title: Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.
abstract: Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
authors.forename: Paula
authors.initials: P
authors.lastname: Fernández-Guerra
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
authors.forename: Rune I D
authors.initials: RI
authors.lastname: Birkler
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Centro de Investigación en Red de Enfermedades Raras (CIBERER), IDIPAZ, Universidad Autónoma Madrid Madrid, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Merinero
authors.affiliation: Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Centro de Investigación en Red de Enfermedades Raras (CIBERER), IDIPAZ, Universidad Autónoma Madrid Madrid, Spain.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Gregersen
authors.affiliation: Dpto Biol. Mol., Centro de Diagnóstico de Enfermedades Moleculares (CEDEM), Centro de Biología Molecular Severo Ochoa, UAM-CSIC, Centro de Investigación en Red de Enfermedades Raras (CIBERER), IDIPAZ, Universidad Autónoma Madrid Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Rodríguez-Pombo
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Bross
authors.affiliation: Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University and Aarhus University Hospital Aarhus, Denmark.
authors.forename: Johan
authors.initials: J
authors.lastname: Palmfeldt
keywords: BCKDH; MCAD (ACADM); MSUD; SRM; branched-chain amino acid catabolism; liquid chromatography; maple syrup urine disease; mass spectrometry; selected reaction monitoring; tandem mass spectrometry