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LC-MS data of trypsin-digested human serum albumin treated with different concentrations of nateglinide followed by the addition of acetyl CoA

ABSTRACT: To study the acetylation of human serum albumin (HSA) with acetyl CoA in the presence and absence of Nateglinide, 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. 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 Nateglinide (prepared in DMSO) followed by incubation with 495 micromolar acetyl CoA (prepared in MilliQ water) in separate aliquots for 2 hours at 37 degree Celcius. 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 dgree 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 different raw files obtained are named as follows and uploaded. S1 HSA plus Nateglinide 400 micromolar plus Acetyl CoA 495 micromolar S2 HSA plus Nateglinide 200 micromolar plus Acetyl CoA 495 micromolar S3 HS plus Nateglinide 50 micromolar plus Acetyl CoA 495 micromolar S4 HSA plus Acetyl CoA 495 micromolar S5 HSA only

INSTRUMENT(S): Orbitrap Fusion

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

SUBMITTER: Dibyajyoti Banerjee

PROVIDER: MSV000096481 | MassIVE | Thu Nov 21 00:41:00 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

Project description: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.

Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

Project description:Study question: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? Summary answer: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable. What is known already: There are no data in the literature on what non-declared proteins are present in un-conditioned (fresh media in which no embryos have been cultured) commercial embryo media. Study design, size, duration: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analysed for each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analysed. Participants/materials, setting, methods: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Main results and the role of chance: Using advanced mass spectrometry and high confidence criteria for accepting proteins (p< 0.01), a total of 110 proteins other than HSA were identified. The average serum albumin content was found to be 94% (92% - 97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. Limitations, reasons for caution: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. Wider implications of the findings: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analysing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in un-conditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.

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LC-MS data of trypsin-digested human serum albumin treated with different concentrations of nateglinide followed by the addition of acetyl CoA

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