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To study the acetylation of human serum albumin (HSA) with 2 Naphthyl Acetate (2NA) after digestion with Trypsin and Pepsin

ABSTRACT: This is study done for academic purpose and funded by ICMR, New Delhi, India, sanctioned project titled:- Pseudoesterase activity of albumin as a determinant for serum cholesterol in rabbits. Project File No. 5/4/1-25/2020-NCD-1. Sample processing protocol: For this, 1 ml of 1 mg/ml solution of fatty acid free human albumin (SIGMA) in 10 mM Tris HCl buffer, pH 7.38 was incubated with and without 2NA (prepared in Acetonitrile) for 45 mins at 37 degree Celsius. 100 microlitre of each aliquot was denatured by boiling with 10 mM dithiothreitol for 10 mins followed by the addition of Iodoacetamide (90 mM, SIGMA) and incubation for 30 mins in the dark at 37 degree Celsius. These were then subjected to trypsin digestion (2 microgram, Promega, MS grade, prepared in acetic acid) and Pepsin digestion (2 microgram, Promega, MS grade, prepared in acidic water).Further the samples with Trypsin digestion were incubated for overnight, whereas samples to be digested by pepsin were incubated for 2 hours at 37 degree Celsius. The Trypsin digested reaction was stopped by the addition of 1 percent trifluoroacetic acid (TCI, LC-MS grade), whereas pepsin digestion was stopped by heating samples at 95 degree Celsius. The sample clean-up with C18 resins column was performed and the aliquots were then sent for data acquisition. Data Processing protocol: The MaxQuant software (2.0.3.1) was used for the analysis of data. Under group-specific parameters acetyl K, acetyl (N-term), and (acetyl protein N-term) were selected as variable modifications whereas the Carbamidomethyl (C) was considered a fixed modification. The label-free quantification was done and the albumin sequence file (accession: P02768) was taken as a reference for understanding the analysis. For digestion, trypsin/P type was selected with all other parameters considered as default. In case of Pepsin digestion, Pepsin enzyme was added to MaxQuant enzyme file. The albumin sequence file (accession: P02768) was taken as a reference for understanding the analysis. Description of the raw files is as follow. Control_Trypsin is HSA digested with Trypsin, Control_Pepsin is HSA digested with Pepsin, HSA_2NA_Trypsin is HSA incubated with 2NA and then digested by Trypsin and HSA_2NA_Pepsin is HSA incubated with 2NA & digested by Pepsin.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dibyajyoti Banerjee

PROVIDER: MSV000096587 | MassIVE | Wed Dec 04 03:20:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:To study the acetylation of human serum albumin (HSA) with acetyl CoA in the presence and absence of Dabrafenib, the LC-MS study was carried out. This is a part of ICMR, New Delhi, India, sanctioned project titled:- Pseudoesterase activity of albumin as a determinant for serum cholesterol in rabbits. Project File No. 5/4/1-25/2020-NCD-1. Sample processing protocol: For this, 1 ml of 1 mg/ml solution of fatty acid free human albumin (SIGMA) in 10 mM Tris HCl buffer, pH 7.38 was incubated for 1 hour with different concentrations of Dabrafenib (prepared in DMSO) followed by incubation with 495 micromolar acetyl CoA (prepared in MilliQ water) in separate aliquots for 2 hours at 37 degree Celsius. Control albumin solution was added with appropriate DMSO and MilliQ water and incubated under similar conditions. 100 microlitre of each aliquot was denatured by boiling with 10 mM dithiothreitol for 10 mins followed by the addition of Iodoacetamide (90 mM, SIGMA) and incubation for 30 mins in the dark at 37 degree Celsius. These were then subjected to trypsin digestion (2 microgram, Promega, MS grade, prepared in acetic acid) and kept overnight at 37 degree Celsius. The reaction was stopped by the addition of 1 percent trifluoroacetic acid (TCI, LC-MS grade). The sample clean-up with C18 resins column was performed and the aliquots were then sent for data acquisition. Data Processing protocol: The MaxQuant software (2.0.3.1) was used for the analysis of data. Under group-specific parameters acetyl K, acetyl (N-term), and (acetyl protein N-term) were selected as variable modifications whereas the Carbamidomethyl (C) was considered a fixed modification. The label-free quantification was done and the albumin sequence file (accession: P02768) was taken as a reference for understanding the analysis. For digestion, trypsin/P type was selected with all other parameters considered as default. The albumin sequence file (accession: P02768) was taken as a reference for understanding the analysis. The different raw files obtained are named as follows and uploaded. S1= HSA plus Dabrafenib 400 micromolar plus Acetyl CoA 495 micromolar S2= HSA plus Dabrafenib 200 micromolar plus Acetyl CoA 495 micromolar S3= HS plus Dabrafenib 50 micromolar plus Acetyl CoA 495 micromolar S4= HSA plus Acetyl CoA 495 micromolar S5= HSA only

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To study the acetylation of human serum albumin (HSA) with 2 Naphthyl Acetate (2NA) after digestion with Trypsin and Pepsin

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