An untargeted analytical workflow based on Kendrick mass defect filtering reveals dysregulations in acylcarnitines in prostate cancer tissue.

BACKGROUND: Metabolomics is nowadays considered one the most powerful analytical for the discovery of metabolic dysregulations associated with the insurgence of cancer, given the reprogramming of the cell metabolism to meet the bioenergetic and biosynthetic demands of the malignant cell. Notwithstanding, several challenges still exist regarding quality control, method standardization, data processing, and compound identification. Therefore, there is a need for effective and straightforward approaches for the untargeted analysis of structurally related classes of compounds, such as acylcarnitines, that have been widely investigated in prostate cancer research for their role in energy metabolism and transport and ß-oxidation of fatty acids. RESULTS: In the present study, an innovative analytical platform was developed for the straightforward albeit comprehensive characterization of acylcarnitines based on high-resolution mass spectrometry, Kendrick mass defect filtering, and confirmation by prediction of their retention time in reversed-phase chromatography. In particular, a customized data processing workflow was set up on Compound Discoverer software to enable the Kendrick mass defect filtering, which allowed filtering out more than 90 % of the initial features resulting from the processing of 25 tumoral and adjacent non-malignant prostate tissues collected from patients undergoing radical prostatectomy. Later, a partial least square-discriminant analysis model validated by repeated double cross-validation was built on the dataset of 74 annotated acylcarnitines, with classification rates higher than 93 % for both groups, and univariate statistical analysis helped elucidate the individual role of the annotated metabolites. SIGNIFICANCE: Hydroxylation of short- and medium-chain minor acylcarnitines appeared to be a significant variable in describing tissue differences, suggesting the hypothesis that the neoplastic growth is linked to oxidation phenomena on selected metabolites and reinforcing the need for effective methods for the annotation of minor metabolites.

Preparation of Monolith for Online Extraction and LC-MS Analysis of ß-Estradiol in Serum Via a Simple Multicomponent Reaction.

Multicomponent reactions offer efficient and environmentally friendly strategies for preparing monoliths suitable for applications in analytical chemistry. In the described study, a multicomponent reaction was utilized for the one-pot miniaturized preparation of a poly(propargyl amine) polymer inside commercial silica-lined PEEK tubing. The reaction involved only small amounts of reagents and was characterized by atom economy. The resulting monolithic column was incorporated into an autosampler system for the online extraction and cleanup of ß-estradiol from human serum. Sample pretreatment was simplified to a simple dilution with methanol and centrifugation to remove proteins. The resulting platform included LC-MS analysis in multiple reaction monitoring for quantitative analysis of ß-estradiol. The method was validated in serum, demonstrating practical applicability for the monitoring of fertile women. Recoveries were above 94%, and LOD and LOQ values at 0.008 and 0.18 ng mL-1, respectively. The developed platform proved to be competitive with previous methods for solid-phase microextraction of ß-estradiol in serum, with comparable recovery and sensitivity but with the advantage of nearly complete automation. The environmental impact of the process was evaluated as acceptable due to the miniaturization of the monolith synthesis and the automation of extraction. The drawback associated with the LC-MS technique can be reduced by the inclusion of additional analytes in a single investigation. The work demonstrates that multicomponent reactions are versatile, economical, and possibly a green methodology for producing reversed-phase and mixed-mode sorbents, enabling miniaturization of the entire analytical procedure from the preparation of extraction sorbents to analysis.

One-phase extraction coupled with photochemical reaction allows the in-depth lipid characterization of hempseeds by untargeted lipidomics.

Due to their valuable nutritional content, several hemp-derived products from hempseeds have recently been placed in the market as food and food ingredients. In particular, the lipid composition of hempseeds has raised interest for their rich content in biologically active polyunsaturated fatty acids with an optimum ratio of omega-3 and omega-6 compounds. At present, however, the overall polar lipidome composition of hempseeds remains largely unknown. In the present work, an analytical platform was developed for the extraction, untargeted HRMS-based analysis, and detailed annotation of the lipid species. First, five one- and two-phase solid-liquid extraction protocols were tested and compared on a hempseed pool sample to select the method that allowed the overall highest efficiency as well as easy coupling with lipid derivatization by photochemical [2 + 2] cycloaddition with 6-azauracil. Underivatized lipids were annotated employing a data processing workflow on Compound Discoverer software that was specifically designed for polar lipidomics, whereas inspection of the MS/MS spectra of the derivatized lipids following the aza-Paternò-Büchi reaction allowed pinpointing the regiochemistry of carbon-carbon double bonds. A total of 184 lipids were annotated, i.e., 26 fatty acids and 158 phospholipids, including minor subclasses such as N-acylphosphatidylethanolamines. Once the platform was set up, the lipid extracts from nine hempseed samples from different hemp strains were characterized, with information on the regiochemistry of free and conjugated fatty acids. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of hempseeds.

