Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA008126

Local Sample ID:	26C
Subject ID:	SU000168
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Sample Preparation:

Sampleprep ID:	SP000168
Sampleprep Summary:	MMP and muscle fractions were isolated from frozen samples using differential centrifugation (18, 19). Biopsy samples were homogenized with protease and phosphatase inhibitors (Halt; Thermo Fisher Scientific) and centrifuged to pellet myofibrillar (MYO) proteins. The supernatant was centrifuged to pellet mitochondrial (MITO) proteins, and the final supernatant was deproteinated with cold ethanol (1:9, v/v) and then centrifuged to pellet sarcoplasmic (SARC) proteins. Aliquots from MMP, MYO, SARC, and MITO were acid hydrolyzed, and free amino acids were purified using cation exchange columns and then were dried. Plasma phenylalanine enrichment was determined using gas chromatography (GC) and mass spectrometry (MS) as described previously (19). Samples were derivatized to a heptafluorobutyryl isobutyl ester and identified with a Micromass Quattro Micro triple quadrupole GC-MS system (Waters) operating under negative ion chemical ionization using isobutane as the reactant gas. Selected ion monitoring of m/z 399.2 and 403.2 M + 2 and M + 6 fragments of phenylalanine and the l-[ring-13C6]phenylalanine, respectively, was performed.

Sampleprep ID:

SP000168

Sampleprep Summary:

MMP and muscle fractions were isolated from frozen samples using differential centrifugation (18, 19). Biopsy samples were homogenized with protease and phosphatase inhibitors (Halt; Thermo Fisher Scientific) and centrifuged to pellet myofibrillar (MYO) proteins. The supernatant was centrifuged to pellet mitochondrial (MITO) proteins, and the final supernatant was deproteinated with cold ethanol (1:9, v/v) and then centrifuged to pellet sarcoplasmic (SARC) proteins. Aliquots from MMP, MYO, SARC, and MITO were acid hydrolyzed, and free amino acids were purified using cation exchange columns and then were dried.
Plasma phenylalanine enrichment was determined using gas chromatography (GC) and mass spectrometry (MS) as described previously (19). Samples were derivatized to a heptafluorobutyryl isobutyl ester and identified with a Micromass Quattro Micro triple quadrupole GC-MS system (Waters) operating under negative ion chemical ionization using isobutane as the reactant gas. Selected ion monitoring of m/z 399.2 and 403.2 M + 2 and M + 6 fragments of phenylalanine and the l-[ring-13C6]phenylalanine, respectively, was performed.