Mario Cindrić

Processed meat products represent a major challenge for proteomic species identification due to extensive thermal treatment and protein structural changes. In this study, species-specific peptides in pork, chicken, and bovine meat products were identified using a directed fragmentation-assisted de novo sequencing workflow that combines 4-formylbenzene-1,3-disulfonic acid (FBDA) peptide derivatization, dual-polarity data-independent mass spectrometry (DIA-MS), and Protein Acrobat de novo sequencing software. Comparative analysis of non-fractionated and strong cation exchange (SCX)-fractionated pork luncheon samples improved peptide and protein identification after fractionation, with 312 peptides and 115 protein groups detected exclusively in fractionated samples. Species-specific peptides were predominantly assigned to conserved muscle-related proteins, including myosin, troponin, and tropomyosin, while sequence variability enabled reliable species discrimination despite protein conservation across species. To evaluate applicability for food fraud detection, mixed meat samples containing 10% chicken in pork or bovine matrices were analyzed, reflecting potential economically motivated adulteration through substitution with lower-cost meat components. Several chicken-specific peptides remained detectable in both mixtures, demonstrating robustness of the FBDA-assisted peptide sequencing combined with SCX fractionation and DIA-MS for detection of adulteration in complex processed food matrices. These findings establish a mass spectrometry-driven orthogonal method to ELISA testing for fast, reliable and accurate metaproteome analysis of highly processed food.

The aim of this study is to investigate the occurrence of melamine and cyanuric acid in milk, baby food, and protein supplements collected in Croatia. A total of 56 samples were collected during 2022 and 2023 from retail stores in Zagreb, Croatia. Sample preparation involved acetonitrile extraction, followed by analysis using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Cyanuric acid concentrations above the limit of quantification (LOQ) were found in five milk samples (33.3% detection frequency), with a range from 0.26 to 0.39 mg/kg and a mean concentration of 0.31 mg/kg. In protein supplements, melamine was detected above the LOQ in six samples (23% detection frequency), with a mean concentration of 0.30 mg/kg and concentrations ranging from 0.20 to 0.57 mg/kg. No concentrations above the LOQ were found in baby food samples. All detected values were below the EU maximum limits (2.5 mg/kg for general food and 1.0 mg/kg for baby food). The accuracy and reliability of the method were verified using certified reference material. This is the first study to confirm the presence of melamine and cyanuric acid in protein supplements and milk on the Croatian market. The detected levels do not indicate a potential health risk to consumers.

Due to widespread use of silver nanoparticles (AgNPs), the assessment of their potential harm to microalgal photosynthesis is crucial, as microalgae, together with cyanobacteria, contribute to approximately 50% of global oxygen production. This study investigated photosynthetic pigments, photosynthetic rate, chlorophyll a fluorescence, and the expression of photosynthesis-related genes and proteins in green alga Chlorella vulgaris after 72 h exposure to citrate- and cetyltrimethylammonium bromide (CTAB)-stabilized AgNPs, as well as silver ions (AgNO₃), at concentrations allowing 75% cell survival (EC₂₅). All treatments impaired photosynthetic performance. The most pronounced decreases in chlorophyll fluorescence parameters and photosynthetic rate, alongside elevated energy dissipation, were observed after exposure to AgNP-CTAB and AgNO₃. AgNP-citrate had milder effects and induced compensatory responses, reflected in an increased performance index and upregulation of photosynthesis-related proteins. AgNP-CTAB induced the strongest downregulation of gene and protein expression, likely due to its higher EC₂₅ concentration and cationic surface promoting interaction with photosynthetic structures. Although AgNO₃ caused fewer molecular changes, it significantly disrupted photosynthetic function, suggesting a direct effect of Ag⁺ ions on photosynthesis-related proteins. Overall, the results highlight the role of AgNPs’ surface coatings and dosage in determining their phytotoxicity, with photosystem disruption and oxidative stress emerging as key mechanisms of action.

Fisheries and aquaculture play a crucial role in global food security, yet species mislabeling remains a persistent challenge, undermining consumer trust and market transparency. Proper food labeling is essential for protecting public health due to the presence of unknown toxic or allergenic substances and preventing illegally sourced products from entering the market. Despite extensive research across Europe, seafood mislabeling in Croatia has remained unexplored. This study aims to provide the first comprehensive assessment of seafood labeling accuracy in Croatia, where fisheries are integral to the coastal economies and tourism. Using DNA barcoding of the COI gene, 109 seafood samples were collected over two years from various sources, including restaurants, markets, and fishing vessels, and analyzed for potential mislabeling. Results revealed a mislabeling rate of 3% among fish samples and 20% among cephalopods, with notable substitutions, such as the yellowfin tuna mislabeled as bigeye tuna and Bluefin tuna and the European squid mislabeled as Patagonian squid. Additionally, 38.5% of samples were partially labeled, while 32% lacked clear country-of-origin information, complicating traceability. While the findings align with the mislabeling rates in other European countries, this study underscores the ongoing challenges in seafood labeling compliance. Establishing standardized monitoring protocols will be essential for improving comparability and effectively addressing seafood fraud.

Adenine nucleotides (ANs)—adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP)—are essential for energy transfer and the supply of countless processes within cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index has broad diagnostic potential, as reduced ATP levels are associated to various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of ANs from a single drop of capillary blood. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. An optimized µ-SPE method, coupled with RP-HPLC and a run time of 30 min, provides a reliable analytical framework for adenine nucleotide analysis of diverse biological samples. AN concentrations measured in capillary blood samples were 1393.1 µM, 254.8 µM, and 76.9 µM for ATP, ADP, and AMP molecules aligning with values reported in the literature. Overall, this study presents a streamlined and precise approach for analyzing ANs from microliters of blood, offering promising applications in clinical diagnostics.

Adenine nucleotides (AN): adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), and adenosine 5’-monophosphate (AMP) are essential for energy transfer and supply of countless processes within the cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index can be used as a clinical health indicator with broad diagnostic potential, as a decrease in ATP levels is associated with various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of AN from just a drop of capillary blood obtained from a healthy volunteer. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. By removing interfering substances, we achieved high chromatographic selectivity and resolution, facilitating the quantification of AN. The analyzed AN concentrations from capillary blood correspond to the values found in the literature, which corroborates reliability of the developed extraction method. Overall, this study presents a streamlined and precise approach for analyzing AN from microliters of blood that offers promising applications in clinical diagnostics.

Our research objective was to investigate the use of 4’-formylbenzo-18-crown-6-ether (4fb18C6) for the covalent labelling of peptides for unambiguous peptide identification. Specifically, 23 peptides were analysed using a coupled system of liquid chr...

Propolis is a natural mixture of honeybee-released and plant-derived compounds produced by honeybees. Poplar propolis is rich in bioactive polyphenolic compounds, and due to its many health benefits, it is commonly used as a food supplement or functional food ingredient. However, it is the only honeybee product whose proteome hasn’t been analyzed. Here, we report a first proteome analysis of poplar-type propolis, a challenging glue-type resinous sample for protein characterization. Raw propolis mixture was precipitated with cold acetone to obtain the protein fraction. Proteins were digested with trypsin, and generated peptides were analyzed on nano-ESI-qTOF SYNAPT G2-Si mass spectrometer (MS) by data-independent acquisition (DIA) and data-dependent acquisition (DDA). Identified peptides and inferred proteins suggest the presence of new bioactive molecules as components of propolis. The poplar-type propolis proteome is composed of a mixture of proteins from the Apis and Populus genera. This is the first-ever report of the proteome of any type of propolis.