Occurrence of per- and polyfluorinated alkyl substances in wastewater treatment plants in Northern Italy.

Wastewater treatment plants are known to be relevant input sources of per- and polyfluoroalkyl substances (PFAS) in the aquatic environment. This study aimed to investigate the occurrence, fate, and seasonal variability of twenty-five PFAS in four municipal wastewater treatment plants (WWTP A, B, C, and D) surrounding the city of Milan (Northern, Italy). Composite 24-h wastewater samples were collected in July and October 2021 and May and February 2022 from influents and effluents of the four WWTPs. PFAS were detected at concentrations ranging between 24.1 and 66.9 µg L-1 for influent and 13.4 and 107 µg L-1 for effluent wastewater samples. Perfluoropentanoic acid was the most abundant (1.91-30.0 µg L-1) in influent samples, whereas perfluorobutane sulfonic acid predominated (0.80-66.1 µg L-1) in effluent samples. In sludge, PFOA was detected in plant A at concentrations in the range of 96.6-165 ng kg-1 dw in primary sludge samples and 98.6-440 ng kg-1 dw in secondary treatment sludge samples. The removal efficiency of total PFAS varied between 6 % and 96 %. However, an increase of PFAS concentrations was observed from influents to effluents for plant D (during July and October), plant A (during October and May), and plant C (during May) indicating that biotransformation of PFAS precursors can occur during biological treatments. This was supported by the observed increase in concentrations of PFOA from primary to secondary treatment sludge samples in plant A. Moreover, the plant operating at shorter hydraulic retention times (plant D) showed lower removal efficiency (<45 %). Seasonal variation of PFAS in influent and effluent appears rather low and more likely due to pulse release instead of seasonal factors.

Biotic transformation products of sulfonamides in environmental water samples: High-resolution mass spectrometry-based tentative identification by a suspect screening approach.

The presence of pharmaceuticals in the aquatic environment is mainly due to their release from the effluents of the wastewater treatment plants (WWTPs), which are unable to completely remove them and their transformation products (TPs). Sulfonamides (SAs) are a synthetic antibacterial class used for the treatment of both human and animal infections; they have often been reported in surface water, thus contributing to the antibiotic resistance emergency. Monitoring SA TPs should be important as well because they could still exert some pharmaceutical activity; however, many TPs are still unknown since several transformation processes are possible (e. g. human and animal metabolism, WWTP activities, environmental factors etc.). In this work, three of the most used SAs, i.e., sulfamethoxazole (SMX), sulfapyridine (SPY), and sulfadiazine (SDZ), were incubated for 20 days in a batch reactor with activated sludge under controlled conditions. Then, the water sample was extracted and analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry in the data dependent acquisition (DDA) mode. Starting from the literature data, the possible transformation pathways were studied, and for each SA, a list of TPs was hypothesized and used for the identification. The raw data files were processed with Compound Discoverer, and 44 TPs (18, 13, and 13 TPs for SMX, SPY, and SDZ, respectively), including multiple TPs, were manually validated. To overcome the limitation of the DDA, the identified TPs were used in an inclusion list to analyze WWTP samples by a suspect screening approach. In this way, 4 SMX TPs and 5 SPY TPs were tentatively identified together with their parent compounds. Among these TPs, 5 of 9 were acetylated forms, in agreement with previous literature reporting that acetylation is the predominant SA transformation.

Evaluation of the carotenoid and fat-soluble vitamin profile of industrial hemp inflorescence by liquid chromatography coupled to mass spectrometry and photodiode-array detection.

Industrial hemp (Cannabis sativa L.) is a plant matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes. Detailed characterization of hemp composition is needed for future research that further exploits the beneficial effects of hemp compounds on human health. Among minor constituents, carotenoids and fat-soluble vitamins have largely been neglected to date despite carrying out several biological activities and regulatory functions. In the present paper, 22 target carotenoids and fat-soluble vitamins were analyzed in the inflorescences of seven Italian industrial hemp varieties cultivated outdoor. The analytes were extracted by cold saponification to avoid artifacts and analyzed by high-performance liquid chromatography coupled with Selected reaction monitoring mass spectrometry. Phytoene, phytofluene, and all-trans-ß-carotene were the most abundant in all analyzed samples (31-55 µg g-1, 11.6-29 µg g-1, and 7.3-53 µg g-1, respectively). Besides the target analytes, liquid chromatography coupled with photodiode-array detection allowed us to tentatively identify several other carotenoids based on their retention behavior and UV-vis spectra with the support of theoretical rules and data in the literature. To the best of our knowledge, this is the first comprehensive characterization of carotenoids and fat-soluble vitamins in industrial hemp inflorescence.

