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions" in Analytical Chemistry, 89, no. 9 (2017):4847-4854,
https://doi.org/10.1021/acs.analchem.6b04466 . .

TY  - JOUR
AU  - Nišavić, Marija
AU  - Masnikosa, Romana
AU  - Butorac, Ana
AU  - Perica, Kristina
AU  - Rilak, Ana
AU  - Korićanac, Lela
AU  - Hozić, Amela
AU  - Petković, Marijana
AU  - Cindrić, Mario
PY  - 2016
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/1124
AB  - Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4 Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4 Cl-tpy)(dach)Cl](+) (2) (where 4 Cl-tpy = 4-chloro-2,2:6,2 -terpyridine, en = 1,2-diaminoethane and dach = 1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77 +/- 0.14 and 6.95 +/- 0.43 mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue. (C) 2016 Elsevier Inc. All rights reserved.
T2  - Journal of Inorganic Biochemistry
T1  - Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin
VL  - 159
SP  - 89
EP  - 95
DO  - 10.1016/j.jinorgbio.2016.02.034
ER  - 

@article{
author = "Nišavić, Marija and Masnikosa, Romana and Butorac, Ana and Perica, Kristina and Rilak, Ana and Korićanac, Lela and Hozić, Amela and Petković, Marijana and Cindrić, Mario",
year = "2016",
abstract = "Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4 Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4 Cl-tpy)(dach)Cl](+) (2) (where 4 Cl-tpy = 4-chloro-2,2:6,2 -terpyridine, en = 1,2-diaminoethane and dach = 1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77 +/- 0.14 and 6.95 +/- 0.43 mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue. (C) 2016 Elsevier Inc. All rights reserved.",
journal = "Journal of Inorganic Biochemistry",
title = "Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin",
volume = "159",
pages = "89-95",
doi = "10.1016/j.jinorgbio.2016.02.034"
}

Nišavić, M., Masnikosa, R., Butorac, A., Perica, K., Rilak, A., Korićanac, L., Hozić, A., Petković, M.,& Cindrić, M.. (2016). Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin. in Journal of Inorganic Biochemistry, 159, 89-95.
https://doi.org/10.1016/j.jinorgbio.2016.02.034

Nišavić M, Masnikosa R, Butorac A, Perica K, Rilak A, Korićanac L, Hozić A, Petković M, Cindrić M. Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin. in Journal of Inorganic Biochemistry. 2016;159:89-95.
doi:10.1016/j.jinorgbio.2016.02.034 .

Nišavić, Marija, Masnikosa, Romana, Butorac, Ana, Perica, Kristina, Rilak, Ana, Korićanac, Lela, Hozić, Amela, Petković, Marijana, Cindrić, Mario, "Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin" in Journal of Inorganic Biochemistry, 159 (2016):89-95,
https://doi.org/10.1016/j.jinorgbio.2016.02.034 . .

TY  - JOUR
AU  - Butorac, Ana
AU  - Mekic, Meliha Solak
AU  - Hozić, Amela
AU  - Diminic, Janko
AU  - Gamberger, Dragan
AU  - Nišavić, Marija
AU  - Cindrić, Mario
PY  - 2016
UR  - https://vinar.vin.bg.ac.rs/handle/123456789/1183
AB  - RATIONALE: One of the most challenging tasks of proteomics is peptide de novo sequencing. 4-Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high-quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in highthroughput de novo sequencing. METHODS: We developed a SPITC peptide derivatization procedure under acidic conditions (pH LT = 5). Derivatized peptides were analyzed by matrix-assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS: The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine-containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS: The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes. Copyright (C) 2016 John Wiley and Sons, Ltd.
T2  - Rapid Communications in Mass Spectrometry
T1  - Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing
VL  - 30
IS  - 14
SP  - 1687
EP  - 1694
DO  - 10.1002/rcm.7594
ER  - 

@article{
author = "Butorac, Ana and Mekic, Meliha Solak and Hozić, Amela and Diminic, Janko and Gamberger, Dragan and Nišavić, Marija and Cindrić, Mario",
year = "2016",
abstract = "RATIONALE: One of the most challenging tasks of proteomics is peptide de novo sequencing. 4-Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high-quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in highthroughput de novo sequencing. METHODS: We developed a SPITC peptide derivatization procedure under acidic conditions (pH LT = 5). Derivatized peptides were analyzed by matrix-assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS: The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine-containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS: The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes. Copyright (C) 2016 John Wiley and Sons, Ltd.",
journal = "Rapid Communications in Mass Spectrometry",
title = "Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing",
volume = "30",
number = "14",
pages = "1687-1694",
doi = "10.1002/rcm.7594"
}

Butorac, A., Mekic, M. S., Hozić, A., Diminic, J., Gamberger, D., Nišavić, M.,& Cindrić, M.. (2016). Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing. in Rapid Communications in Mass Spectrometry, 30(14), 1687-1694.
https://doi.org/10.1002/rcm.7594

Butorac A, Mekic MS, Hozić A, Diminic J, Gamberger D, Nišavić M, Cindrić M. Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing. in Rapid Communications in Mass Spectrometry. 2016;30(14):1687-1694.
doi:10.1002/rcm.7594 .

Butorac, Ana, Mekic, Meliha Solak, Hozić, Amela, Diminic, Janko, Gamberger, Dragan, Nišavić, Marija, Cindrić, Mario, "Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing" in Rapid Communications in Mass Spectrometry, 30, no. 14 (2016):1687-1694,
https://doi.org/10.1002/rcm.7594 . .