Integrated proteomic and metabolomic analyses of the mitochondrial neurodegenerative disease MELAS

Abstract

MELAS (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes) is a progressive neurodegenerative disease caused by pathogenic mitochondrial DNA variants. The pathogenic mechanism of MELAS remains enigmatic due to the exceptional clinical...

Protein Contaminants Matter: Building Universal Protein Contaminant Libraries for DDA and DIA Proteomics

“…The routine bottom-up proteomics workflow was conducted for contaminant-only samples, HEK cells, and mouse brain lysates as described previously. , Briefly, disulfide bonds were reduced using 5 mM Tris­(2-carboxyethyl)­phosphine (TCEP) for 30 min, 15 mM iodoacetamide for 30 min in dark, and 5 mM TCEP for 10 min on a ThermoMixer shaking at 1,200 rpm at 37 °C. Protein digestions were conducted using various enzymes (contaminant-only samples) and trypsin/Lys-C (HEK and mouse samples) for 18 h at 37 °C on the ThermoMixer and quenched with 10% trifluoroacetic acid until pH < 3.…”

Protein Contaminants Matter: Building Universal Protein Contaminant Libraries for DDA and DIA Proteomics

“…The routine bottom-up proteomic workflow was conducted for contaminant-only samples, HEK cells and mouse brain lysates as described previously. 30,31 Briefly, disulfide bonds were reduced using 5 mM Tris(2-carboxylethyl)phosphine (TCEP) for 30 min, 15 mM of iodoacetamide for 30 min in dark, and 5 mM TCEP for 10 min on a ThermoMixer shaking at 1,200 rpm at 37 ºC.…”

“…Contaminant FASTA library has been widely used for DDA proteomics, but is rarely included in DIA data analysis. 26,30,37,38 Since DIA uses a much wider precursor isolation window (4-15 Da) compared to DDA (0.4-2 Da), contaminant peptides in DIA are more likely to be coeluted and co-fragmented with other peptides. If not addressed properly, contaminant peptides can cause false identifications of peptides/proteins.…”

“…The routine bottom-up proteomic workflow was conducted for contaminant-only samples, HEK cells and mouse brain lysates as described previously. 35,36 Briefly, disulfide bonds were reduced using 5 mM Tris(2-carboxylethyl)phosphine (TCEP) for 30 min, 15 mM of iodoacetamide for 30 min in dark, and 5 mM TCEP for 10 min on a ThermoMixer shaking at 1,200 rpm at 37 ºC. Protein digestions were conducted using various enzymes (contaminant-only samples) and Trypsin/Lys-C (HEK and mouse samples) for 18 hours at 37 ºC on a ThermoMixer, and quenched with 10% trifluoroacetic acid until pH < 3.…”

Combining multiple acquisition modes and computational data annotation for structural characterization in traditional Chinese medicine: Miao Nationality medicine Qijiao Shengbai Capsule as a case study

“…Therefore, integrated DIA and DDA acquisition modes, which could produce complementary mass spectrometry information from each other, were applied for the comprehensive characterization of compounds from herbal medicines. 10 Moreover, considering that natural herbal medicines oen contained the identical structures of isomers with similar chromatographic retention behaviors that could hardly be separated or detected by common modes of the instrument, the ion mobility model was introduced as an additional dimension of ions separation based on their charge state, size and shape. 11 The exploitation of mass spectrometry data in different scan modes offers the opportunity to analyze chemical components from multiple perspectives.…”