Untargeted analysis of environmental contaminants in surface snow samples of Svalbard Islands by liquid chromatography-high resolution mass spectrometry.

In recent years, there is increasing attention on the contaminants of emerging concern (CECs), which include plasticizers, flame retardants, industrial chemicals, pharmaceuticals, and personal care products, since they have been detected even far away from pollution sources. The polar regions are not exempt from the presence of anthropogenic contaminants, and they are employed as a model for understanding the pollutant fate and impact. During the 2021 spring campaign, sixteen surface snow samples were collected close to the research station of Ny-Ålesund located on the Spitsbergen Island of the Norwegian Svalbard Archipelago. The samples were extracted by solid-phase extraction and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) following an untargeted approach. Compound tentative identification was obtained with the aid of the software Compound Discoverer, using both mass spectral database search and manual validation. Among the 114 compounds identified with a high confidence level in the snow samples, >80 have some commercial or industrial use (drugs, plasticizers, fragrances, etc.), therefore they could be of anthropogenic origin. Nonetheless, a clear contamination trend did not appear in the snow samples collected on eight different days during one month. The comparison with aerosol samples collected in the same area did not help identifying the source, either, since only a few compounds were in common, and they were mainly of natural origin. As such, the analysis of aerosol sample did not support possible long-range transport, also considering that compounds were detected mostly in the coarse fraction.

Investigating the Short Peptidome Profile of Italian Dry-Cured Ham at Different Processing Times by High-Resolution Mass Spectrometry and Chemometrics.

Short peptides have been spiking interest owing to their significant contribution to the taste and functional properties of dry-cured ham. In this study, a suspect screening approach based on high-resolution mass spectrometry was employed for the comprehensive characterization of the short endogenous peptidome in dry-cured ham samples at different processing stages (14, 22, and 34 months). After careful manual spectra interpretation, a chemometric approach based on principal component analysis was employed for highlighting the differences between the three sets of samples. A total of 236 short peptide sequences was tentatively identified, including 173 natural short peptides and 63 sequences containing non-proteinogenic amino acids, the highest number ever reported for endogenous sequences in dry-cured ham. Samples in the latest processing stages presented a generally higher abundance of dipeptides, indicating residual proteolytic activity. Moreover, the several annotated modified short peptides, mainly pyroglutamination and lactoyl conjugation, allowed hypothesizing several reactions occurring over time. For the first time, several lactoyl-dipeptides were tentatively identified in dry-cured ham samples with maximum concentration in the late processing stage samples. The presented results significantly contribute to the understanding of the reaction involving short peptides that affect the sensory and functional properties of dry-cured ham.

Detailed investigation of the composition and transformations of phenolic compounds in fresh and fermented Vaccinium floribundum berry extracts by high-resolution mass spectrometry and bioinformatics.

INTRODUCTION: Blueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet. OBJECTIVES: In the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content. MATERIALS AND METHODS: V. floribundum native and fermented berries were extracted and analyzed by UHPLC-HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification. RESULTS: In total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds. CONCLUSION: Andean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

High-Resolution Mass Spectrometry and Chemometrics for the Detailed Characterization of Short Endogenous Peptides in Milk By-Products.

The process of cheese-making has long been part of human food culture and nowadays dairy represents a large sector of the food industry. Being the main byproduct of cheese-making, the revalorization of milk whey is nowadays one of the primary goals in alignment with the principles of the circular economy. In the present paper, a deep and detailed investigation of short endogenous peptides in milk and its byproducts (whole whey, skimmed whey, and whey permeate) was carried out by high-resolution mass spectrometry, with a dedicated suspect screening data acquisition and data analysis approach. A total of 79 short peptides was tentatively identified, including several sequences already known for their exerted biological activities. An unsupervised chemometric approach was then employed for highlighting the differences in the short peptide content among the four sets of samples. Whole and skimmed whey showed not merely a higher content of short bioactive peptides compared to whole milk, but also a peculiar composition of peptides that are likely generated during the process of cheese-making. The results clearly demonstrate that whey represents a valuable source of bioactive compounds and that the set-up of processes of revalorization of milk byproducts is a promising path in the obtention of high revenue-generating products from dairy industrial waste.

