Eluates of HA-captured proteins derived from each biological replicate were prepared for mass spectrometry analysis using the FASP (filter-aided sample preparation) method47 (link), with the following modifications. Proteins were reduced with 10 mM Tris-(2-carboxyethyl) phosphine (TCEP), alkylated with 50 mM iodoacetamide, then digested with 1 μg sequence-grade modified trypsin gold (Promega) in 50 mM NH4HCO3 and incubated overnight at 37 °C. Peptides were eluted with 50 mM NH4HCO3 in two 40 μl sequential washes and acidified in 1% formic acid (FA, final concentration).
Sequence grade modified trypsin gold
Manufactured by Promega
Sourced in Germany
Sequence-grade modified trypsin gold is a high-purity proteolytic enzyme used for the digestion of proteins in preparation for mass spectrometry-based proteomic analysis. It is optimized for efficient and specific cleavage at arginine and lysine residues, generating peptides suitable for downstream identification and quantification.
Automatically generated - may contain errors
4 protocols using sequence grade modified trypsin gold
1
Trypsin-based Protein Digestion Protocol
2
Tryptic Digestion and Mass Spectrometry
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Equal amounts of lung lysates (~200 μg) or eluates from SOCS5 immunoprecipitates were subjected to tryptic digest using the FASP protein digestion kit (Protein Discovery) (Wiśniewski et al., 2009 (link)), with the following modifications. Protein material was reduced with TCEP (5 mM final) and digested overnight with 2 μg sequence-grade modified trypsin Gold (Promega) in 50 mM NH4HCO3 at 37°C. Peptides were eluted with 50 mM NH4HCO3 in two 40 μL sequential washes and acidified in 1% formic acid (final). Mass spectrometric analysis was performed as described in Appendix 1.
3
Proteomic Analysis of Diverse Oilseeds
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Acetonitrile (LC–MS grade) and formic acid (MS grade) were obtained from Sigma-Aldrich (Schnelldorf, Germany). Sequence-grade modified trypsin gold, lyophilized, was obtained from Promega GmbH (Mannheim, Germany). Reversed-phase Sep-Pak C18 Plus cartridges, sorbent weight 360 mg/0.7 mL, were obtained from Waters (Milford, MA, USA). All other chemicals were purchased from Sigma-Aldrich, at the best available purity grade.
The material for the study consisted of 10 selected oilseeds, namely, coconut (Cocos nucifera L.), evening primrose (Oenothera biennis L.), hemp (Cannabis sativa L.), flax (Linum usitatissimum L.), milk thistle (Silybum marianum L.), nigella/black cumin (Nigella sativa or N. indica), pumpkin (Cucurbita pepo L.), rapeseed (Brassica napus L.), sesame (Sesamum indicum L.), and sunflower (Helianthus annuus L.). The seeds were obtained from the Polish company SemCo Sp. z o.o. (Szamotuły near Poznań) which specializes in the production of oils. Coconut shreds were purchased at a supermarket. Seeds were stored at 4 °C until further proteomic analysis.
The material for the study consisted of 10 selected oilseeds, namely, coconut (Cocos nucifera L.), evening primrose (Oenothera biennis L.), hemp (Cannabis sativa L.), flax (Linum usitatissimum L.), milk thistle (Silybum marianum L.), nigella/black cumin (Nigella sativa or N. indica), pumpkin (Cucurbita pepo L.), rapeseed (Brassica napus L.), sesame (Sesamum indicum L.), and sunflower (Helianthus annuus L.). The seeds were obtained from the Polish company SemCo Sp. z o.o. (Szamotuły near Poznań) which specializes in the production of oils. Coconut shreds were purchased at a supermarket. Seeds were stored at 4 °C until further proteomic analysis.
4
Identification of Meat and Oilseed Species
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Acetonitrile (LC-MS grade) and formic acid (MS grade) were obtained from Sigma-Aldrich (Schnelldorf, Germany). Sequence-grade modified trypsin gold, lyophilized, was obtained from Promega GmbH (Mannheim, Germany). Reversed-phase Sep-Pak C18 Plus cartridges, sorbent weight 360 mg/0.7 mL, were obtained from Waters (Milford, MA, USA). All other chemicals were purchased from Sigma-Aldrich at the best available purity grade.
The samples for the study (n = 18) consisted of five variants of pork meatballs differing in the quantity of hemp cake, as well as food products purchased at stationary and on-line stores. The commercial products contained various oilseeds.Table 1 presents the meat and oilseed species composition of the analysed samples. The samples were analysed in triplicate. Proteins and peptides derived from guinea fowl (Numida meleagris), rabbit (Oryctolagus cuniculus), pig (Sus scrofa), chicken (Gallus gallus), coconut (Cocos nucifera L.), hemp (Cannabis sativa L.), flax (Linum usitatissimum L.), milk thistle (Silybum marianum L.), nigella/black cumin (Nigella sativa or N. indica), pumpkin (Cucurbita pepo L.), rapeseed (Brassica napus L.), sesame (Sesamum indicum L.), and sunflower (Helianthus annuus L.) were examined in the present study.
The samples for the study (n = 18) consisted of five variants of pork meatballs differing in the quantity of hemp cake, as well as food products purchased at stationary and on-line stores. The commercial products contained various oilseeds.
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