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.