Characterization of the Trans-Epithelial Transport of Green Tea (C. sinensis) Catechin Extracts with In Vitro Inhibitory Effect against the SARS-CoV-2 Papain-like Protease Activity.

This work describes an untargeted analytical approach for the screening, identification, and characterization of the trans-epithelial transport of green tea (Camellia sinensis) catechin extracts with in vitro inhibitory effect against the SARS-CoV-2 papain-like protease (PLpro) activity. After specific catechin extraction, a chromatographic separation obtained six fractions were carried out. The fractions were assessed in vitro against the PLpro target. Fraction 5 showed the highest inhibitory activity against the SARS-CoV-2 PLpro (IC50 of 0.125 µg mL-1). The untargeted characterization revealed that (-)-epicatechin-3-gallate (ECG) was the most abundant compound in the fraction and the primary molecule absorbed by differentiated Caco-2 cells. Results indicated that fraction 5 was approximately 10 times more active than ECG (IC50 value equal to 11.62 ± 0.47 µg mL-1) to inhibit the PLpro target. Overall, our findings highlight the synergistic effects of the various components of the crude extract compared to isolated ECG.

Fully Automatized Detection of Phosphocholine-Containing Lipids through an Isotopically Labeled Buffer Modification Workflow.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.

Targeted and untargeted characterization of underivatized policosanols in hemp inflorescence by liquid chromatography-high resolution mass spectrometry.

The paper describes the development of a targeted quantitative method for the analysis of policosanols in hemp inflorescence. Policosanols are long chain aliphatic alcohols, with carbon chains typically in the range 20-36, with interesting biological activities. These compounds are typically separated by gas chromatography and only a few methods employ liquid chromatography for policosanols. In both cases, methods always include the derivatization of policosanols. In this study, policosanols were separated by ultra-high performance liquid chromatography without any derivatization and detected using high resolution mass spectrometry by formation of lithiated adducts. The procedure was optimized and a quantitative method was validated for the most abundant policosanols (with C24, C26, C27, C28, and C30 chain lengths) in industrial hemp inflorescence extracts. The method was used for the quantitative analysis of policosanols in five hemp types. Hemp wax was found rich in these compounds, especially C26 and C28 policosanols, which may prove useful for revalorization of wax by-products. Finally, the acquired data were also used to expand the search to the untargeted qualitative analysis of policosanols using Compound Discoverer. The untargeted method allowed the annotation of underivatized policosanols up to C33.

Production and Characterization of Medium-Sized and Short Antioxidant Peptides from Soy Flour-Simulated Gastrointestinal Hydrolysate.

Soybeans (Glycine max) are an excellent source of dietary proteins and peptides with potential biological activities, such as antihypertensive, anti-cholesterol, and antioxidant activity; moreover, they could prevent cancer. Also, soy contains all the essential amino acids for nutrition; therefore, it represents an alternative to animal proteins. The goal of this paper was the comprehensive characterization of medium-sized and short peptides (two to four amino acids) obtained from simulated gastrointestinal digestion. Two different analytical approaches were employed for peptide characterization, namely a common peptidomic analysis for medium-sized peptides and a suspect screening analysis for short peptides, employing an inclusion list of exact m/z values of all possible amino acid combinations. Moreover, fractionation by preparative reversed-phase liquid chromatography was employed to simplify the starting protein hydrolysate. Six fractions were collected and tested for antioxidative activity by an innovative antioxidant assay on human gastric adenocarcinoma AGS cell lines. The two most active fractions (2 and 3) were then characterized by a peptidomic approach and database search, as well as by a suspect screening approach, in order to identify potential antioxidant amino acid sequences. Some of the peptides identified in these two fractions have been already reported in the literature for their antioxidant activity.

Profiling and quantitative analysis of underivatized fatty acids in Chlorella vulgaris microalgae by liquid chromatography-high resolution mass spectrometry.

Chlorella vulgaris is a popular microalga used for biofuel production; nevertheless, it possesses a strong cell wall that hinders the extraction of molecules, especially lipids within the cell wall. For tackling this issue, we developed an efficient and cost-effective method for optimal lipid extraction. Microlaga cell disruption by acid hydrolysis was investigated comparing different temperatures and reaction times; after hydrolysis, lipids were extracted with n-hexane. The best recoveries were obtained at 140°C for 90 min. The microalgae were then analyzed by an untargeted approach based on liquid chromatography with high-resolution mass spectrometry, providing the tentative identification of 28 fatty acids. First, a relative quantification on the untargeted data was performed using peak area as a surrogate of analyte abundance. Then, a targeted quantitative method was validated for the tentatively identified fatty acids, in terms of recovery (78-100%), intra- and interday relative standard deviations (<10 and <9%, respectively) and linearity (R2  > 0.98). The most abundant fatty acids were palmitic, palmitoleic, oleic, linoleic, linolenic, and stearic acids.

