title: Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.
abstract: Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.
authors.affiliation: Metabolomics Resource Core, Mayo Clinic, 200 First St SW, Joseph 5-194, Rochester, MN, 55905, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Hines
authors.forename: G Charles
authors.initials: GC
authors.lastname: Ford
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Klaus
authors.forename: Brian A
authors.initials: BA
authors.lastname: Irving
authors.forename: Beverly L
authors.initials: BL
authors.lastname: Ford
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Johnson
authors.forename: Ian R
authors.initials: IR
authors.lastname: Lanza
authors.forename: K Sreekumaran
authors.initials: KS
authors.lastname: Nair
mesh_terms: D000328:Adult; D000368:Aged; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D008875:Middle Aged; D009124:Muscle Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry*
chemical_list: D009124:Muscle Proteins
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