This study aimed to characterize the walnut oleosome proteins by a shot-gun approach, with a focus on the effect of processing on protein fraction. The method developed in a micro HPLC-HRMS system allowed the identification of hundreds of proteins including those specifically associated to the oil-bodies (oleosins, caleosins, and steroleosins).
1 Introduction The walnuts oil bodies (OBs), also known as oleosomes, are spherical organelles (0.5-2.5 ?m in diameter) used by plants to store the energy needed for germination and growth [1]. In nuts, OBs surround larger structures, called protein bodies (PBs), that contain the proteins involved in the embryogenic program of the seed (mainly legumins and vicilins) [2]. At the molecular level, only English walnut (Juglans regia) genome was fully sequenced, which was published in 2016 [3], while a curated database was released in UniProt KB in February 2018. This knowledge at the DNA level has allowed a global proteomic analysis of raw English walnut to be performed [4]. However, because of their localization, their low abundance and the difficulty of their extraction, OB proteins have not yet been fully characterized. Considering the already demonstrated effect of processing on the OB structure of hazelnut [5], a comprehensive proteomic characterization on raw, boiled and roasted walnuts by using the shot-gun proteomic approach was performed. 2. Experimental The walnut protein mixtures were digested O/N at 37 °C under shaking with modified porcine proteomic grade trypsin (1 ?g/?L) at a 1:20 trypsin-protein ratio. The analyses were performed using an Orbitrap Q Exactive Plus, coupled to a UHPLC binary pump system. The stationary phase was a BioBasic(TM) C18 HPLC Column (1 × 150 mm, 5 ?m; Thermo Scientific). The mobile phases were 0.1 % (v/v) FA in MilliQ water (A) and 0.1 % (v/v) FA in ACN (B). and they were eluted at a flow-rate of 68.0 ?L/min at increasing concentrations of solvent B, that is, from 5 % to 45 %, over 57 min, and from 45 % to 50 % in 2 min. The injection volume was 2.0 ?L. The oven temperature was set at 55 °C. The autosampler was set at 6 °C. Mass spectra were acquired in Full MS-ddMS2 mode. The instrument was set up so that Full MS spectra were acquired in an m/z scan range of 150-1600, with an isolation window of 2 m/z. All the Data Dependent Analysis (DDA) files were searched using MaxQuant (https://maxquant.org) v. 2.0.3.0 against the UniProt Juglans database (reviewed and unreviewed, 63749 entries). 3. Results The shot-gun sub-proteome analysis allowed 82, 256 and 119 proteins to be identified in the raw, boiled and roasted walnuts, respectively. The 60 shared proteins (Fig. 1), among the three conditions, include all the oleosome characterizing proteins: one caleosin, one steroleosin and eight oleosins (corresponding to all those indexed in the UniProt KB walnut database). The higher LFQ intensities observed for the caleosins, the steroleosin and the oleosins after boiling, compared to the raw and roasted samples (Fig. 2), suggested that this processing was able to promote some disaggregation phenomena thus improving the protein recovery. Moreover, when considering proteins exclusively present in one condition, the boiled sample is the richest with 149 proteins being identified. A Gene Ontology analysis was carried out to cluster the proteins by three functional categories: biological process (BP), cellular component (CC) and molecular function (MF) (Fig. 3). The "lipid storage" and "lipid metabolic process" were the predominant subcategories for the BP category. These results are consistent with the finding that the walnut kernel is an energy reservoir [6]. In the CC category, the largest subcategory was the "integral component of the membrane", while only for the boiled sample many proteins belonging to "cytoplasm" and "cytosol" subcategories were found; this is probably correlated with the thermal processing, as already demonstrated for hazelnut roasting by Lamberti et al. [5]. 4. Conclusion The shot-gun approach showed an unexpected complexity of the walnut oleosome sub proteome suggesting that the different types of processing may affect the extraction and separation of OB associated proteins, even though to different extents.