Analytical Methodologies for Lipidomics in Hemp Plant.

The chemical composition of Cannabis sativa L. has been extensively studied for tens of years, but little is known about its lipidome. This chapter describes an analytical workflow for polar lipid determination in hemp. After extraction, lipids are enriched and isolated by graphitized carbon black sorbent, and the isolated lipid is analyzed by liquid chromatography (LC) coupled with high resolution mass spectrometry, leading to identification of many lipid species. We have developed a semi-automated platform using commercially available Lipostar software for lipid identification. Our approach affords the identification of 189 polar lipids in hemp extract, including sulfolipids and phospholipids. The number of the identified lipid species is by far the highest ever reported for Cannabis sativa.

In-depth cannabis fatty acid profiling by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry.

Industrial hemp (Cannabis sativa L.) represents an important plant, used for a variety of uses including pharmaceutical and nutraceutical purposes. As such, a detailed characterization of the composition of this plant could help future research to further exploit the beneficial effects of hemp compounds on the human health. Among the many compounds of hemp, fatty acids represent an interesting class of minor components, which has been overlooked so far. In this work, an untargeted approach based on liquid-chromatography coupled to a high-resolution mass spectrometry and a dedicated structure-based workflow for raw data interpretation was employed for the characterization of fatty acids from hemp inflorescences. A simple method, without any chemical derivatization, was developed for extraction and characterization of fatty acids leading to the tentative identification of 39 fatty acid species in the five hemp samples. A quantitative analysis on the untargeted data was initially performed, using peak areas as surrogate of analyte abundance for relative quantitation. Five fatty acids resulted the most abundant in all hemp samples, with ca. 90% of the total peak area. For these compounds a targeted quantitative method was validated, indicating that the most abundant ones were linolenic acid (1.39-7.95 mg g-1) and linoleic acid (1.04-7.87 mg g-1), followed by palmitic acid (3.74-6.08 mg g-1), oleic acid (0.91-4.73 mg g-1) and stearic acid (0.64-2.25 mg g-1).

Degradation of the polar lipid and fatty acid molecular species in extra virgin olive oil during storage based on shotgun lipidomics.

Among the bioactive compounds present in extra-virgin olive oil, polar lipids and free fatty acids are minor compounds with well-known nutritional values and have been studied for traceability and adulteration investigations as well. In the present paper, the simultaneous characterization of polar lipids and free fatty acids in a pool of fifteen EVOO samples was achieved by means of reversed phase C18 analysis coupled to negative polarity high-resolution mass spectrometry. A total of 24 polar lipids, comprising 19 phospholipids and 5 sulfolipids, and 27 free fatty acids were tentatively identified, including several odd-chain and very long-chain fatty acids at trace levels. Moreover, a one-month study of lipid degradation on simulated storage conditions was carried out thanks to the set-up of a dedicated approach for degradation product analysis which was implemented of Compound Discoverer software. By virtue of the customized data processing workflow, more than forty compounds were tentatively identified, including compounds deriving from hydrolysis and oxidation reactions. Finally, by analysis of peak area trends, phosphoester hydrolyses of polar heads of phospholipids emerged as the fastest reactions, followed by glycerol ester hydrolyses and oxidative processes.

Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle.

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices.

A rapid and innovative extraction and enrichment method for the metaproteomic characterization of dissolved organic matter in groundwater samples.

Metaproteomic analysis of aquifer systems provides valuable information on the microbial populations, their influence on drinking water quality, and the effect on human health. In the present paper, an extraction and enrichment method by C18 extra-wide pore cartridge was developed, optimized, and applied for the first time to the metaproteomic characterization of dissolved organic matter in groundwater samples. In particular, three elution procedures were tested and compared on water spiked with a yeast protein extract to maximize the recovery of proteins from a complex matrix. The maximum protein recovery was obtained by the use of two sequential elution buffers, one employing a denaturing agent and the other one containing an acidified organic solvent. A comprehensive metaproteomic analysis of the dissolved organic matter of groundwater was then performed by nano-high performance liquid chromatography coupled to high-resolution mass spectrometry. A total of 239 proteins was identified; in agreement with the current knowledge on proteins in aquifer systems, most identified sequences derived from bacteria, protobacteria, and ciliates. The paper is the first metaproteomic study applied to groundwater samples with particular emphasis on the need for sample pretreatment to obtain comprehensive information on the proteome in dissolved organic matter.