APPLICATION OF A µHPLC-HRMS SYSTEM FOR THE PROTEOMIC SHOT-GUN ANALYSIS OF THE WALNUT OLEOSOME
Cirrincione S;Cavallarin L;Lamberti C;Giuffrida M G
2023
Abstract
1 Introduction The walnuts oil bodies (OBs), also known as oleosomes, are spherical organelles (0.5-2.5 ?m in diameter) used by plants to store the energy needed for germination and growth [1]. In nuts, OBs surround larger structures, called protein bodies (PBs), that contain the proteins involved in the embryogenic program of the seed (mainly legumins and vicilins) [2]. At the molecular level, only English walnut (Juglans regia) genome was fully sequenced, which was published in 2016 [3], while a curated database was released in UniProt KB in February 2018. This knowledge at the DNA level has allowed a global proteomic analysis of raw English walnut to be performed [4]. However, because of their localization, their low abundance and the difficulty of their extraction, OB proteins have not yet been fully characterized. Considering the already demonstrated effect of processing on the OB structure of hazelnut [5], a comprehensive proteomic characterization on raw, boiled and roasted walnuts by using the shot-gun proteomic approach was performed. 2. Experimental The walnut protein mixtures were digested O/N at 37 °C under shaking with modified porcine proteomic grade trypsin (1 ?g/?L) at a 1:20 trypsin-protein ratio. The analyses were performed using an Orbitrap Q Exactive Plus, coupled to a UHPLC binary pump system. The stationary phase was a BioBasic(TM) C18 HPLC Column (1 × 150 mm, 5 ?m; Thermo Scientific). The mobile phases were 0.1 % (v/v) FA in MilliQ water (A) and 0.1 % (v/v) FA in ACN (B). and they were eluted at a flow-rate of 68.0 ?L/min at increasing concentrations of solvent B, that is, from 5 % to 45 %, over 57 min, and from 45 % to 50 % in 2 min. The injection volume was 2.0 ?L. The oven temperature was set at 55 °C. The autosampler was set at 6 °C. Mass spectra were acquired in Full MS-ddMS2 mode. The instrument was set up so that Full MS spectra were acquired in an m/z scan range of 150-1600, with an isolation window of 2 m/z. All the Data Dependent Analysis (DDA) files were searched using MaxQuant (https://maxquant.org) v. 2.0.3.0 against the UniProt Juglans database (reviewed and unreviewed, 63749 entries). 3. Results The shot-gun sub-proteome analysis allowed 82, 256 and 119 proteins to be identified in the raw, boiled and roasted walnuts, respectively. The 60 shared proteins (Fig. 1), among the three conditions, include all the oleosome characterizing proteins: one caleosin, one steroleosin and eight oleosins (corresponding to all those indexed in the UniProt KB walnut database). The higher LFQ intensities observed for the caleosins, the steroleosin and the oleosins after boiling, compared to the raw and roasted samples (Fig. 2), suggested that this processing was able to promote some disaggregation phenomena thus improving the protein recovery. Moreover, when considering proteins exclusively present in one condition, the boiled sample is the richest with 149 proteins being identified. A Gene Ontology analysis was carried out to cluster the proteins by three functional categories: biological process (BP), cellular component (CC) and molecular function (MF) (Fig. 3). The "lipid storage" and "lipid metabolic process" were the predominant subcategories for the BP category. These results are consistent with the finding that the walnut kernel is an energy reservoir [6]. In the CC category, the largest subcategory was the "integral component of the membrane", while only for the boiled sample many proteins belonging to "cytoplasm" and "cytosol" subcategories were found; this is probably correlated with the thermal processing, as already demonstrated for hazelnut roasting by Lamberti et al. [5]. 4. Conclusion The shot-gun approach showed an unexpected complexity of the walnut oleosome sub proteome suggesting that the different types of processing may affect the extraction and separation of OB associated proteins, even though to different extents.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
